H3K27me3 is a histone modification associated with transcriptional repression that plays a pivotal role in differentiation and development in plants and animals. H2A.Z, a histone variant of H2A, is often colocalized with H3K27me3 in Arabidopsis. Previous genome-wide studies have raised the possibility that H2A.Z contributes to H3K27me3 homeostasis in plants. To examine the contribution of H2A.Z to H3K27me3, we made use of hta9 hta11 plants, which are defective in two of three genes that code for H2A.Z. Use of the quantitative siQ-ChIP pipeline to examine H2A.Z enrichment reveals that hta9 hta11 plants exhibit a two-fold global reduction of H2A.Z abundance. Concurrent analysis of histone protein abundance in the chromatin of hta9 hta11 plants by mass spectrometry confirms a global reduction of H2A.Z and also reveals an altered relative abundance of H2A.Z variants. A similar characterization of H3K27me3 reveals an altered normalized distribution of this histone modification in the genome of hta9 hta11 plants. There is a slight decrease in the global abundance of H3K27me3 at non-expressed genes in hta9 hta11 plants, and hundreds of individual loci exhibit reduced H3K27me3 enrichment. Further, genes marked with H3K27me3 are substantially overrepresented among genes exhibiting differential expression in hta9 hta11 plants. Intriguingly, our analyses raise the possibility that ectopic expression of the transcriptional regulator FUS3 in hta9 hta11 seedlings contributes to altered H3K27me3 enrichment of target genes. Our combined analyses reveal that perturbation of H2A.Z is sufficient to alter H3K27me3 homeostasis in a locus-specific fashion and are consistent with the possibility of a functional relationship between these conserved chromatin features in plants.

Quantitative disease resistance (QDR) is the most common form of disease resistance in crops, but it is challenging to understand at the cellular level due to the involvement of many genes and biological processes. Ralstonia solanacearum, the causal agent of bacterial wilt disease, is a destructive plant pathogen of Solanaceous species that is best controlled by quantitatively resistant varieties, but few QDR genes are known. We previously found that a tomato auxin pathway mutant known as diageotropica (dgt) has enhanced resistance to R. solanacearum. Here, we show that, as with wild-type quantitatively resistant tomato plants, resistance in dgt is the result of multiple mechanisms. Mock-inoculated dgt roots have endogenously higher levels of the plant defense hormone salicylic acid (SA). However, the SA-deficient double mutant dgtNahG is still resistant to R. solanacearum, indicating that SA-independent pathways are also required for resistance. Scanning electron microscopy revealed that R. solanacearum colonization of the root xylem is delayed in dgt. We found an increased number of lignified xylem cells and altered root vasculature anatomy in dgt, and dgt root length was not impacted by R. solanacearum treatment. Similar to the wilt-resistant wild-type tomato Hawaii7996, RNA sequencing results suggested that dgt may tolerate R. solanacearum-induced water stress better than the wilt-susceptible parent. Thus, resistance in dgt is due to several pathways, including preactivated SA defenses, physical barriers in the xylem, and an ability to tolerate water stress. The pleiotropic nature of this single mutation appears to mimic quantitative resistance. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Mediator complex coordinates regulatory input for transcription driven by RNA polymerase II in eukaryotes. reduced epidermal fluorescence4-3 (ref4-3) is a semidominant mutation that results in a single amino acid substitution in the Mediator tail subunit Med5b. Previous characterization of ref4-3 revealed altered expression of a variety of loci in Arabidopsis, including those contributing to phenylpropanoid biosynthesis. Examination of existing RNA-seq data indicated that loci enriched for the transcriptionally repressive chromatin modification H3K27me3 are overrepresented among genes that are misregulated in ref4-3. We used ChIP-seq and RNA-seq to examine the possibility that perturbation of H3K27me3 homeostasis in ref4-3 plants contributed to altered transcript levels. We observed that ref4-3 results in a modest global reduction of H3K27me3 at enriched loci and that this reduction is not dependent on gene expression; however, altered H3K27me3 was not strongly predictive of altered expression in ref4-3 plants. Instead, our analyses revealed a substantial enrichment of targets of the MYC2 transcriptional regulator among genes that exhibit decreased expression in ref4-3. Consistent with previous characterization of ref4-3, we observed that ref4-3-dependent decreased expression of MYC2 targets can be suppressed by loss of another Mediator tail subunit, MED25. This observation is consistent with previous biochemical characterization of MYC2. Our data highlight the diverse and distinct impacts that a single amino acid change in the tail subunit of Mediator can have on transcriptional circuits and raise the prospect that Mediator directly contributes to H3K27me3 homeostasis in plants.

