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Meiotic mapping, a linkage-based method for analyzing the recombinant progeny of a cross, has long been a cornerstone of genetic research. The yeast Saccharomyces cerevisiae is a powerful system because it is possible to isolate and cultivate the four products (spores) of a single meiotic event. However, the throughput of this process has historically been limited by the process of identifying tetrads in a heterogeneous population of vegetative cells, tetrads, and dyads followed by manual separation (dissection) of the spores contained in a tetrad. To date, methods that facilitate high throughput characterization and isolation of meiotic progeny have relied on genetic engineering. Here, we characterize the ability of the fluorescent dye DiBAC(4)(5) to stain yeast tetrads and dyads as well as to adhere to spores following bulk tetrad disruption. Applications include quantitative assays of sporulation rates and efficiency by flow cytometry as well as enrichment of intact tetrads, dyads, or disrupted spores by FACS in strains that have not been genetically modified.

Dysregulated oxidative stress plays a major role in cancer pathogenesis and some types of cancer cells are particularly vulnerable to inhibiting cellular antioxidant capacity. Glutamate-cysteine ligase (GCL) is the first and rate-limiting step in the synthesis of the major cellular antioxidant glutathione (GSH). Developing a GCL inhibitor may be an attractive therapeutic strategy for certain cancer types that are particularly sensitive to oxidative stress. In this study, we reveal a cysteine-reactive covalent ligand EN25 that covalently targets an allosteric cysteine C114 on GCLM, the modifier subunit of GCL, leading to inhibition of GCL activity, lowering of cellular GSH levels, and impaired cell viability in ARID1A-deficient cancer cells that are particularly vulnerable to glutathione depletion, but not in ARID1A-positive cancer cells. Our studies uncover a novel potential ligandable site within GCLM that can be targeted to inhibit the GSH synthesis in cancer cells to target vulnerable cancer cell types.

BackgroundPathogenic variants inPHGDH, PSAT1, andPSPHcause a set of rare, autosomal recessive diseases known as serine biosynthesis defects. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu–Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately in the form of infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, as childhood disease with intellectual disability. However, because L-serine supplementation, especially if started early, can ameliorate and in some cases even prevent symptoms, knowledge of pathogenic variants is highly actionable.MethodsRecently, our laboratory established a yeast-based assay for humanPSAT1function. We have now applied it at scale to assay the functional impact of 1,914 SNV-accessible amino acid substitutions. In addition to assaying the functional impact of individual variants in yeast haploid cells, we can assay pairwise combinations ofPSAT1alleles that recapitulate human genotypes, including compound heterozygotes, in yeast diploids.ResultsResults of our assays of individual variants (in haploid yeast cells) agree well with clinical interpretations and protein structure-function relationships, supporting the use of our data as functional evidence under the ACMG interpretation guidelines. Results from our diploid assay successfully distinguish patient genotypes from those of healthy carriers and agree well with disease severity. Finally, we present a linear model that uses individual allele measurements (in haploid yeast cells) to accurately predict the biallelic function (in diploid yeast cells) of~1.8 million allele combinations corresponding to potential human genotypes.ConclusionsTaken together, our work provides an example of how large-scale functional assays in model systems can be powerfully applied to the study of a rare disease.

Deleterious mutations in the X-linked gene encoding ornithine transcarbamylase (OTC) cause the most common urea cycle disorder, OTC deficiency. This rare, but highly actionable disease can present with severe neonatal onset in males or with later onset in either sex. Neonatal onset patients appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma and death, outcomes ameliorated by rapid diagnosis and treatment. Existing biochemical assays have limitations, including the sensitivity of the citrulline assays used in newborn screening panels. With prior knowledge of variant pathogenicity, DNA sequence-based diagnostics would provide an alternative screening method. Here, we develop a high throughput functional assay for human OTC and measure the impact of 1,570 variants, 84% of all SNP-accessible missense mutations. Our assay scores agree well with existing clinical significance calls, distinguishing known benign from pathogenic variants and variants with neonatal onset from late-onset disease presentation. Further, use of an intronless expression construct allows us to measure the impact of amino acid changes at splice sites independent of their effect on splicing, thereby separating the contribution of splicing and protein coding changes to aid the analysis of molecular mechanisms underlying pathogenicity. Finally, we assess the utility of our functional data onOTCvariant curation by using the current ACMG/AMP guidelines to reclassify variants. Inclusion of our data as PS3/BS3 substantially improves variant interpretation. Thus, our dataset is of high clinical utility and illustrates the power of functional assays to inform interpretation of existing and novel genetic variation.

