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Human beings must often perform multiple tasks concurrently or in rapid succession. Laboratory research has revealed striking limitations in the ability to dual task by asking participants to identify two target objects that are inserted into a rapid stream of irrelevant items. Under a variety of conditions, identification of the second target (T2) is impaired for a short period of time following presentation of the first target (T1). Several theories have been developed to account for this “attentional blink” (AB), but none makes a specific prediction about how processing of T1 might impact an observer’s ability to ignore a salient distractor that accompanies T2. Using event-related potentials (ERPs) to track target and distractor processing, we show that healthy young adults are capable of suppressing a salient visual-search distractor (D2) while dual tasking (as measured by the PD component, which has been associated with suppression) but struggle to do so shortly after the appearance of T1. In fact, the impairment was more severe for distractor processing than it was for target processing (as measured by the N2pc component). Whereas, the T2-elicited N2pc was merely delayed during the AB, the distractor PD was reduced in magnitude and was found to be statistically absent. We conclude that the inhibitory control processes that are typically engaged to prevent distraction are unavailable while an observer is busy processing a target that appeared earlier.

Homologs of the mutagenic Escherichia coli DNA polymerase V (pol V) are encoded by numerous pathogens and mobile elements. We have used Rum pol (RumA′2B), from the integrative conjugative element (ICE), R391, as a model mobile element-encoded polymerase (MEPol). The highly mutagenic Rum pol is transferred horizontally into a variety of recipient cells, including many pathogens. Moving between species, it is unclear if Rum pol can function on its own or requires activation by host factors. Here, we show that Rum pol biochemical activity requires the formation of a physical mutasomal complex, Rum Mut, containing RumA′2B-RecA-ATP, with RecA being donated by each recipient bacteria. For R391, Rum Mut specific activities in vitro and mutagenesis rates in vivo depend on the phylogenetic distance of host-cell RecA from E. coli RecA. Rum pol is a highly conserved and effective mobile catalyst of rapid evolution, with the potential to generate a broad mutational landscape that could serve to ensure bacterial adaptation in antibiotic-rich environments leading to the establishment of antibiotic resistance.

Nav1.6 immunostaining of proprioceptive sensory neurons receptor endings

Kv3.3 immunostaining of proprioceptive sensory neurons receptor endings and cell bodies

Cancer and chemotherapy interaction does not lead to electrophysiological evidence of degeneration

Supplemental Figure Legends

Exemplar Graphical Bayesian Model Validation

Heat-map distribution of significantly differentially expressed genes specific to neuron neuronal excitability

Immunohistochemical and morphological analyses of healthy and tumor bearing colons

Uneven ladder walking of an ApcPirc/++control rat.

List of Parameters used for Neurophysiological Analyses

List of gene clusters

GSEA results

ASIC2 immunostaining of proprioceptive sensory neurons receptor endings

Relationship between DEG and biologic processes

vGlut1 immunostaining of proprioceptive sensory neurons receptor endings.

Steps outlining parallel independent transcriptome analyses

For the constellation of neurologic disorders known as chemotherapy-induced peripheral neuropathy, mechanistic understanding and treatment remain deficient. Here, we present the first evidence that chronic sensory neuropathy depends on nonlinear interactions between cancer and chemotherapy. Global transcriptional profiling of dorsal root ganglia revealed differential expression, notably in regulators of neuronal excitability, metabolism, and inflammatory responses, all of which were unpredictable from effects observed with either chemotherapy or cancer alone. Systemic interactions between cancer and chemotherapy also determined the extent of deficits in sensory encoding and ion channel protein expression by single mechanosensory neurons, with the potassium ion channel Kv3.3 emerging as one potential contributor to sensory neuron dysfunction. Validated measures of sensorimotor behavior in awake, behaving animals revealed dysfunction after chronic chemotherapy treatment was exacerbated by cancer. Notably, errors in precise forelimb placement emerged as a novel behavioral deficit unpredicted by our previous study of chemotherapy alone. These original findings identify novel contributors to peripheral neuropathy and emphasize the fundamental dependence of neuropathy on the systemic interaction between chemotherapy and cancer.Significance:These findings highlight the need to account for pathobiological interactions between cancer and chemotherapy as a major contributor to neuropathy and will have significant and immediate impact on future investigations in this field.

Steps of In Vivo Electrophysiology Analysis

Uneven ladder walking of an ApcWT+control rat.