DEGs analysis in high (Gleason grade score{greater than or equal to}8) and low (Gleason grade score<8) grade PCa tumors

<p>DEGs analysis in enzalutamide sensitive- (C4-2) and resistant- (C4-2R) PCa cell lines</p>

<p>Supplementary Materials and Methods and Figure legends.</p>

<p>DEGs analysis in enzalutamide sensitive- (C4-2) and resistant- (C4-2R) PCa cell lines</p>

<p>Table S4: Genomic pathways enrichment analysis. Table S5: Tumor formation with gradient dilution of GFP+ and GFP- C4-2 cells in nude mice. Table S6: Multinomial logistic regression analysis in clinical specimens.</p>

<p>Figure S1. Analysis of correlation between AR and β-catenin in PCa. Figure S2. The Wnt/β-catenin pathway is activated in cell lines and human specimens. Figure S3. Treatment with ICG001 overcomes enzalutamide resistance in PCa. Figure S4. Activation of the Wnt/β-catenin pathway contributes to enzalutamide resistance. Figure S5. Different gene signature in human PCa specimens and correlation between β-catenin and survival probability.</p>

The current dynamic information environment allows readers to access a vast amount of content, but of varied quality. Information professionals increasingly should promote students' scientific news literacy and critical thinking skills. The challenge for instructors, however, is to assess and provide constructive feedback on writing assignments, especially in large-scale online classes. Online peer-assessment tools can facilitate reliable self- and peer-assessment in scientific literacy education at scale. This mixed method study empirically examines differences between self- and peer-assessment and inter-rater reliability among peer assessors and investigates patterns of discrepancies in grading.

Reversible phosphorylation is a pervasive regulatory event in cellular physiology controlled by reciprocal actions of protein kinases and phosphatases. Determining the inherent substrate specificity of kinases and phosphatases is essential for understanding their cellular roles. Synthetic peptides have long served as substrate proxies for defining intrinsic kinase and phosphatase specificities. Here, we describe a high throughput protocol to simultaneously measure specificity constants (kcat/KM) of many synthetic peptide substrates in a single pool using label-free quantitative mass spectrometry. The generation of specificity constants from a single pooled reaction provides a rigorous and rapid comparison of substrate variants to help define an enzyme’s specificity. Equally applicable to kinases and phosphatases, as well as other enzyme classes, the protocol consists of three general steps: (1) reaction of enzyme with pooled peptide substrates, each ideally with a unique mass and at concentrations well below KM, (2) analysis of reaction products using liquid chromatography-coupled mass spectrometry (LC-MS), and (3) automated extraction and integration of elution peaks for each substrate/product pair. We incorporate an ionization correction strategy allowing direct calculation of reaction progress, and subsequently kcat/KM, from substrate and product peak areas in a single sample, obviating the need for stable isotope labeling. Peptide consumption is minimal, and high peptide purity and accurate concentrations are not required. Access to a high-resolution LC-MS system is the only nonstandard equipment need. We present an analysis pipeline consisting entirely of established open-source software tools, and demonstrate proof of principle with the highly selective cell cycle phosphatase Cdc14 from Saccharomyces cerevisiae.

Plants produce several hundreds of thousands of secondary metabolites that are important for adaptation to various environmental conditions. Although different groups of secondary metabolites are synthesized through unique biosynthetic pathways, plants must orchestrate their production simultaneously. Phenylpropanoids and glucosinolates are two classes of secondary metabolites that are synthesized through apparently independent biosynthetic pathways. Genetic evidence has revealed that the accumulation of glucosinolate intermediates limits phenylpropanoid production in a Mediator Subunit 5 (MED5) dependent manner. To elucidate the molecular mechanism underlying this process, we analyzed the transcriptomes of a suite of glucosinolate-deficient mutants using RNAseq and identified mis-regulated genes that are rescued by the disruption of MED5. The expression of a group of Kelch Domain F-Box genes ( KFB s) that function in PAL degradation is affected in glucosinolate biosynthesis mutants and the disruption of these KFBs restores phenylpropanoid deficiency, dwarfism and sterility in the mutants. Our study suggests that glucosinolate/phenylpropanoid metabolic crosstalk involves the transcriptional regulation of KFB genes that initiate the degradation of the enzyme phenylalanine ammonia-lyase, which catalyzes the first step of the phenylpropanoid biosynthesis pathway. Nevertheless, KFB mutant plants remain partially sensitive to glucosinolate pathway mutations, suggesting that other mechanisms that link the two pathways also exist.