Background and Purpose: The AFFINITY trial (Assessment of Fluoxetine in Stroke Recovery) reported that oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and seizures. After trial medication was ceased at 6 months, survivors were followed to 12 months post-randomization. This preplanned secondary analysis aimed to determine any sustained or delayed effects of fluoxetine at 12 months post-randomization. Methods: AFFINITY was a randomized, parallel-group, double-blind, placebo-controlled trial in adults (n=1280) with a clinical diagnosis of stroke in the previous 2 to 15 days and persisting neurological deficit who were recruited at 43 hospital stroke units in Australia (n=29), New Zealand (4), and Vietnam (10) between 2013 and 2019. Participants were randomized to oral fluoxetine 20 mg once daily (n=642) or matching placebo (n=638) for 6 months and followed until 12 months after randomization. The primary outcome was function, measured by the modified Rankin Scale, at 6 months. Secondary outcomes for these analyses included measures of the modified Rankin Scale, mood, cognition, overall health status, fatigue, health-related quality of life, and safety at 12 months. Results: Adherence to trial medication was for a mean 167 (SD 48) days and similar between randomized groups. At 12 months, the distribution of modified Rankin Scale categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio, 0.93 [95% CI, 0.76–1.14]; P =0.46). Compared with placebo, patients allocated fluoxetine had fewer recurrent ischemic strokes (14 [2.18%] versus 29 [4.55%]; P =0.02), and no longer had significantly more falls (27 [4.21%] versus 15 [2.35%]; P =0.08), bone fractures (23 [3.58%] versus 11 [1.72%]; P =0.05), or seizures (11 [1.71%] versus 8 [1.25%]; P =0.64) at 12 months. Conclusions: Fluoxetine 20 mg daily for 6 months after acute stroke had no delayed or sustained effect on functional outcome, falls, bone fractures, or seizures at 12 months poststroke. The lower rate of recurrent ischemic stroke in the fluoxetine group is most likely a chance finding. Registration: URL: http://www.anzctr.org.au/ ; Unique identifier: ACTRN12611000774921.

Background: Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population.Methods: AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921.Findings: Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [Interpretation: Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke.Funding: National Health and Medical Research Council of Australia.

Myocardial infarction remains the leading cause of heart failure. The adult human heart lacks the capacity to undergo endogenous regeneration. New blood vessel growth is integral to regenerative medicine necessitating a comprehensive understanding of the pathways that regulate vascular regeneration. We sought to define the transcriptomic dynamics of coronary endothelial cells following ischaemic injuries in the developing and adult mouse and human heart and to identify new mechanistic insights and targets for cardiovascular regeneration.We carried out a comprehensive meta-analysis of integrated single cell RNA-sequencing data of coronary vascular endothelial cells from the developing and adult mouse and human heart spanning healthy and acute and chronic ischaemic cardiac disease. We identified species-conserved gene regulatory pathways aligned to endogenous neovascularisation. We annotated injury-associated temporal shifts of the endothelial transcriptome and validated four genes: VEGF-C, KLF4, EGR1 and ZFP36. Moreover, we showed that ZFP36 regulates human coronary endothelial cell proliferation and defined that VEGF-C administration in vivo enhances clonal expansion of the cardiac vasculature post-myocardial infarction. Finally, we constructed a coronary endothelial cell meta-atlas, CrescENDO, to empower future in-depth research to target pathways associated with coronary neovascularisation.We present a high-resolution single cell meta-atlas of healthy and injured coronary endothelial cells in the mouse and human heart, revealing a suite of novel targets with great potential to promote vascular regeneration, and providing a rich resource for therapeutic development.Myocardial infarction is the leading cause of heart failure, a condition with high morbidity and mortality. Vascular regeneration is vital to tissue survival and functional restoration while a comprehensive understanding of the underpinning mechanisms is lacking. We show that the meta-analysis of single cell RNA-sequencing data excels at deciphering multi-faceted vascular responses conserved between mouse and human following ischaemic injury. We envision that these data will inform future strategies and accelerate therapeutic development for promoting vascular regeneration in the ischaemic heart.

We identify spectral similarities between asteroids and meteorites. We identify spectral matches between 500 asteroid spectra and over 1,000 samples of RELAB meteorite spectra over 0.45-2.5 microns. We reproduce many major and previously known meteorite-asteroid connections and find possible new, more rare or less-established connections. Well-established connections include: ordinary chondrites (OC) with S-complex asteroids; pristine CM carbonaceous chondrites with Ch-type asteroids and heated CMs with C-type asteroids; HED meteorites with V-types; enstatite chondrites with Xc-type asteroids; CV meteorites with K-type asteroids; Brachinites, Pallasites and R chondrites with olivine-dominated A-type asteroids. We find a trend from Q, Sq, S, Sr to Sv correlates with LL to H, with Q-types matching predominately to L and LL ordinary chondrites, and Sr and Sv matching predominantly with L and H ordinary chondrites. Ordinary chondrite samples that match to the X-complex, all measurements of slabs and many labeled as dark or black (shocked) OCs. We find carbonaceous chondrite samples having spectral slopes large enough to match D-type asteroid spectra. In many cases the asteroid type to meteorite type links are not unique. While there are well established matches between an asteroid class and meteorite class, there are less common but still spectrally compatible matches between many asteroid types and meteorite types. This result emphasizes the diversity of asteroid and meteorite compositions and highlights the degeneracy of classification by spectral features alone. Recent and upcoming spacecraft missions will shed light on the compositions of many of the asteroid classes, particularly those without diagnostic features, (C-, B-, X-, and D-types), with measurements of Ceres, Ryugu, Bennu, Psyche, and C-, P-, and D-types as part of the Lucy mission., Comment: Accepted for publication in Icarus. 38 pages, 8 figures

