Genetic variation at mouse and human ribosomal DNA influences associated epigenetic states.

BACKGROUND: Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. A systematic analysis of how this variation impacts epigenetic states and expression of the rDNA has thus far not been performed. RESULTS: Using a combination of long- and short-read sequencing, we establish that 45S rDNA units in the C57BL/6J mouse strain exist as distinct genetic haplotypes that influence the epigenetic state and transcriptional output of any given unit. DNA methylation dynamics at these haplotypes are dichotomous and life-stage specific: at one haplotype, the DNA methylation state is sensitive to the in utero environment, but refractory to post-weaning influences, whereas other haplotypes entropically gain DNA methylation during aging only. On the other hand, individual rDNA units in human show limited evidence of genetic haplotypes, and hence little discernible correlation between genetic and epigenetic states. However, in both species, adjacent units show similar epigenetic profiles, and the overall epigenetic state at rDNA is strongly positively correlated with the total rDNA copy number. Analysis of different mouse inbred strains reveals that in some strains, such as 129S1/SvImJ, the rDNA copy number is only approximately 150 copies per diploid genome and DNA methylation levels are < 5%. CONCLUSIONS: Our work demonstrates that rDNA-associated genetic variation has a considerable influence on rDNA epigenetic state and consequently rRNA expression outcomes. In the future, it will be important to consider the impact of inter-individual rDNA (epi)genetic variation on mammalian phenotypes and diseases.

Identification of the transcription factor MAZ as a regulator of erythropoiesis.

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.

Effect of the Ductal Carcinoma In Situ Margin Consensus Guideline Implementation on Re-Excision Rates, Satisfaction, and Cost.

INTRODUCTION: The 2016 consensus guideline on margins for breast-conserving surgery (BCS) with whole-breast irradiation (WBI) for ductal carcinoma in situ (DCIS) recommended 2 mm margins to decrease local recurrence rates. We examined re-excision rates, cost, and patient satisfaction before and after guideline implementation. METHODS: From an Institutional Review Board-approved database, patients with DCIS who underwent BCS with over 1 year of follow-up at one academic institution and one community cancer center were evaluated. Two groups were compared based on when they received treatment, i.e. before (pre-consensus [PRE]) and after November 2016 (post consensus [POST]), with respect to outcome and cost parameters. RESULTS: After consensus guideline implementation, re-excision rate (32.1% vs. 20.0%) and mastectomy conversion (8.3% vs. 2.3%) significantly increased, although total resection volume, operative cost per patient, and satisfaction with breast scores did not differ. Not all patients with <2 mm margins were re-excised, although the re-excision rate among this subset significantly increased (62.4% vs. 31.3%). On multivariable analysis controlling for age, estrogen receptor status, WBI use, and margin status, surgery after consensus guideline publication was independently associated with a higher re-excision rate (odds ratio [OR] 1.97, 95% confidence interval [CI] 1.08-3.59, p = 0.03) and a higher rate of conversion to mastectomy (OR 6.84, 95% CI 1.67-28.00, p = 0.007). CONCLUSIONS: Implementation of the 2016 margin consensus guideline for DCIS resulted in an increase in re-excisions and mastectomy conversions at two institutions. Research is needed for operative tools and strategies to decrease DCIS re-excision rates.

The DNA methylome of human sperm is distinct from blood with little evidence for tissue-consistent obesity associations.

Epidemiological research suggests that paternal obesity may increase the risk of fathering small for gestational age offspring. Studies in non-human mammals indicate that such associations could be mediated by DNA methylation changes in spermatozoa that influence offspring development in utero. Human obesity is associated with differential DNA methylation in peripheral blood. It is unclear, however, whether this differential DNA methylation is reflected in spermatozoa. We profiled genome-wide DNA methylation using the Illumina MethylationEPIC array in a cross-sectional study of matched human blood and sperm from lean (discovery n = 47; replication n = 21) and obese (n = 22) males to analyse tissue covariation of DNA methylation, and identify obesity-associated methylomic signatures. We found that DNA methylation signatures of human blood and spermatozoa are highly discordant, and methylation levels are correlated at only a minority of CpG sites (~1%). At the majority of these sites, DNA methylation appears to be influenced by genetic variation. Obesity-associated DNA methylation in blood was not generally reflected in spermatozoa, and obesity was not associated with altered covariation patterns or accelerated epigenetic ageing in the two tissues. However, one cross-tissue obesity-specific hypermethylated site (cg19357369; chr4:2429884; P = 8.95 × 10-8; 2% DNA methylation difference) was identified, warranting replication and further investigation. When compared to a wide range of human somatic tissue samples (n = 5,917), spermatozoa displayed differential DNA methylation across pathways enriched in transcriptional regulation. Overall, human sperm displays a unique DNA methylation profile that is highly discordant to, and practically uncorrelated with, that of matched peripheral blood. We observed that obesity was only nominally associated with differential DNA methylation in sperm, and therefore suggest that spermatozoal DNA methylation is an unlikely mediator of intergenerational effects of metabolic traits.