PURPOSE. Although zebrafish rods begin to develop as early as 2 days postfertilization (dpf), they are not deemed anatomically mature and functional until 15 to 21 dpf. A recent study detected a small electroretinogram (ERG) from rods in a cone mutant called no optokinetic response f (nof) at 5 dpf, suggesting that young rods are functional. Whether they can mediate behavioral responses in larvae is unknown. METHODS. We first confirmed rod function by measuring nof ERGs under photopic and scotopic illumination at 6 dpf. We evaluated the role of rods in visual behaviors using two different assays: the visual-motor response (VMR) and optokinetic response (OKR). We measured responses from wild-type (WT) larvae and nof mutants under photopic and scotopic illuminations at 6 dpf. RESULTS. Nof mutants lacked a photopic ERG. However, after prolonged dark adaptation, they displayed scotopic ERGs. Compared with WT larvae, the nof mutants displayed reduced VMRs. The VMR difference during light onset gradually diminished with decreased illumination and became nearly identical at lower light intensities. Additionally, light-adapted nof mutants did not display an OKR, whereas dark-adapted nof mutants displayed scotopic OKRs. CONCLUSIONS. Because the nof mutants lacked a photopic ERG but displayed scotopic ERGs after dark adaptation, the mutants clearly had functional rods. WT larvae and the nof mutants displayed comparable scotopic light-On VMRs and scotopic OKRs after dark adaptation, suggesting that these responses were driven primarily by rods. Together, these observations indicate that rods contribute to zebrafish visual behaviors as early as 6 dpf.

Lightning talks of EduHPC are a venue where HPC educators discuss work in progress. In this paper we summarize nine diverse lightning talks, divided in three categories: 1) Training initiatives, describing training programs or degree enhancements carried out in a diverse set of institutions. 2) Tools, describing specific pedagogical resources to be used as aids in the classroom, and 3) Miscellaneous, introducing fun examples and general ideas that can be used to enrich teaching and mentoring settings.

Prostate cancer is the second leading cause of cancer death among men in the United States. The androgen receptor (AR) antagonist enzalutamide is a Food and Drug Administration-approved drug for treatment of patients with late-stage prostate cancer and is currently under clinical study for early-stage prostate cancer treatment. After a short positive response period, tumors will develop drug resistance. In this study using RNA-Seq and bioinformatics analyses, we observed that NOTCH signaling is a deregulated pathway in enzalutamide-resistant cells. NOTCH2 and c-MYC gene expression positively correlated with AR expression in samples from patient with hormone refractory disease in which AR expression levels correspond to those typically observed in enzalutamide resistance. Cleaved NOTCH1, HES1 (Hes family BHLH transcription factor 1), and c-MYC protein expression levels are elevated in two enzalutamide-resistant cell lines, MR49F and C4-2R, indicating NOTCH signaling activation. Moreover, inhibition of the overexpressed ADAM metallopeptidase domain 10 (ADAM10) in the resistant cells induces an exclusive reduction in cleaved NOTCH1 expression. Furthermore, exposure of enzalutamide-resistant cells to both PF-03084014 and enzalutamide increased cell death, decreased colony formation ability, and resensitized cells to enzalutamide. Knockdown of NOTCH1 in C4-2R increased enzalutamide sensitivity by decreasing cell proliferation and increasing cleaved PARP expression. In a 22RV1 xenograft model, PF-03084014 and enzalutamide decreased tumor growth through reducing cell proliferation and increasing apoptosis. These results indicate that NOTCH1 signaling may contribute to enzalutamide resistance in prostate cancer, and inhibition of NOTCH signaling can resensitize resistant cells to enzalutamide.

Abstract Enzalutamide is a second-generation nonsteroidal antiandrogen clinically approved for the treatment of castration-resistant prostate cancer (CRPC), yet resistance to endocrine therapy has limited its success in this setting. Although the androgen receptor (AR) has been associated with therapy failure, the mechanisms underlying this failure have not been elucidated. Bioinformatics analysis predicted that activation of the Wnt/β-catenin pathway and its interaction with AR play a major role in acquisition of enzalutamide resistance. To validate the finding, we show upregulation of β-catenin and AR in enzalutamide-resistant cells, partially due to reduction of β-TrCP–mediated ubiquitination. Although activation of the Wnt/β-catenin pathway in enzalutamide-sensitive cells led to drug resistance, combination of β-catenin inhibitor ICG001 with enzalutamide inhibited expression of stem-like markers, cell proliferation, and tumor growth synergistically in various models. Analysis of clinical datasets revealed a molecule pattern shift in different stages of prostate cancer, where we detected a significant correlation between AR and β-catenin expression. These data identify activation of the Wnt/β-catenin pathway as a major mechanism contributing to enzalutamide resistance and demonstrate the potential to stratify patients with high risk of said resistance. Significance: Wnt/β-catenin inhibition resensitizes prostate cancer cells to enzalutamide. Cancer Res; 78(12); 3147–62. ©2018 AACR.