Plant metabolism is more complex relative to individual microbes. In single‐celled microbes, transcriptional regulation by single transcription factors (TFs) is sufficient to shift primary metabolism. Corresponding genome‐level transcriptional regulatory maps of metabolism reveal the underlying design principles responsible for these shifts as a model in which master regulators largely coordinate specific metabolic pathways. Plant primary and specialized metabolism occur within innumerable cell types, and their reactions shift depending on internal and external cues. Given the importance of plants and their metabolites in providing humanity with food, fiber, and medicine, we set out to develop a genome‐scale transcriptional regulatory map of Arabidopsis metabolic genes. A comprehensive set of protein–DNA interactions between Arabidopsis thaliana TFs and gene promoters in primary and specialized metabolic pathways were mapped. To demonstrate the utility of this resource, we identified and functionally validated regulators of the tricarboxylic acid (TCA) cycle. The resulting network suggests that plant metabolic design principles are distinct from those of microbes. Instead, metabolism appears to be transcriptionally coordinated via developmental‐ and stress‐conditional processes that can coordinate across primary and specialized metabolism. These data represent the most comprehensive resource of interactions between TFs and metabolic genes in plants., A transcription factor‐promoter interaction network of primary and specialized metabolism in Arabidopsis thaliana reveals transcriptional coordination across multiple metabolic pathways that is developmental and stress condition‐dependent.

Mixed lineage kinase domain‐like (MLKL) is the executioner in the caspase‐independent form of programmed cell death called necroptosis. Receptor‐interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis‐specific multi‐mono‐ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin‐insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane‐located deubiquitylating enzyme. Oligomerization‐induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome‐ and lysosome‐dependent turnover. Using a MLKL‐DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto‐activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL‐induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.

To determine the prevalence of Mycoplasmataceae species in pregnant women and evaluate their association with immune system mediators.Women were prospectively enrolled between 16-22 weeks' gestation. Vaginal swabs were self-collected and analyzed with PCR forOne-hundred-twenty women were enrolled and had complete lab data available. Colonization was 20.8, 2.5, 10.0, and 48.3% for MH, MG, UU, and UP, respectively. Women with any Mycoplasmataceae were more likely to be younger, of the Black race, and have public insurance. There were no significant differences in immune mediators between women with vaginal Mycoplasmataceae versus those without. After controlling for confounders, women with MH and/or MG had significantly elevated levels of IL-1β compared to women without MH or MG (estimate = 1.12; 95% CI = 0.33, 1.93). There were no other significant differences in immune mediators in women with MH and/or MG compared to those without.Colonization rates were highest for UP and lowest for MG. Higher IL-1β levels were seen in the presence of MH and/or MG, indicating that these less frequently encountered organisms may incite a stronger host response. There were no other significant differences in immune mediator levels.

SummaryIn single-celled microbes, transcriptional regulation by single transcription factors is sufficient to shift primary metabolism. Corresponding genome-level transcriptional regulatory maps of metabolism reveal the underlying design principles responsible for these shifts as a model in which master regulators largely coordinate specific metabolic pathways. Relative to individual microbes, plant metabolism is more complex. Primary and specialized metabolism occur within innumerable cell types, and their reactions shift depending on internal and external cues. Given the importance of plants and their metabolites in providing humanity with food, fiber and medicine, we set out to develop a genome-scale transcriptional regulatory map of Arabidopsis metabolic genes. A comprehensive set of protein-DNA interactions between Arabidopsis thaliana transcription factors and promoters of primary metabolism and specialized metabolism were mapped. To demonstrate the utility of this resource, we identified and functionally validated regulators of the TCA cycle. The resulting network suggests that plant metabolic design principles are distinct from that of microbes. Instead, metabolism appears to be transcriptionally coordinated via developmental- and stress-conditional processes that can coordinate across primary and specialized metabolism. These data represent the most comprehensive resource of interactions between TFs and metabolic genes in plants.

