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Higher-order genomic organization supports the activation of histone genes in response to cell cycle regulatory cues that epigenetically mediates stringent control of transcription at the G1/S-phase transition. Histone locus bodies (HLBs) are dynamic, non-membranous, phase-separated nuclear domains where the regulatory machinery for histone gene expression is organized and assembled to support spatiotemporal epigenetic control of histone genes. HLBs provide molecular hubs that support synthesis and processing of DNA replication-dependent histone mRNAs. These regulatory microenvironments support long-range genomic interactions among non-contiguous histone genes within a single topologically associating domain (TAD). HLBs respond to activation of the cyclin E/CDK2/NPAT/HINFP pathway at the G1/S transition. HINFP and its coactivator NPAT form a complex within HLBs that controls histone mRNA transcription to support histone protein synthesis and packaging of newly replicated DNA. Loss of HINFP compromises H4 gene expression and chromatin formation, which may result in DNA damage and impede cell cycle progression. HLBs provide a paradigm for higher-order genomic organization of a subnuclear domain that executes an obligatory cell cycle-controlled function in response to cyclin E/CDK2 signaling. Understanding the coordinately and spatiotemporally organized regulatory programs in focally defined nuclear domains provides insight into molecular infrastructure for responsiveness to cell signaling pathways that mediate biological control of growth, differentiation phenotype, and are compromised in cancer.

The 9.4T scanner in Maastricht is a whole-body magnet with head gradients and parallel RF transmit capability. At the time of the design, it was conceptualized to be one of the best fMRI scanners in the world, but it has also been used for anatomical and diffusion imaging. 9.4T offers increases in sensitivity and contrast, but the technical ultra-high field (UHF) challenges, such as field inhomogeneities and constraints set by RF power deposition, are exacerbated compared to 7T. This article reviews some of the 9.4T work done in Maastricht. Functional imaging experiments included blood oxygenation level-dependent (BOLD) and blood-volume weighted (VASO) fMRI using different readouts. BOLD benefits from shorter T2* at 9.4T while VASO from longer T1. We show examples of both ex vivo and in vivo anatomical imaging. For many applications, pTx and optimized coils are essential to harness the full potential of 9.4T. Our experience shows that, while considerable effort was required compared to our 7T scanner, we could obtain high-quality anatomical and functional data, which illustrates the potential of MR acquisitions at even higher field strengths. The practical challenges of working with a relatively unique system are also discussed.

When microbes grow in foreign nutritional environments, selection may enrich mutations in unexpected pathways connecting growth and homeostasis. An evolution experiment designed to identify beneficial mutations inBurkholderia cenocepaciacaptured six independent nonsynonymous substitutions in the essential genetilS, which modifies tRNAIle2by adding a lysine to the anticodon for faithful AUA recognition. Further, five additional mutants acquired mutations in tRNAIle2, which strongly suggests that disrupting the TilS:tRNAIle2interaction was subject to strong positive selection. Mutated TilS incurred greatly reduced enzymatic function but retained capacity for tRNAIle2binding. Yet both mutant sets outcompeted wild-type by decreasing lag phase duration by ∼3.5 hours. We hypothesized that lysine demand could underlie fitness in the experimental conditions. As predicted, supplemental lysine complemented the ancestral fitness deficit, but so did additions of several other amino acids. Mutant fitness advantages were also specific to rapid growth on galactose using oxidative overflow metabolism that generates redox imbalance, not resources favoring more balanced metabolism. Remarkably, 13tilSmutations also evolved in the Long-Term Evolution Experiment withE. coli, including four fixed mutations. These results suggest that TilS or unknown binding partners contribute to improved growth under conditions of rapid sugar oxidation at the predicted expense of translational accuracy.

S1. RUNX1 mRNA is decreased during breast cancer progression. S2. RUNX1 does not change cell proliferation. S3. RUNX1 represses tumor growth in mammary fat pad. S4. Gate for MCF10AT1 sorting. S5. CD24high Cells have high RUNX1 expression in MCF10AT1 cells. S6. Loss of RUNX1 promotes stemness in MCF10A and MCF7 cells. S7. Overexpression RUNX1 in MCF10CA1a cells does not change BCSC population. S8. Zeb1 is expressed at low level in MCF10CA1a cells. S9. Schematic diagram of ChIP qPCR primers and amplicons over Zeb1 for ChIP-qPCR. S10. Knockdown Zeb1 in MCF10AT1 ns cells does not change cancer stem cell phenotypes.

Breast cancer remains the most common malignant disease in women worldwide. Despite advances in detection and therapies, studies are still needed to understand the mechanisms underlying this cancer. Cancer stem cells (CSC) play an important role in tumor formation, growth, drug resistance, and recurrence. Here, it is demonstrated that the transcription factor RUNX1, well known as essential for hematopoietic differentiation, represses the breast cancer stem cell (BCSC) phenotype and suppresses tumor growth in vivo. The current studies show that BCSCs sorted from premalignant breast cancer cells exhibit decreased RUNX1 levels, whereas ectopic expression of RUNX1 suppresses tumorsphere formation and reduces the BCSC population. RUNX1 ectopic expression in breast cancer cells reduces migration, invasion, and in vivo tumor growth (57%) in mouse mammary fat pad. Mechanistically, RUNX1 functions to suppress breast cancer tumor growth through repression of CSC activity and direct inhibition of ZEB1 expression. Consistent with these cellular and biochemical results, clinical findings using patient specimens reveal that the highest RUNX1 levels occur in normal mammary epithelial cells and that low RUNX1 expression in tumors is associated with poor patient survival.Implications:The key finding that RUNX1 represses stemness in several breast cancer cell lines points to the importance of RUNX1 in other solid tumors where RUNX1 may regulate CSC properties.

Background and Objectives A key strategy for resolving the antibiotic resistance crisis is the development of new drugs with antimicrobial properties. The engineered cationic antimicrobial peptide WLBU2 (also known as PLG0206) is a promising broad-spectrum antimicrobial compound that has completed Phase I clinical studies. It has activity against Gram-negative and Gram-positive bacteria including infections associated with biofilm. No definitive mechanisms of resistance to WLBU2 have been identified. Methodology Here, we used experimental evolution under different levels of mutation supply and whole genome sequencing (WGS) to detect the genetic pathways and probable mechanisms of resistance to this peptide. We propagated populations of wild-type and hypermutator Pseudomonas aeruginosa in the presence of WLBU2 and performed WGS of evolved populations and clones. Results Populations that survived WLBU2 treatment acquired a minimum of two mutations, making the acquisition of resistance more difficult than for most antibiotics, which can be tolerated by mutation of a single target. Major targets of resistance to WLBU2 included the orfN and pmrB genes, previously described to confer resistance to other cationic peptides. More surprisingly, mutations that increase aggregation such as the wsp pathway were also selected despite the ability of WLBU2 to kill cells growing in a biofilm. Conclusions and implications The results show how experimental evolution and WGS can identify genetic targets and actions of new antimicrobial compounds and predict pathways to resistance of new antibiotics in clinical practice.