Objective: This paper describes a project to revise an existing research data management (RDM) course to include instruction in computer skills with robust data science tools. Setting: A Carnegie R1 university. Brief Description: Graduate student researchers need training in the basic concepts of RDM. However, they generally lack experience with robust data science tools to implement these concepts holistically. Two library instructors fundamentally redesigned an existing research RDM course to include instruction with such tools. The course was divided into lecture and lab sections to facilitate the increased instructional burden. Learning objectives and assessments were designed at a higher order to allow students to demonstrate that they not only understood course concepts but could use their computer skills to implement these concepts. Results: Twelve students completed the first iteration of the course. Feedback from these students was very positive, and they appreciated the combination of theoretical concepts, computer skills and hands-on activities. Based on student feedback, future iterations of the course will include more “flipped” content including video lectures and interactive computer tutorials to maximize active learning time in both lecture and lab.

Selective, tissue-specific gene expression is facilitated by the epigenetic modification H3K27me3 (trimethylation of lysine 27 on histone H3) in plants and animals. Much remains to be learned about how H3K27me3-enriched chromatin states are constructed and maintained. Here, we identify a genetic interaction in Arabidopsis thaliana between the chromodomain helicase DNA binding chromatin remodeler PICKLE (PKL), which promotes H3K27me3 enrichment, and the SWR1-family remodeler PHOTOPERIOD INDEPENDENT EARLY FLOWERING1 (PIE1), which incorporates the histone variant H2A.Z. Chromatin immunoprecipitation-sequencing and RNA-sequencing reveal that PKL, PIE1, and the H3K27 methyltransferase CURLY LEAF act in a common gene expression pathway and are required for H3K27me3 levels genome-wide. Additionally, H3K27me3-enriched genes are largely a subset of H2A.Z-enriched genes, further supporting the functional linkage between these marks. We also found that recombinant PKL acts as a prenucleosome maturation factor, indicating that it promotes retention of H3K27me3. These data support the existence of an epigenetic pathway in which PIE1 promotes H2A.Z, which in turn promotes H3K27me3 deposition. After deposition, PKL promotes retention of H3K27me3 after DNA replication and/or transcription. Our analyses thus reveal roles for H2A.Z and ATP-dependent remodelers in construction and maintenance of H3K27me3-enriched chromatin in plants.

Eukaryotes use a temporally regulated process, known as the replication timing program, to ensure that their genomes are fully and accurately duplicated during S phase. Replication timing programs are predictive of genomic features and activity and are considered to be functional readouts of chromatin organization. Although replication timing programs have been described for yeast and animal systems, much less is known about the temporal regulation of plant DNA replication or its relationship to genome sequence and chromatin structure. We used the thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to describe, at high-resolution, the genome-wide replication timing program for Arabidopsis (Arabidopsis thaliana) Col-0 suspension cells. We identified genomic regions that replicate predominantly during early, mid, and late S phase, and correlated these regions with genomic features and with data for chromatin state, accessibility, and long-distance interaction. Arabidopsis chromosome arms tend to replicate early while pericentromeric regions replicate late. Early and mid-replicating regions are gene-rich and predominantly euchromatic, while late regions are rich in transposable elements and primarily heterochromatic. However, the distribution of chromatin states across the different times is complex, with each replication time corresponding to a mixture of states. Early and mid-replicating sequences interact with each other and not with late sequences, but early regions are more accessible than mid regions. The replication timing program in Arabidopsis reflects a bipartite genomic organization with early/mid-replicating regions and late regions forming separate, noninteracting compartments. The temporal order of DNA replication within the early/mid compartment may be modulated largely by chromatin accessibility.

Current antifungal therapies have limited effectiveness in treating invasive fungal infections. Furthermore, the development of new antifungal is currently unable to keep pace with the urgent demand for safe and effective new drugs. Auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, inhibits growth of a diverse array of clinical isolates of fungi and represents a new antifungal agent with a previously unexploited mechanism of action. In addition to auranofin's potent antifungal activity against planktonic fungi, this drug significantly reduces the metabolic activity of Candida cells encased in a biofilm. Unbiased chemogenomic profiling, using heterozygous S. cerevisiae deletion strains, combined with growth assays revealed three probable targets for auranofin's antifungal activity-mia40, acn9, and coa4. Mia40 is of particular interest given its essential role in oxidation of cysteine rich proteins imported into the mitochondria. Biochemical analysis confirmed auranofin targets the Mia40-Erv1 pathway as the drug inhibited Mia40 from interacting with its substrate, Cmc1, in a dose-dependent manner similar to the control, MB-7. Furthermore, yeast mitochondria overexpressing Erv1 were shown to exhibit resistance to auranofin as an increase in Cmc1 import was observed compared to wild-type yeast. Further in vivo antifungal activity of auranofin was examined in a Caenorhabditis elegans animal model of Cryptococcus neoformans infection. Auranofin significantly reduced the fungal load in infected C. elegans. Collectively, the present study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antifungal agent and may offer a safe, effective, and quick supplement to current approaches for treating fungal infections.