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor significance of this event have been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin insoluble cell fraction comprising plasma/organellar membranes and protein aggregates. This ubiquitylated form is diminished by a plasma membrane located deubiquitylating enzyme. MLKL is ubiquitylated on at least 4 separate lysine residues once oligomerized, and this correlates with proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activity independent of necroptosis signalling in mouse and human cells. Therefore, besides its role in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, ubiquitylation also contributes to the restraint of basal levels of activated MLKL to avoid errant cell death.

The sessile lifestyle of plants requires accurate physiology adjustments to be able to thrive in a changing environment. Plants integrate environmental timing signals to control developmental and stress responses. Here, we identified Far1 Related Sequence (FRS) 7 and FRS12, two transcriptional repressors that accumulate in short-day conditions, as regulators of Arabidopsis glucosinolate (GSL) biosynthesis. Loss of function of FRS7 and FRS12 results in plants with increased amplitudes of diurnal expression of GSL pathway genes. Protein interaction analyses revealed that FRS7 and FRS12 recruit the NOVEL INTERACTOR OF JAZ (NINJA) to assemble a transcriptional repressor complex. Genetic and molecular evidence demonstrated that FRS7, FRS12 and NINJA jointly regulate the expression of GSL biosynthetic genes, and thus constitute a molecular mechanism that modulates specialized metabolite accumulation.

Background Wide-field calcium imaging (WFCI) allows for monitoring of cortex-wide neural dynamics in mice. When applied to the study of sleep, WFCI data are manually scored into the sleep states of wakefulness, non-REM (NREM) and REM by use of adjunct EEG and EMG recordings. However, this process is time-consuming and often suffers from low inter- and intra-rater reliability and invasiveness. Therefore, an automated sleep state classification method that operates on WFCI data alone is needed. New Method A hybrid, two-step method is proposed. In the first step, spatial-temporal WFCI data is mapped to multiplex visibility graphs (MVGs). Subsequently, a two-dimensional convolutional neural network (2D CNN) is employed on the MVGs to be classified as wakefulness, NREM and REM. Results Sleep states were classified with an accuracy of 84% and Cohen’s κ of 0.67. The method was also effectively applied on a binary classification of wakefulness/sleep (accuracy=0.82, κ=0.62) and a four-class wakefulness/sleep/anesthesia/movement classification (accuracy=0.74, κ=0.66). Gradient-weighted class activation maps revealed that the CNN focused on short- and long-term temporal connections of MVGs in a sleep state-specific manner. Sleep state classification performance when using individual brain regions was highest for the posterior area of the cortex and when cortex-wide activity was considered. Comparison with Existing Method On a 3-hour WFCI recording, the MVG-CNN achieved a κ of 0.65, comparable to a κ of 0.60 corresponding to the human EEG/EMG-based scoring. Conclusions The hybrid MVG-CNN method accurately classifies sleep states from WFCI data and will enable future sleep-focused studies with WFCI.

How a plant regulates the relationship between plant growth and plant defense is critical for understanding plant fitness or yield. Yet, little about the required complex underlying interactions are understood.... Plants integrate internal and external signals to finely coordinate growth and defense for maximal fitness within a complex environment. A common model suggests that growth and defense show a trade-offs relationship driven by energy costs. However, recent studies suggest that the coordination of growth and defense likely involves more conditional and intricate connections than implied by the trade-off model. To explore how a transcription factor (TF) network may coordinate growth and defense, we used a high-throughput phenotyping approach to measure growth and flowering in a set of single and pairwise mutants previously linked to the aliphatic glucosinolate (GLS) defense pathway. Supporting a link between growth and defense, 17 of the 20 tested defense-associated TFs significantly influenced plant growth and/or flowering time. The TFs’ effects were conditional upon the environment and age of the plant, and more critically varied across the growth and defense phenotypes for a given genotype. In support of the coordination model of growth and defense, the TF mutant’s effects on short-chain aliphatic GLS and growth did not display a simple correlation. We propose that large TF networks integrate internal and external signals and separately modulate growth and the accumulation of the defensive aliphatic GLS.

Recent medical imaging technologies, specifically functional magnetic resonance imaging (fMRI), have advanced the diagnosis of neurological and neurodevelopmental disorders by allowing scientists and physicians to observe the activity within and between different regions of the brain. Deep learning methods have frequently been implemented to analyze images produced by such technologies and perform disease classification tasks, however, current state-of-the-art approaches do not take advantage of all the information offered by fMRI scans. In this paper, we propose a deep multimodal model that learns a joint representation from two types of connectomic data offered by fMRI scans. Incorporating two functional imaging modalities in an automated end-to-end autism diagnosis system will offer a more comprehensive picture of the neural activity, and thus allow for more accurate diagnoses. Our multimodal training strategy achieves a classification accuracy of 74% and a recall of 95%, as well as an F1 score of 0.805, and its overall performance is superior to using only one type of functional data.

