Natural Killer Cell Dysfunction in Premenopausal BRCA1 Mutation Carriers: A Potential Mechanism for Ovarian Carcinogenesis.

BACKGROUND: Tissue-specificity for fimbrial fallopian tube ovarian carcinogenesis remains largely unknown in BRCA1 mutation carriers. We aimed to assess the cell autonomous and cell-nonautonomous implications of a germline BRCA1 mutation in the context of cancer immunosurveillance of CD3- CD56+ natural killer (NK) cells. METHODS: Premenopausal BRCA1 mutation carriers versus age-matched non-carriers were compared. Daily urinary 5ß-pregnanediol levels were used to determine progesterone metabolomics across an ovarian cycle. Using peripherally acquired NK cells the cell-mediated cytotoxicity of tumor targets (OVCAR-3, K-562) was determined using live cellular impedance (xCELLigence®) and multicolor flow cytometry. Hypoxia-inducible factor 1-alpha (HIF-1α) immunohistochemistry of cancer-free fallopian tube specimens allowed a comparison of proximal versus distal portions. Utilizing these findings the role of environmental factors relevant to the fimbrial fallopian tube (progesterone, hypoxia) on NK cell functional activity were studied in an ovarian phase-specific manner. RESULTS: BRCA1 mutation carriers demonstrate a differential progesterone metabolome with a phase-specific reduction of peripheral NK cell functional activity. Progesterone exposure further impairs NK cell-mediated cytotoxicity in a dose-dependent manner, which is reversed with the addition of mifepristone (1.25 µM). The fimbrial fallopian tube demonstrated significantly higher HIF-1α staining, particularly in BRCA1 mutation carriers, reflecting a site-specific 'hypoxic niche'. Exposure to hypoxic conditions (1% O2) can further impair tumor cytotoxicity in high-risk carriers. CONCLUSIONS: Phase-specific differential NK cell activity in BRCA1 mutation carriers, either systemically or locally, may favor site-specific pre-invasive carcinogenesis. These cumulative effects across a reproductive lifecycle in high-risk carriers can have a detrimental effect further supporting epidemiological evidence for ovulation inhibition.

FacZ is a GpsB-interacting protein that prevents aberrant division-site placement in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive pathogen responsible for antibiotic-resistant infections. To identify vulnerabilities in cell envelope biogenesis that may overcome resistance, we enriched for S. aureus transposon mutants with defects in cell surface integrity or cell division by sorting for cells that stain with propidium iodide or have increased light-scattering properties, respectively. Transposon sequencing of the sorted populations identified more than 20 previously uncharacterized factors impacting these processes. Cells inactivated for one of these proteins, factor preventing extra Z-rings (FacZ, SAOUHSC_01855), showed aberrant membrane invaginations and multiple FtsZ cytokinetic rings. These phenotypes were suppressed in mutants lacking the conserved cell-division protein GpsB, which forms an interaction hub bridging envelope biogenesis factors with the cytokinetic ring in S. aureus. FacZ was found to interact directly with GpsB in vitro and in vivo. We therefore propose that FacZ is an envelope biogenesis factor that antagonizes GpsB function to prevent aberrant division events in S. aureus.

MICA: a multi-omics method to predict gene regulatory networks in early human embryos.

Recent advances in single-cell omics have transformed characterisation of cell types in challenging-to-study biological contexts. In contexts with limited single-cell samples, such as the early human embryo inference of transcription factor-gene regulatory network (GRN) interactions is especially difficult. Here, we assessed application of different linear or non-linear GRN predictions to single-cell simulated and human embryo transcriptome datasets. We also compared how expression normalisation impacts on GRN predictions, finding that transcripts per million reads outperformed alternative methods. GRN inferences were more reproducible using a non-linear method based on mutual information (MI) applied to single-cell transcriptome datasets refined with chromatin accessibility (CA) (called MICA), compared with alternative network prediction methods tested. MICA captures complex non-monotonic dependencies and feedback loops. Using MICA, we generated the first GRN inferences in early human development. MICA predicted co-localisation of the AP-1 transcription factor subunit proto-oncogene JUND and the TFAP2C transcription factor AP-2γ in early human embryos. Overall, our comparative analysis of GRN prediction methods defines a pipeline that can be applied to single-cell multi-omics datasets in especially challenging contexts to infer interactions between transcription factor expression and target gene regulation.