While overall cancer mortality has steadily decreased in recent decades, cancer health disparities among racial and ethnic population groups persist. Here we studied the relationship between cancer survival disparities (CSD), genetic ancestry (GA), and tumor molecular signatures across 33 cancers in a cohort of 9,818 patients. GA correlated with race and ethnicity but showed observable differences in effects on CSD, with significant associations identified in four cancer types: breast invasive carcinoma (BRCA), head and neck squamous cell carcinoma (HNSCC), kidney renal clear cell carcinoma (KIRC), and skin cutaneous carcinoma (SKCM). Differential gene expression and methylation between ancestry groups associated cancer-related genes with CSD, of which, seven protein-coding genes [progestin and adipoQ receptor family member 6 (PAQR6), Lck-interacting transmembrane adaptor 1 (LIME1), Sin3A-associated protein 25 (SAP25), MAX dimerization protein 3 (MXD3), coiled-coil glutamate rich protein 2 (CCER2), refilin A (RFLNA), and cathepsin W (CTSW)] significantly interacted with GA and exacerbated observed survival disparities. These findings indicated that regulatory changes mediated by epigenetic mechanisms have a greater contribution to CSD than population-specific mutations. Overall, we uncovered various molecular mechanisms through which GA might impact CSD, revealing potential population-specific therapeutic targets for groups disproportionately burdened by cancer.Significance:This large-cohort, multicancer study identifies four cancer types with cancer survival disparities and seven cancer-related genes that interact with genetic ancestry and contribute to disparities.

LD1 analysis of all neuronal parameters

Uneven ladder walking of ApcPirc/++OX rats.

Effects of physiologic compensation in ApcWT+control and ApcPirc/++OX neurons.

Longitudinal change in animal weight

Model of molecular determinants of neuronal signaling of sensory neurons

Supplementary Table from Association of Genetic Ancestry and Molecular Signatures with Cancer Survival Disparities: A Pan-Cancer Analysis

'Kruschke style' hierarchical model graphical diagram

Supplementary Data from Association of Genetic Ancestry and Molecular Signatures with Cancer Survival Disparities: A Pan-Cancer Analysis

Supplementary Figures 1-5, Supplementary Tables 1-2 from Rapid Mass Spectrometric Metabolic Profiling of Blood Sera Detects Ovarian Cancer with High Accuracy

Background: Ovarian cancer diagnosis is problematic because the disease is typically asymptomatic, especially at the early stages of progression and/or recurrence. We report here the integration of a new mass spectrometric technology with a novel support vector machine computational method for use in cancer diagnostics, and describe the application of the method to ovarian cancer.Methods: We coupled a high-throughput ambient ionization technique for mass spectrometry (direct analysis in real-time mass spectrometry) to profile relative metabolite levels in sera from 44 women diagnosed with serous papillary ovarian cancer (stages I-IV) and 50 healthy women or women with benign conditions. The profiles were input to a customized functional support vector machine–based machine-learning algorithm for diagnostic classification. Performance was evaluated through a 64-30 split validation test and with a stringent series of leave-one-out cross-validations.Results: The assay distinguished between the cancer and control groups with an unprecedented 99% to 100% accuracy (100% sensitivity and 100% specificity by the 64-30 split validation test; 100% sensitivity and 98% specificity by leave-one-out cross-validations).Conclusion: The method has significant clinical potential as a cancer diagnostic tool. Because of the extremely low prevalence of ovarian cancer in the general population (∼0.04%), extensive prospective testing will be required to evaluate the test's potential utility in general screening applications. However, more immediate applications might be as a diagnostic tool in higher-risk groups or to monitor cancer recurrence after therapeutic treatment.Impact: The ability to accurately and inexpensively diagnose ovarian cancer will have a significant positive effect on ovarian cancer treatment and outcome. Cancer Epidemiol Biomarkers Prev; 19(9); 2262–71. ©2010 AACR.

Immunostaining of sensory neurons in dorsal root ganglia

Kv3.3 Immunostaining of neuromuscular junction

Pathway analysis and GSEA based comparison of the independent effects of cancer and chemotherapy show convergence of mitochondrial dysfunction

Neuronal signaling responses to slow and fast naturalistic stimulus in ApcWT+control and ApcPirc/++OX neurons.

Supplementary Figure 2 from Hyaluronan Deficiency in Tumor Stroma Impairs Macrophage Trafficking and Tumor Neovascularization

Figure S6. Proposed pathway of H1.3 inhibiting H19 and ovarian cancer cell growth. Proposed pathway of H1.3 inhibiting H19 and ovarian cancer cell growth.