PREMISE OF THE STUDY: Previous phylogenetic studies employing molecular markers have yielded various insights into the evolutionary history across Brassicales, but many relationships between families remain poorly supported or unresolved. A recent phylotranscriptomic approach utilizing 1155 nuclear markers obtained robust estimates for relationships among 14 of 17 families. Here we report a complete family-level phylogeny estimated using the plastid genome. METHODS: We conducted phylogenetic analyses on a concatenated data set comprising 44,926 bp from 72 plastid genes for species distributed across all 17 families. Our analysis includes three additional families, Tovariaceae, Salvadoraceae, and Setchellanthaceae, that were omitted in the previous phylotranscriptomic study. KEY RESULTS: Our phylogenetic analyses obtained fully resolved and strongly supported estimates for all nodes across Brassicales. Importantly, these findings are congruent with the topology reported in the phylotranscriptomic study. This consistency suggests that future studies could utilize plastid genomes as markers for resolving relationships within some notoriously difficult clades across Brassicales. We used this new phylogenetic framework to verify the placement of the At-α event near the origin of Brassicaceae, with median date estimates of 31.8 to 42.8 million years ago and restrict the At-β event to one of two nodes with median date estimates between 85 to 92.2 million years ago. These events ultimately gave rise to novel chemical defenses and are associated with subsequent shifts in net diversification rates. CONCLUSIONS: We anticipate that these findings will aid future comparative evolutionary studies across Brassicales, including selecting candidates for whole-genome sequencing projects.

Nitrogen is an essential macronutrient for plant growth and basic metabolic processes. The application of nitrogen-containing fertilizer increases yield, which has been a substantial factor in the green revolution

© 2015 The Authors. Efficiently obtaining and utilizing energy and elements is critical for an organism to maximize its fitness. Optimizing these processes requires precise regulation and coordination of an organism's metabolic networks in response to diverse environmental conditions and developmental stages. Metabolic regulation is often considered to largely occur by allosteric feedback where the metabolites directly influence the enzymes function. Recent work is showing that there is also an extensive role for transcriptional control of the enzyme encoding genes to construct the metabolic network in response to developmental and environmental stimuli. Within this review, we go through the extensive evidence of how transcription can coordinate the necessary metabolic shifts required to coordinate a plants metabolism with its development and environment. Additionally, we discuss evidence that the metabolites not only feed-back regulate the enzymes but also the upstream transcriptional processes, possibly to stabilize the system.

Coevolutionary interactions are thought to have spurred the evolution of key innovations and driven the diversification of much of life on Earth. However, the genetic and evolutionary basis of the innovations that facilitate such interactions remains poorly understood. We examined the coevolutionary interactions between plants (Brassicales) and butterflies (Pieridae), and uncovered evidence for an escalating evolutionary arms-race. Although gradual changes in trait complexity appear to have been facilitated by allelic turnover, key innovations are associated with gene and genome duplications. Furthermore, we show that the origins of both chemical defenses and of molecular counter adaptations were associated with shifts in diversification rates during the arms-race. These findings provide an important connection between the origins of biodiversity, coevolution, and the role of gene and genome duplications as a substrate for novel traits.

STUDY OBJECTIVE To examine whether a set of virtual reality (VR) surgical simulation drills have correlative validity when compared with the validated Robotic Objective Structured Assessment of Technical Skills (R-OSATS) dry lab drills. DESIGN A prospective methods comparison study (Canadian Task Force classification II-2). SETTING A teaching hospital. PARTICIPANTS Thirty current residents, fellows, and faculty from the Departments of Obstetrics and Gynecology, Urology, and General Surgery. INTERVENTIONS Participants completed 5 VR drills on the da Vinci Skills Simulator and 5 dry lab drills. Participants were randomized to the order of completion. MEASUREMENTS AND MAIN RESULTS VR drills were scored automatically by the simulator. Dry lab drills were recorded, reviewed by 3 blinded experts, and scored using the R-OSATS assessment tool. Spearman correlation coefficients were calculated comparing simulator scores and R-OSATS scores for the same surgeon. The correlation for overall summary scores between VR and dry lab drills was strong (r = 0.83; p

Yeast one-hybrid assays are an in vitro gene-centered approach to map transcription factor-DNA interactions. Here we describe this method and adaptations to screen for interactions between plant transcriptional regulators and their targets. Of particular note, the use of yeast one-hybrid assays fills in an important gap in available methodologies. When one is interested in a specific biological process of interest, the yeast one-hybrid assay is the only method that allows researchers to identify upstream regulators of the biological process of interest. This technique can be also used to further validate physical protein-DNA interactions or as a hypothesis-generating tool. In this method, promoters or DNA regions of interest are cloned and transformed into yeast and tested for interaction against a collection of transcription factors (TFs). Yeast one-hybrid screens are adaptable to the question the researcher is asking and the tools and components available. In this chapter we will describe large-scale and high-throughput Y1H screening; however, this can easily be scaled down for smaller studies.