Identification of FacZ as a division site placement factor in Staphylococcus aureus.

Staphylococcus aureus is a gram-positive pathogen responsible for life-threatening infections that are difficult to treat due to antibiotic resistance. The identification of new vulnerabilities in essential processes like cell envelope biogenesis represents a promising avenue towards the development of anti-staphylococcal therapies that overcome resistance. To this end, we performed cell sorting-based enrichments for S. aureus mutants with defects in envelope integrity and cell division. We identified many known envelope biogenesis factors as well as a large collection of new factors with roles in this process. Mutants inactivated for one of the hits, the uncharacterized SAOUHSC_01855 protein, displayed aberrant membrane invaginations and multiple FtsZ cytokinetic ring structures. This factor is broadly distributed among Firmicutes, and its inactivation in B. subtilis similarly caused division and membrane defects. We therefore renamed the protein FacZ (Firmicute-associated coordinator of Z-rings). In S. aureus, inactivation of the conserved cell division protein GpsB suppressed the division and morphological defects of facZ mutants. Additionally, FacZ and GpsB were found to interact directly in a purified system. Thus, FacZ is a novel antagonist of GpsB function with a conserved role in controlling division site placement in S. aureus and other Firmicutes.

Antiprogestins reduce epigenetic field cancerization in breast tissue of young healthy women.

BACKGROUND: Breast cancer is a leading cause of death in premenopausal women. Progesterone drives expansion of luminal progenitor cells, leading to the development of poor-prognostic breast cancers. However, it is not known if antagonising progesterone can prevent breast cancers in humans. We suggest that targeting progesterone signalling could be a means of reducing features which are known to promote breast cancer formation. METHODS: In healthy premenopausal women with and without a BRCA mutation we studied (i) estrogen and progesterone levels in saliva over an entire menstrual cycle (n = 20); (ii) cancer-free normal breast-tissue from a control population who had no family or personal history of breast cancer and equivalently from BRCA1/2 mutation carriers (n = 28); triple negative breast cancer (TNBC) biopsies and healthy breast tissue taken from sites surrounding the TNBC in the same individuals (n = 14); and biopsies of ER+ve/PR+ve stage T1-T2 cancers and healthy breast tissue taken from sites surrounding the cancer in the same individuals (n = 31); and (iii) DNA methylation and DNA mutations in normal breast tissue (before and after treatment) from clinical trials that assessed the potential preventative effects of vitamins and antiprogestins (mifepristone and ulipristal acetate; n = 44). RESULTS: Daily levels of progesterone were higher throughout the menstrual cycle of BRCA1/2 mutation carriers, raising the prospect of targeting progesterone signalling as a means of cancer risk reduction in this population. Furthermore, breast field cancerization DNA methylation signatures reflective of (i) the mitotic age of normal breast epithelium and (ii) the proportion of luminal progenitor cells were increased in breast cancers, indicating that luminal progenitor cells with elevated replicative age are more prone to malignant transformation. The progesterone receptor antagonist mifepristone reduced both the mitotic age and the proportion of luminal progenitor cells in normal breast tissue of all control women and in 64% of BRCA1/2 mutation carriers. These findings were validated by an alternate progesterone receptor antagonist, ulipristal acetate, which yielded similar results. Importantly, mifepristone reduced both the TP53 mutation frequency as well as the number of TP53 mutations in mitotic-age-responders. CONCLUSIONS: These data support the potential usage of antiprogestins for primary prevention of poor-prognostic breast cancers. TRIAL REGISTRATION: Clinical trial 1 Mifepristone treatment prior to insertion of a levonorgestrel releasing intrauterine system for improved bleeding control - a randomized controlled trial, clinicaltrialsregister.eu, 2009-009014-40 ; registered on 20 July 2009. Clinical trial 2 The effect of a progesterone receptor modulator on breast tissue in women with BRCA1 and 2 mutations, clinicaltrials.gov, NCT01898312 ; registered on 07 May 2013. Clinical trial 3 A pilot prevention study of the effects of the anti- progestin Ulipristal Acetate (UA) on surrogate markers of breast cancer risk, clinicaltrialsregister.eu, 2015-001587-19 ; registered on 15 July 2015.

Susceptibility to hormone-mediated cancer is reflected by different tick rates of the epithelial and general epigenetic clock.