Constructing Normalcy in Dementia Care: Carers' Perceptions of Their Roles and the Supports They Need.

BACKGROUND AND OBJECTIVES: People with dementia are critically dependent on their carers when accessing and utilizing health care. To inform health care development and delivery, we aimed to explore carers' perceptions of their role in caring for a family member with dementia and to identify carers' skills and attributes and factors impacting on care. RESEARCH DESIGN AND METHODS: We used semistructured interviews to collect data from 25 carers supporting older adults with dementia. Data were thematically analyzed and the paradigm model was used to guide theory development. RESULTS: "Constructing normalcy" was central to all carers did, impacted by stage of life and relationship status and driven by a holistic focus on their care-recipient's quality of life. Goals guiding care were: keeping the peace; facilitating participation, happiness and independence; and ensuring safety. Enablers included: social contact; knowledge; and quality social services. Barriers included health and legal issues; symptoms of dementia; and reduced knowledge. These goals kept the peace and reduced stress for the cared-for person, but often at the cost of unrelenting responsibility and loss of carers' original roles. Discussion and Implications: As carers are so critical to the access and uptake of health care of those with dementia, health professionals and services need to support carers in their quest to construct normalcy. Our findings provide guidance to assist in ensuring appropriate support and understanding of carers work in order to optimize dementia health care delivery.

DNA methylation at the crossroads of gene and environment interactions.

DNA methylation is an epigenetic mark involved in regulating genome function and is critical for normal development in mammals. It has been observed that the developmental environment can lead to permanent changes in gene expression and DNA methylation, at least at 'metastable epialleles'. These are defined as regions of the genome that show a variable epigenetic state that is established early in development and maintained through subsequent cell divisions. However, the majority of the known genome does not behave in this manner. Here, we use the developmental origins of adult disease hypothesis to understand environmental epigenomics. Some challenges to studying how DNA methylation is influenced by the environment include identifying DNA methylation changes associated with an environmental exposure in tissues with a complex cellular composition and at genomic regions for which DNA methylation is dynamically regulated in a cell-type specific manner. We also offer a perspective of how emerging technologies may be useful for dissecting the functional contribution of exposure-associated epigenetic changes and highlight recent evidence that suggests that genomic regions that are absent from genome assemblies may be unappreciated hotspots for environmental modulation of the epigenetic state.

Strain-Specific Metabolic Requirements Revealed by a Defined Minimal Medium for Systems Analyses of Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive pathogenic bacterium that colonizes an estimated one-third of the human population and can cause a wide spectrum of disease, ranging from superficial skin infections to life-threatening sepsis. The adaptive mechanisms that contribute to the success of this pathogen remain obscure partially due to a lack of knowledge of its metabolic requirements. Systems biology approaches can be extremely useful in predicting and interpreting metabolic phenotypes; however, such approaches rely on a chemically defined minimal medium as a basis to investigate the requirements of the cell. In this study, a chemically defined minimal medium formulation, termed synthetic minimal medium (SMM), was investigated and validated to support growth of three S. aureus strains: LAC and TCH1516 (USA300 lineage), as well as D592 (USA100 lineage). The formulated SMM was used in an adaptive laboratory evolution experiment to probe the various mutational trajectories of all three strains leading to optimized growth capabilities. The evolved strains were phenotypically characterized for their growth rate and antimicrobial susceptibility. Strains were also resequenced to examine the genetic basis for observed changes in phenotype and to design follow-up metabolite supplementation assays. Our results reveal evolutionary trajectories that arose from strain-specific metabolic requirements. SMM and the evolved strains can also serve as important tools to study antibiotic resistance phenotypes of S. aureusIMPORTANCE As researchers try to understand and combat the development of antibiotic resistance in pathogens, there is a growing need to thoroughly understand the physiology and metabolism of the microbes. Staphylococcus aureus is a threatening pathogen with increased antibiotic resistance and well-studied virulence mechanisms. However, the adaptive mechanisms used by this pathogen to survive environmental stresses remain unclear, mostly due to the lack of information about its metabolic requirements. Defining the minimal metabolic requirements for S. aureus growth is a first step toward unraveling the mechanisms by which it adapts to metabolic stresses. Here, we present the development of a chemically defined minimal medium supporting growth of three S. aureus strains, and we reveal key genetic mutations contributing to improved growth in minimal medium.