Yeast one-hybrid assays are an in vitro gene-centered approach to map transcription factor-DNA interactions. Here we describe this method and adaptations to screen for interactions between plant transcriptional regulators and their targets. Of particular note, the use of yeast one-hybrid assays fills in an important gap in available methodologies. When one is interested in a specific biological process of interest, the yeast one-hybrid assay is the only method that allows researchers to identify upstream regulators of the biological process of interest. This technique can be also used to further validate physical protein-DNA interactions or as a hypothesis-generating tool. In this method, promoters or DNA regions of interest are cloned and transformed into yeast and tested for interaction against a collection of transcription factors (TFs). Yeast one-hybrid screens are adaptable to the question the researcher is asking and the tools and components available. In this chapter we will describe large-scale and high-throughput Y1H screening; however, this can easily be scaled down for smaller studies.

Ribonucleotide reductase (RNR) plays a critical role in catalyzing the biosynthesis and maintaining the intracellular concentration of 4 deoxyribonucleoside triphosphates (dNTPs). Unbalanced or deficient dNTP pools cause serious genotoxic consequences. Autophagy is the process by which cytoplasmic constituents are degraded in lysosomes to maintain cellular homeostasis and bioenergetics. However, the role of autophagy in regulating dNTP pools is not well understood. Herein, we reported that starvation- or rapamycin-induced autophagy was accompanied by a decrease in RNR activity and dNTP pools in human cancer cells. Furthermore, downregulation of the small subunit of RNR (RRM2) by siRNA or treatment with the RNR inhibitor hydroxyurea substantially induced autophagy. Conversely, cancer cells with abundant endogenous intracellular dNTPs or treated with dNTP precursors were less responsive to autophagy induction by rapamycin, suggesting that autophagy and dNTP pool levels are regulated through a negative feedback loop. Lastly, treatment with si-RRM2 caused an increase in MAP1LC3B, ATG5, BECN1, and ATG12 transcript abundance in xenografted Tu212 tumors in vivo. Together, our results revealed a previously unrecognized reciprocal regulation between dNTP pools and autophagy in cancer cells.

This essay will reflect on Santa Clara University's (SCU) forays into experiential learning around food justice through the Bronco Urban Gardens (BUG) program. BUG works with urban schools and a community center in San Jose, California, using a garden-based education approach. This program emerged out of our student garden, The Forge. University student farms and gardens provide opportunities for students to learn how to grow, manage, and market food. At Santa Clara University, our half-acre (0.2 hectare) garden plays that role. However, because of our institution's commitment to social justice and a strong network of community partners, our campus garden has blossomed into a larger food justice outreach program. We will first discuss the motivation behind experiential learning for social justice and reflect on its connection to food justice. We then focus on several observations, challenges, and questions that have emerged out of our BUG experiences. Some of those observations involve the challenge of working with students and community partners where the interests of both groups must be served. We also explore what food justice means in this context, and what it means when a program expands beyond the committed few to an entire student body. By engaging in food justice with low-income communities of color through innovative campus programs such as BUG, our students are likely to see the food system from a very different vantage point than if they stayed on campus, resulting in deep learning experiences and also benefits for communities.

Ribonucleotide reductase small subunit B (RRM2B) is a stress response protein that protects normal human fibroblasts from oxidative stress. However, the underlying mechanism that governs this function is not entirely understood. To identify factors that interact with RRM2B and mediate anti-oxidation function, large-scale purification of human Flag-tagged RRM2B complexes was performed. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1, PYCR2) were identified by mass spectrometry analysis as components of RRM2B complexes. Silencing of both PYCR1 and PYCR2 by expressing short hairpin RNAs induced defects in cell proliferation, partial fragmentation of the mitochondrial network and hypersensitivity to oxidative stress in hTERT-immortalized human foreskin fibroblasts (HFF-hTERT). Moderate overexpression of RRM2B, comparable to stress-induced level, protected cells from oxidative stress. Silencing of both PYCR1 and PYCR2 completely abolished anti-oxidation activity of RRM2B, demonstrating a functional collaboration of these metabolic enzymes in response to oxidative stress.