BACKGROUND: A variety of epigenetic clocks utilizing DNA methylation changes have been developed; these clocks are either tissue-independent or designed to predict chronological age based on blood or saliva samples. Whether discordant tick rates between tissue-specific and general epigenetic clocks play a role in health and disease has not yet been explored. RESULTS: Here we analyze 1941 cervical cytology samples, which contain a mixture of hormone-sensitive cervical epithelial cells and immune cells, and develop the WID general clock (Women's IDentification of risk), an epigenetic clock that is shared by epithelial and immune cells and optimized for cervical samples. We then develop the WID epithelial clock and WID immune clock, which define epithelial- and immune-specific clocks, respectively. We find that the WID-relative-epithelial-age (WID-REA), defined as the difference between the epithelial and general clocks, is significantly reduced in cervical samples from pre-menopausal women with breast cancer (OR 2.7, 95% CI 1.28-5.72). We find the same effect in normal breast tissue samples from pre-menopausal women at high risk of breast cancer and show that potential risk reducing anti-progesterone drugs can reverse this. In post-menopausal women, this directionality is reversed. Hormone replacement therapy consistently leads to a significantly lower WID-REA in cancer-free women, but not in post-menopausal women with breast or ovarian cancer. CONCLUSIONS: Our findings imply that there are multiple epigenetic clocks, many of which are tissue-specific, and that the differential tick rate between these clocks may be an informative surrogate measure of disease risk.

Inference of tissue relative proportions of the breast epithelial cell types luminal progenitor, basal, and luminal mature.

Single-cell analysis has revolutionised genomic science in recent years. However, due to cost and other practical considerations, single-cell analyses are impossible for studies based on medium or large patient cohorts. For example, a single-cell analysis usually costs thousands of euros for one tissue sample from one volunteer, meaning that typical studies using single-cell analyses are based on very few individuals. While single-cell genomic data can be used to examine the phenotype of individual cells, cell-type deconvolution methods are required to track the quantities of these cells in bulk-tissue genomic data. Hormone receptor negative breast cancers are highly aggressive, and are thought to originate from a subtype of epithelial cells called the luminal progenitor. In this paper, we show how to quantify the number of luminal progenitor cells as well as other epithelial subtypes in breast tissue samples using DNA and RNA based measurements. We find elevated levels of cells which resemble these hormone receptor negative luminal progenitor cells in breast tumour biopsies of hormone receptor negative cancers, as well as in healthy breast tissue samples from BRCA1 (FANCS) mutation carriers. We also find that breast tumours from carriers of heterozygous mutations in non-BRCA Fanconi Anaemia pathway genes are much more likely to be hormone receptor negative. These findings have implications for understanding hormone receptor negative breast cancers, and for breast cancer screening in carriers of heterozygous mutations of Fanconi Anaemia pathway genes.

Comodularity and detection of co-communities.

This paper introduces the notion of comodularity, to cocluster observations of bipartite networks into co-communities. The task of coclustering is to group together nodes of one type with nodes of another type, according to the interactions that are the most similar. The measure of comodularity is introduced to assess the strength of co-communities, as well as to arrange the representation of nodes and clusters for visualization, and to define an objective function for optimization. We demonstrate the usefulness of our proposed methodology on simulated data, and with examples from genomics and consumer-product reviews.

Fusion of single-cell transcriptome and DNA-binding data, for genomic network inference in cortical development.

BACKGROUND: Network models are well-established as very useful computational-statistical tools in cell biology. However, a genomic network model based only on gene expression data can, by definition, only infer gene co-expression networks. Hence, in order to infer gene regulatory patterns, it is necessary to also include data related to binding of regulatory factors to DNA. RESULTS: We propose a new dynamic genomic network model, for inferring patterns of genomic regulatory influence in dynamic processes such as development. Our model fuses experiment-specific gene expression data with publicly available DNA-binding data. The method we propose is computationally efficient, and can be applied to genome-wide data with tens of thousands of transcripts. Thus, our method is well suited for use as an exploratory tool for genome-wide data. We apply our method to data from human fetal cortical development, and our findings confirm genomic regulatory patterns which are recognised as being fundamental to neuronal development. CONCLUSIONS: Our method provides a mathematical/computational toolbox which, when coupled with targeted experiments, will reveal and confirm important new functional genomic regulatory processes in mammalian development.

CrvA and CrvB form a curvature-inducing module sufficient to induce cell-shape complexity in Gram-negative bacteria.