Surprising synergy of dual translation inhibition vs. Acinetobacter baumannii and other multidrug-resistant bacterial pathogens.

BACKGROUND: Multidrug-resistant (MDR) Acinetobacter baumannii infections have high mortality rates and few treatment options. Synergistic drug combinations may improve clinical outcome and reduce further emergence of resistance in MDR pathogens. Here we show an unexpected potent synergy of two translation inhibitors against the pathogen: commonly prescribed macrolide antibiotic azithromycin (AZM), widely ignored as a treatment alternative for invasive Gram-negative pathogens, and minocycline, among the current standard-of-care agents used for A. baumannii. METHODS: Media-dependent activities of AZM and MIN were evaluated in minimum inhibitory concentration assays and kinetic killing curves, alone or in combination, both in standard bacteriologic media (cation-adjusted Mueller-Hinton Broth) and more physiologic tissue culture media (RPMI), with variations of bicarbonate as a physiologic buffer. Synergy was calculated by fractional inhibitory concentration index (FICI). Therapeutic benefit of combining AZM and MIN was tested in a murine model of A. baumannii pneumonia. AZM + MIN synergism was probed mechanistically by bacterial cytological profiling (BCP), a quantitative fluorescence microscopy technique that identifies disrupted bacterial cellular pathways on a single cell level, and real-time kinetic measurement of translation inhibition via quantitative luminescence. AZM + MIN synergism was further evaluated vs. other contemporary high priority MDR bacterial pathogens. FINDINGS: Although two translation inhibitors are not expected to synergize, each drug complemented kinetic deficiencies of the other, speeding the initiation and extending the duration of translation inhibition as verified by FICI, BCP and kinetic luminescence markers. In an MDR A. baumannii pneumonia model, AZM + MIN combination therapy decreased lung bacterial burden and enhanced survival rates. Synergy between AZM and MIN was also detected vs. MDR strains of Gram-negative Klebsiella pneumoniae and Pseudomonas aeruginosa, and the leading Gram-positive pathogen methicillin-resistant Staphylococcus aureus. INTERPRETATION: As both agents are FDA approved with excellent safety profiles, clinical investigation of AZM and MIN combination regimens may immediately be contemplated for optimal treatment of A. baumannii and other MDR bacterial infections in humans. FUND: National Institutes of Health U01 AI124326 (JP, GS, VN) and U54 HD090259 (GS, VN). IC was supported by the UCSD Research Training Program for Veterinarians T32 OD017863.

A prospective, multi-institution assessment of irreversible electroporation for treatment of locally advanced pancreatic adenocarcinoma: initial outcomes from the AHPBA pancreatic registry.

BACKGROUND: The optimal treatment and management of locally advanced pancreatic cancer (LAPC) remains unclear and controversial. This study aimed to report the initial outcomes of the AHPBA Registry and evaluate the reproducibility of existing evidence that the addition of Irreversible Electroporation (IRE), a nonthermal ablative treatment, confers survival benefits beyond standard therapeutic options for patients with LAPC. METHODS: From December 2015 to October 2017, patients with LAPC were treated with open-technique IRE following the AHPBA Registry Protocols. Patient demographics, long-term outcomes, and adverse events were recorded. Survival analyses were performed using Kaplan-Meier (KM) curves for overall survival (OS), progression free survival (PFS) and time to progression (TTP). RESULTS: A total of 152 patients underwent successful IRE. Morbidity and mortality were 18% and 2% respectively, with 19 (13%) patients experiencing severe adverse events. Nine (6%) patients presented with local recurrence. Median TTP, PFS, and OS from diagnosis were 27.3 months, 22.8 months, and 30.7 months respectively. CONCLUSION: The combination of IRE with established multiagent therapy is safe and demonstrates encouraging survival among patients with LAPC. IRE is associated with a low rate of serious adverse events and has been optimized for more widespread adoption through the standardized protocols available through the AHPBA registry.

Deciphering the Role of the Non-Coding Genome in Regulating Gene-Diet Interactions.

Protein encoding genes constitute a small fraction of mammalian genomes. In addition to the protein coding genes, there are other functional units within the genome that are transcribed, but not translated into protein, the so called non-coding RNAs. There are many types of non-coding RNAs that have been identified and shown to have important roles in regulating gene expression either at the transcriptional or post-transcriptional level. A number of recent studies have highlighted that dietary manipulation in mammals can influence the expression or function of a number of classes of non-coding RNAs that contribute to the protein translation machinery. The identification of protein translation as a common target for nutritional regulation underscores the need to investigate how this may mechanistically contribute to phenotypes and diseases that are modified by nutritional intervention. Finally, we describe the state of the art and the application of emerging '-omics' technologies to address the regulation of protein translation in response to diet.