© 2014 American Society of Plant Biologists. All rights reserved. A key unanswered question in plant biology is how a plant regulates metabolism to maximize performance across an array of biotic and abiotic environmental stresses. In this study, we addressed the potential breadth of transcriptional regulation that can alter accumulation of the defensive glucosinolate metabolites in Arabidopsis (Arabidopsis thaliana). A systematic yeast one-hybrid study was used to identify hundreds of unique potential regulatory interactions with a nearly complete complement of 21 promoters for the aliphatic glucosinolate pathway. Conducting high-throughput phenotypic validation, we showed that .75% of tested transcription factor (TF) mutants significantly altered the accumulation of the defensive glucosinolates. These glucosinolate phenotypes were conditional upon the environment and tissue type, suggesting that these TFs may allow the plant to tune its defenses to the local environment. Furthermore, the pattern of TF/promoter interactions could partially explain mutant phenotypes. This work shows that defense chemistry within Arabidopsis has a highly intricate transcriptional regulatory system that may allow for the optimization of defense metabolite accumulation across a broad array of environments.

B-cells are important in the development of type 1 diabetes, but their role is not completely defined. Although B-cells produce autoantibodies, these are not thought to be pathogenic; however, their antigen-presenting function is postulated to be critical. To examine the relative importance of these functions of B-cells, we have generated nonobese diabetic (NOD) B-cell–deficient mice that express a transgene encoding a mutant heavy chain immunoglobulin transgene on the cell surface but cannot secrete immunoglobulins (mIgs). This allowed us to dissect the importance of the relative roles of antigen presentation, dissociated from antibody production. We found that the expression of the mIg transgene increased insulitis and the incidence of diabetes compared with transgene-negative NOD B-cell–deficient mice, indicating that the ability to produce antibodies is not necessary for B-cells to have some effect on the development of diabetes. However, diabetes was not restored to the level seen in normal NOD mice. This may relate to reduced ability to activate an islet-specific T-cell repertoire, presumably due to the reduced islet-specific B-cell repertoire. Our results implicate a specific antigen-presenting function for B-cells.

 Premise of the study: Cruciferous vegetables, many of which are in the genus Brassica (Brassicaceae), are prized for their nutritive value and have been cultivated for thousands of years. There are numerous wild northwestern Mediterranean species in the tribe Brassiceae, and it is therefore assumed this center of diversity is also the region of origin. Within the tribe, the Nigra and Oleracea clades contain the three diploid Brassica crops, B. oleracea , B. rapa , and B. nigra. These three species hybridized in the past to form the tetraploid crop species B. juncea , B. carinata , and B. napus . Collectively, these crop Brassicas have been thought to be closely related because they can still hybridize.  Methods: Using a combination of molecular phylogenetics, diversifi cation analysis, and historical biogeography, we evaluated the relationships and origins of four nested clades: the tribe Brassiceae, the Nigra-Oleracea clade, the core Oleracea (includes B. oleracea + B. rapa and their respective wild relatives), and Brassica oleracea and relatives.  Key results: We found evidence that the tribe originated around the intersection forming between the Arabian Peninsula and Saharan Africa approximately 24 million years ago (Mya). Our data also suggest that the maternal genomes of the three diploid crop Brassicas are not closely related and that the Nigra-Oleracea clade diverged 20 Mya. Finally, our analyses indicate that the core Oleracea lineage giving rise to B. oleracea + B. rapa originated ≈ 3 Mya in the northeastern Mediterranean, from where ancestors of B. oleracea spread through Europe and B. rapa to Asia.  Conclusions: These results challenge previous hypotheses about the biogeographic origins of the tribe Brassiceae and the crop Brassica species and appear to be correlated with major geological and climatic events in the Mediterranean basin.

Camelina sativa is a Brassicaceae oilseed species being explored as a biofuel and industrial oil crop. A growing number of studies have indicated that the turnover of phosphatidylcholine plays an important role in the synthesis and modification of triacylglycerols. This study manipulated the expression of a patatin-related phospholipase AIIIδ (pPLAIIIδ) in camelina to determine its effect on seed oil content and plant growth. Constitutive overexpression of pPLAIIIδ under the control of the constitutive cauliflower mosaic 35S promoter resulted in a significant increase in seed oil content and a decrease in cellulose content. In addition, the content of major membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine, in 35S::pPLAIIIδ plants was increased. However, these changes in 35S::pPLAIIIδ camelina were associated with shorter cell length, leaves, stems, and seed pods and a decrease in overall seed production. When pPLAIIIδ was expressed under the control of the seed specific, β-conglycinin promoter, the seed oil content was increased without compromising plant growth. The results suggest that pPLAIIIδ alters the carbon partitioning by decreasing cellulose content and increasing oil content in camelina.