Bacterial species have diverse cell shapes that enable motility, colonization and virulence. The cell wall defines bacterial shape and is primarily built by two cytoskeleton-guided synthesis machines, the elongasome and the divisome. However, the mechanisms producing complex shapes, like the curved-rod shape of Vibrio cholerae, are incompletely defined. Previous studies have reported that species-specific regulation of cytoskeleton-guided machines enables formation of complex bacterial shapes such as cell curvature and cellular appendages. In contrast, we report that CrvA and CrvB are sufficient to induce complex cell shape autonomously of the cytoskeleton in V. cholerae. The autonomy of the CrvAB module also enables it to induce curvature in the Gram-negative species Escherichia coli, Pseudomonas aeruginosa, Caulobacter crescentus and Agrobacterium tumefaciens. Using inducible gene expression, quantitative microscopy and biochemistry, we show that CrvA and CrvB circumvent the need for patterning via cytoskeletal elements by regulating each other to form an asymmetrically localized, periplasmic structure that binds directly to the cell wall. The assembly and disassembly of this periplasmic structure enables dynamic changes in cell shape. Bioinformatics indicate that CrvA and CrvB may have diverged from a single ancestral hybrid protein. Using fusion experiments in V. cholerae, we find that a synthetic CrvA/B hybrid protein is sufficient to induce curvature on its own, but that expression of two distinct proteins, CrvA and CrvB, promotes more rapid curvature induction. We conclude that morphological complexity can arise independently of cell-shape specification by the core cytoskeleton-guided synthesis machines.

Vibrio cholerae filamentation promotes chitin surface attachment at the expense of competition in biofilms.

Collective behavior in spatially structured groups, or biofilms, is the norm among microbes in their natural environments. Though biofilm formation has been studied for decades, tracing the mechanistic and ecological links between individual cell morphologies and the emergent features of cell groups is still in its infancy. Here we use single-cell-resolution confocal microscopy to explore biofilms of the human pathogen Vibrio cholerae in conditions mimicking its marine habitat. Prior reports have noted the occurrence of cellular filamentation in V. cholerae, with variable propensity to filament among both toxigenic and nontoxigenic strains. Using a filamenting strain of V. cholerae O139, we show that cells with this morphotype gain a profound competitive advantage in colonizing and spreading on particles of chitin, the material many marine Vibrio species depend on for growth in seawater. Furthermore, filamentous cells can produce biofilms that are independent of primary secreted components of the V. cholerae biofilm matrix; instead, filamentous biofilm architectural strength appears to derive at least in part from the entangled mesh of cells themselves. The advantage gained by filamentous cells in early chitin colonization and growth is countered in long-term competition experiments with matrix-secreting V. cholerae variants, whose densely packed biofilm structures displace competitors from surfaces. Overall, our results reveal an alternative mode of biofilm architecture that is dependent on filamentous cell morphology and advantageous in environments with rapid chitin particle turnover. This insight provides an environmentally relevant example of how cell morphology can impact bacterial fitness.

Culture change and mandating quality improvement.

BACKGROUND: A mandatory programme of quality improvement (QI) education was developed for newly qualified Foundation Year 1 (FY1) doctors to complete their curriculum requirements. Their perceptions were evaluated to refine the programme. METHODS: The programme delivered theoretical and experiential learning. Participants were asked to form groups and to come up with their own projects addressing the areas of need that they had identified. The 9-month group project included formal teaching and a formal presentation to the hospital. There was access to facilitation and teaching throughout. Self-rating questionnaires were used to measure the effects of the programme on knowledge and motivation. In a quasi-experimental design, data were compared before and after the programme, and comparison data were collected from the FY1 of the previous year. RESULTS: Across the domains of knowledge, attitudes and motivations, there were no significant differences between the pre-intervention group and the comparison group. Pre- and post-intervention [mean (SD) median] there was a significant increase in overall QI knowledge [2.1 (0.76) 2.0 versus 3.2 (0.62) 3.0; p = 0.000]; however, by the end the FY1s were less motivated to complete the projects [3.9 (0.54) 4.0 versus 3.4 (0.91) 4.0; p = 0.02] and were less positive about them [2.3 (0.69) 2.0 versus 3.0 (0.77) 3.0; p = 0.005]. They felt that the projects were less important for their professional development [3.89 (0.66) 4.0 versus 3.1 (0.73) 3.0; p = 0.000] and were less likely to generate positive change in the long term [2.72 (0.61) 3.0 versus 3.4 (0.92) 3.5; p = 0.007]. Doctors perceptions [of QI] were evaluated to refine a mandatory programme DISCUSSION: Despite delivering a successful theoretical and experiential QI teaching programme with more trainee involvement, the results suggest that mandatory participation for the completion of training risks losing the innovative and creative force of junior doctors and, for some, reducing it to a tick-box exercise.