Supplementary Figure 3 from Hyaluronan Deficiency in Tumor Stroma Impairs Macrophage Trafficking and Tumor Neovascularization

Figure S1. Overexpression and knockdown of H1.3. A) Generation of OV-3/H1.3 stable cell lines. Western blotting of H1.3 overexpressing clones. OVCAR-3 cells were transfected with a pcDNA-FLAG-H1.3 construct. G418 resistant clones were screened by Western blotting using an anti-FLAG antibody. Coomassie staining shows equal loading of the cell lysates. Lane 1: OVCAR-3; Lane 2: OVCAR-3 transfected with pcDNA3 vector only (designated as OV-3/V.O); Lanes 3-9: OVCAR-3/H1.3 clones. Clones marked with "*" and "#" were used as representative high and low FLAG-H1.3 expressing clones (OV-3/H1.3(H) and OV-3/H1.3(L)). B) Knockdown of H1.3 levels by shRNA in OV-3/H1.3(H) cells. Whole cell lysates were analyzed by Western blotting using an anti-H1.3 antibody. Western blots using an anti-ACTIN antibody were included as loading controls.

Figure S2. Quantitate RT-PCR analysis of selected genes. Upper and lower panels show representative genes downregulated and upregulated, respectively, in OV-3/H1.3(H) as compared with OVCAR-3 cells. Bar: SD.

Table S2. Gene Ontology Categories of Genes with Altered Expression in OV3/H1.3(H) Cells. Gene Ontology Categories of Genes with Altered Expression in OV3/H1.3(H) Cells.

Supplementary Figure 1 from Hyaluronan Deficiency in Tumor Stroma Impairs Macrophage Trafficking and Tumor Neovascularization

Figure S5. ChIP-seq analysis of FLAG-H1.3 in OV-3/H1.3(H) cells. A) Metagene analysis of H1.3 distribution on genes partitioned by gene expression levels (each group containing 20% of genes in genome) over a 10 kb region covering -5 kb to +5 kb of transcription start sites (TSS). On average, H1.3 exhibits the deepest dip at TSS of highly expressed genes (red) but no or minimal dip at TSS of inactive genes (blue). ChIP-seq was performed using anti-FLAG antibody in OV-3/H1.3(H) cells. B) Distribution of H1.3 along H19 (upper) and GAPDH (lower) loci in OV-3/H1.3(H) cells. Y axis: tag counts per 1000 bp window per 10 million mappable reads. IP-IN: normalized signal intensity of ChIP-seq subtracted by that of input-seq.

Ovarian cancer is a deadly gynecologic malignancy for which novel biomarkers and therapeutic targets are imperative for improving survival. Previous studies have suggested the expression pattern of linker histone variants as potential biomarkers for ovarian cancer. To investigate the role of histone H1 in ovarian cancer cells, we characterize individual H1 variants and overexpress one of the major somatic H1 variants, H1.3, in the OVCAR-3 epithelial ovarian cancer cell line. We find that overexpression of H1.3 decreases the growth rate and colony formation of OVCAR-3 cells. We identify histone H1.3 as a specific repressor for the noncoding oncogene H19. Overexpression of H1.3 suppresses H19 expression, and knockdown of H1.3 increases its expression in multiple ovarian epithelial cancer cell lines. Furthermore, we demonstrate that histone H1.3 overexpression leads to increased occupancy of H1.3 at the H19 regulator region encompassing the imprinting control region (ICR), concomitant with increased DNA methylation and reduced occupancy of the insulator protein CTCF at the ICR. Finally, we demonstrate that H1.3 overexpression and H19 knockdown synergistically decrease the growth rate of ovarian cancer cells. Our findings suggest that H1.3 dramatically inhibits H19 expression, which contributes to the suppression of epithelial ovarian carcinogenesis. Cancer Res; 74(22); 6463–73. ©2014 AACR.

Figure S4. Characterization of OVCAR-3/H1.1 cells. A) qChIP analysis of FLAG-H1.1 along H19 regulatory regions in OV-3/H1.1 and OVCAR-3 cells. Dashed line: overlay of FLAG-H1.3 occupancy in OV-3/H1.3 cells (Fig. 4). Bars: SD. B) qChIP analysis of CTCF in OVCAR-3 and OV-3/H1.1(H) cells. C-E) Growth curves (C), MTT assay (D), and colony formation assays (E) of OVCAR-3 and OV-3/H1.1(H) cells. Data are presented as mean {plus minus} SD.