Autoantibodies from patients with antiphospholipid syndrome (APS) recognize an epitope on β2glycoprotein I (β2GPI) only when native β2GPI is adsorbed on surfaces composed of anionic phospholipids or oxidized polystyrene, β2GPI was modified with the crosslinking agent, glutardialdehyde (GDA), which induced exposure of the anti-β2GPI epitope at GDA: β2GPI mol ratios in the range of 500–2000. A second crosslinking agent, dimethyl-suberimidate (DMS), did not expose the epitope, which may be a consequence of its having less tendency than GDA to form intermolecular links. SDS-PAGE experiments demonstrate that GDA does promote extensive intermolecular crosslinking of β2GPI, and DMS does not. Formaldehyde also reacts with the lysine residues of β2GPI, but does not expose the epitope. The circular dichroism spectra of native and modified β2GPI confirm that GDA induces changes in conformation that are qualitatively different from those caused by formaldehyde. These data provide evidence that binding of lysine residues is not a sufficient condition for exposure of the autoepitope, and also support the likelihood that β2GPI antibodies bind only to aggregates of the protein. Thus, by synthesizing an active holoantigen of β2GPI, conditions were defined that are necessary for binding of human autoantibodies. The authors also suggest that treatment of phospholipid-binding proteins with chemical agents might provide a strategy to modify their structure and permit exposure of epitopes, resulting in synthetic antigens for therapeutic and diagnostic use.

The internal transcribed spacers of the nuclear ribosomal RNA gene cluster, termed ITS1 and ITS2, are the most frequently used nuclear markers for phylogenetic analyses across many eukaryotic groups including most plant families. The reasons for the popularity of these markers include: 1.) Ease of amplification due to high copy number of the gene clusters, 2.) Available cost-effective methods and highly conserved primers, 3.) Rapidly evolving markers (i.e. variable between closely related species), and 4.) The assumption (and/or treatment) that these sequences are non-functional, neutrally evolving phylogenetic markers. Here, our analyses of ITS1 and ITS2 for 50 species suggest that both sequences are instead under selective constraints to preserve proper secondary structure, likely to maintain complete self-splicing functions, and thus are not neutrally-evolving phylogenetic markers. Our results indicate the majority of sequence sites are co-evolving with other positions to form proper secondary structure, which has implications for phylogenetic inference. We also found that the lowest energy state and total number of possible alternate secondary structures are highly significantly different between ITS regions and random sequences with an identical overall length and Guanine-Cytosine (GC) content. Lastly, we review recent evidence highlighting some additional problematic issues with using these regions as the sole markers for phylogenetic studies, and thus strongly recommend additional markers and cost-effective approaches for future studies to estimate phylogenetic relationships.

Common genetic variants that increase the risk for Parkinson's disease may differentiate patient subgroups and influence future individualized therapeutic strategies. Herein we show evidence for leucine-rich repeat kinase 2 (LRRK2) c.4883G>C (R1628P) as a risk factor in ethnic Chinese populations. A study of 1,986 individuals from 3 independent centers in Taiwan and Singapore demonstrates that Lrrk2 R1628P increases risk for Parkinson's disease (odds ratio, 1.84; 95% confidence interval, 1.20-2.83; p = 0.006). Haplotype analysis suggests an ancestral founder for carriers approximately 2,500 years ago. These findings support the importance of LRRK2 variants in sporadic Parkinson's disease. Ann Neurol 2008.

ETV6 (ets translocation variant gene 6) TEL (translocation ets leukemia), encoding a transcriptional repressor, is involved in various translocations associated with human malignancies. Strikingly, the nonrearranged ETV6 allele is often deleted or inactivated in cells harboring these translocations. Although ETV6 translocations are infrequent in acute myeloid leukemia (AML), mutations or deregulated expression of ETV6 may contribute to leukemogenesis. To investigate the involvement of ETV6 in AML, we analysed 300 newly diagnosed patients for mutations in the coding region of the gene. Furthermore, we studied protein expression in 77 patients using two ETV6-specific antibodies. Five somatic heterozygous mutations were detected, which affected either the homodimerization- or the DNA-binding domain of ETV6. The proteins translated from the cDNAs of these mutants were unable to repress transcription and showed dominant-negative effects. In addition, we demonstrate that one-third of AML patients have deficient ETV6 protein expression, which is not related to ETV6 mRNA expression levels. In conclusion, we demonstrate that ETV6 abnormalities are not restricted to translocations and occur more frequently in AML than previously thought. Additional comprehensive studies are required to define the clinical consequence of ETV6 loss of function in AML.

We showed that the frequency of a LRRK2 variant (c.4883G > C, R1628P) was higher in Parkinson’s disease (PD) compared to controls (8.4 vs. 3.4%, P = 0.046, OR 2.5, 95% CI 1.1–5.6). In the multivariate logistic regression (with adjustments made for the effect of age, age of onset, and gender), the heterozygous R1628P genotype was associated with an increased risk of PD compared to controls (OR 3.3, 95% CI 1.4– 7.9, P = 0.007). We provided an independent confirmation that the R1628P variant increases the risk of PD among Chinese.