Single-cell Co-expression Subnetwork Analysis.

Single-cell transcriptomic data have rapidly become very popular in genomic science. Genomic science also has a long history of using network models to understand the way in which genes work together to carry out specific biological functions. However, working with single-cell data presents major challenges, such as zero inflation and technical noise. These challenges require methods to be specifically adapted to the context of single-cell data. Recently, much effort has been made to develop the theory behind statistical network models. This has lead to many new models being proposed, and has provided a thorough understanding of the properties of existing models. However, a large amount of this work assumes binary-valued relationships between network nodes, whereas genomic network analysis is traditionally based on continuous-valued correlations between genes. In this paper, we assess several established methods for genomic network analysis, we compare ways that these methods can be adapted to the single-cell context, and we use mixture-models to infer binary-valued relationships based on gene-gene correlations. Based on these binary relationships, we find that excellent results can be achieved by using subnetwork analysis methodology popular amongst network statisticians. This methodology thereby allows detection of functional subnetwork modules within these single-cell genomic networks.

Parenclitic Network Analysis of Methylation Data for Cancer Identification.

We make use of ideas from the theory of complex networks to implement a machine learning classification of human DNA methylation data, that carry signatures of cancer development. The data were obtained from patients with various kinds of cancers and represented as parenclictic networks, wherein nodes correspond to genes, and edges are weighted according to pairwise variation from control group subjects. We demonstrate that for the 10 types of cancer under study, it is possible to obtain a high performance of binary classification between cancer-positive and negative samples based on network measures. Remarkably, an accuracy as high as 93-99% is achieved with only 12 network topology indices, in a dramatic reduction of complexity from the original 15295 gene methylation levels. Moreover, it was found that the parenclictic networks are scale-free in cancer-negative subjects, and deviate from the power-law node degree distribution in cancer. The node centrality ranking and arising modular structure could provide insights into the systems biology of cancer.

A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis.

Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism and function of this curvature were previously unknown. Here, we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm and thus can be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (quantitative analysis of sacculus architecture remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertions into the outer face than the inner face. Furthermore, crvA is quorum regulated, and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis.

The Role of Entropy in Nanoparticle Agglomeration.

Agglomeration processes in non-interacting particle systems can be understood from a thermodynamic point of view. If the enthalpy of agglomeration is negligible, the distribution of agglomeration states adopts the state of highest entropy. Herein, we provide the exact analytical solution to the mole fractions of agglomerates comprising i monomers, xi =2-i .

Tracking of photochemical Ostwald ripening of nanoparticles through voltammetric atom counting.

We report the tracking of atom count in individual nanoparticles during photochemical Ostwald ripening. The nano-impact technique, in conjunction with UV-Vis and TEM analysis, is used to follow the photochemical formation of silver nano-prisms from spherical seed particles. A mechanism of photochemical Ostwald ripening is deduced and key growth stages are identified.

Femtomolar Detection of Silver Nanoparticles by Flow-Enhanced Direct-Impact Voltammetry at a Microelectrode Array.

We report the femtomolar detection of silver (Ag) nanoparticles by direct-impact voltammetry. This is achieved through the use of a random array of microelectrodes (RAM) integrated into a purpose-built flow cell, allowing combined diffusion and convection to the electrode surface. A coupled RAM-flow cell system is implemented and is shown to give reproducible wall-jet type flow characteristics, using potassium ferrocyanide as a molecular redox species. The calibrated flow system is then used to detect and quantitatively size Ag nanoparticles at femtomolar concentrations. Under flow conditions, it is found the nanoparticle impact frequency increases linearly with the volumetric flow rate. The resulting limit of detection is more than 2 orders of magnitude smaller than the previous detection limit for direct-impact voltammetry (900 fM) [J. Ellison et al. Sens. Actuators, B 2014, 200, 47], and is more than 30 times smaller than the previous detection limit for mediated-impact voltammetry (83 fM) [T. M. Alligrant et al. Langmuir 2014, 30, 13462].