Table S1. Genes with Altered Expression in OV3/H1.3(H) Cells. Genes upregulated or downregulated ({greater than or equal to}2-fold) are shown.

Figure S3. H1.3 suppresses H19 expression and cell growth in OV-90 and SK-OV-3 cells. A) Overexpression of H1.3 in OV-90 cells suppresses H19 expression and cell growth. B) Knockdown of H1.3 in SK-OV-3 cells augments H19 expression and cell growth. Ai, Bi) Reverse phase HPLC and mass spectrometry analysis of linker histone H1 variants in OV-90 cells (Ai) and SK-OV-3 cells (Bi). Insets: ESI-TOF mass spectrometry profiles of the indicated peak eluted from HPLC. Aii, Bii) Western blots of histone extracts (Aii) and whole cell lysates (Bii) from indicated cell lines using an anti-H1.3 antibody. Western blots using anti-H3 and anti-ACTIN antibodies were included as the loading controls. Aiii, Biii) Relative expression of the H19 mRNA transcript level in each indicated cell line analyzed by qRT-PCR. H19 expression unit was normalized over GAPDH, and H19 relative expression of each indicated cell line was normalized over respective OV-90 (Aiii) and SK-OV-3 (Biii) cells. Bar: SD. Overexpressed exogenous FLAG-H1 levels were analyzed by immunoblotting using an anti-FLAG antibody (Aiii middle). Equal loading of histone extracts is indicated by anti-H3 Western blots (Aiii lower). Aiv, Biv) Growth curves of indicated cells. Data are presented as mean {plus minus} SD. P

Supplementary Methods, Figure Legends 1-3 from Hyaluronan Deficiency in Tumor Stroma Impairs Macrophage Trafficking and Tumor Neovascularization

Stiffness has been observed to decrease for many cancer cell types as their metastatic potential increases. Although cell mechanics and metastatic potential are related, the underlying molecular factors associated with these phenotypes remain unknown. Therefore, we have developed a workflow to measure the mechanical properties and gene expression of single cells that is used to generate large linked-datasets. The process combines atomic force microscopy to measure the mechanics of individual cells with multiplexed RT-qPCR gene expression analysis on the same single cells. Surprisingly, the genes that most strongly correlated with mechanical properties were not cytoskeletal, but rather were markers of extracellular matrix remodeling, epithelial-to-mesenchymal transition, cell adhesion, and cancer stemness. In addition, dimensionality reduction analysis showed that cell clustering was improved by combining mechanical and gene expression data types. The single cell genomechanics method demonstrates how single cell studies can identify molecular drivers that could affect the biophysical processes underpinning metastasis.

IntroductionAs antibiotic resistance has become more prevalent, the social and economic impacts are increasingly pressing. Indeed, bacteria have developed the SOS response which facilitates the evolution of resistance under genotoxic stress. The transcriptional repressor, LexA, plays a key role in this response. Mutation of LexA to a non-cleavable form that prevents the induction of the SOS response sensitizes bacteria to antibiotics. Achieving the same inhibition of proteolysis with small molecules also increases antibiotic susceptibility and reduces drug resistance acquisition. The availability of multiple LexA crystal structures, and the unique Ser-119 and Lys-156 catalytic dyad in the protein enables the rational design of inhibitors.MethodsWe pursued a binary approach to inhibit proteolysis; we first investigated β-turn mimetics, and in the second approach we tested covalent warheads targeting the Ser-119 residue. We found that the cleavage site region (CSR) of the LexA protein is a classical Type II β-turn, and that published 1,2,3-triazole compounds mimic the β-turn. Generic covalent molecule libraries and a β-turn mimetic library were docked to the LexA C-terminal domain using molecular modelling methods in FlexX and CovDock respectively. The 133 highest-scoring molecules were screened for their ability to inhibit LexA cleavage under alkaline conditions. The top molecules were then tested using a RecA-mediated cleavage assay.ResultsThe β-turn library screen did not produce any hit compounds that inhibited RecA-mediated cleavage. The covalent screen discovered an electrophilic serine warhead that can inhibit LexA proteolysis, reacting with Ser-119 via a nitrile moiety. DiscussionThis research presents a starting point for hit-to-lead optimisation, which could lead to inhibition of the SOS response and prevent the acquisition of antibiotic resistance.