Early life diet conditions the molecular response to post-weaning protein restriction in the mouse.

BACKGROUND: Environmental influences fluctuate throughout the life course of an organism. It is therefore important to understand how the timing of exposure impacts molecular responses. Herein, we examine the responses of two key molecular markers of dietary stress, namely variant-specific methylation at ribosomal DNA (rDNA) and small RNA distribution, including tRNA fragments, in a mouse model of protein restriction (PR) with exposure at pre- and/or post-weaning. RESULTS: We first confirm that pre-weaning PR exposure modulates the methylation state of rDNA in a genotype-dependent manner, whereas post-weaning PR exposure has no such effect. Conversely, post-weaning PR induces a shift in small RNA distribution, but there is no effect in the pre-weaning PR model. Intriguingly, mice exposed to PR throughout their lives show neither of these two dietary stress markers, similar to controls. CONCLUSIONS: The results show that the timing of the insult affects the nature of the molecular response but also, critically, that 'matching' diet exposure either side of weaning eliminates the stress response at the level of rDNA methylation and small RNA in sperm.

Early-life nutrition modulates the epigenetic state of specific rDNA genetic variants in mice.

A suboptimal early-life environment, due to poor nutrition or stress during pregnancy, can influence lifelong phenotypes in the progeny. Epigenetic factors are thought to be key mediators of these effects. We show that protein restriction in mice from conception until weaning induces a linear correlation between growth restriction and DNA methylation at ribosomal DNA (rDNA). This epigenetic response remains into adulthood and is restricted to rDNA copies associated with a specific genetic variant within the promoter. Related effects are also found in models of maternal high-fat or obesogenic diets. Our work identifies environmentally induced epigenetic dynamics that are dependent on underlying genetic variation and establishes rDNA as a genomic target of nutritional insults.

Sexually dimorphic gene expression emerges with embryonic genome activation and is dynamic throughout development.

BACKGROUND: As sex determines mammalian development, understanding the nature and developmental dynamics of the sexually dimorphic transcriptome is important. To explore this, we generated 76 genome-wide RNA-seq profiles from mouse eight-cell embryos, late gestation and adult livers, together with 4 ground-state pluripotent embryonic (ES) cell lines from which we generated both RNA-seq and multiple ChIP-seq profiles. We complemented this with previously published data to yield 5 snap-shots of pre-implantation development, late-gestation placenta and somatic tissue and multiple adult tissues for integrative analysis. RESULTS: We define a high-confidence sex-dimorphic signature of 69 genes in eight-cell embryos. Sex-chromosome-linked components of this signature are largely conserved throughout pre-implantation development and in ES cells, whilst the autosomal component is more dynamic. Sex-biased gene expression is reflected by enrichment for activating and repressive histone modifications. The eight-cell signature is largely non-overlapping with that defined from fetal liver, neither was it correlated with adult liver or other tissues analysed. The number of sex-dimorphic genes increases throughout development. We identified many more dimorphic genes in adult compared to fetal liver. However, approximately two thirds of the dimorphic genes identified in fetal liver were also dimorphic in adult liver. Sex-biased expression differences unique to adult liver were enriched for growth hormone-responsiveness. Sexually dimorphic gene expression in pre-implantation development is driven by sex-chromosome based transcription, whilst later development is characterised by sex dimorphic autosomal transcription. CONCLUSION: This systematic study identifies three distinct phases of sex dimorphism throughout mouse development, and has significant implications for understanding the developmental origins of sex-specific phenotypes and disease in mammals.

Incidence and risk factors for surgically acquired pressure ulcers: a prospective cohort study investigators.

PURPOSE: To assess the incidence of hospital-acquired, surgery-related pressure injury (ulcers) and identify risk factors for these injuries. DESIGN: We used a prospective cohort study to investigate the research question. SUBJECTS AND SETTINGS: The study was conducted at a major metropolitan hospital in Brisbane, Australia. Five hundred thirty-four adult patients booked for any surgical procedure expected to last more than 30 minutes were eligible for inclusion. METHODS: Patients who provided informed consent for study participation were assessed for pressure ulcers, using the European Pressure Ulcer Advisory Panel and National Pressure Ulcer Advisory Panel Guidelines, before entering the operating room and again in the post-anesthetic care unit (PACU). Research nurses and all PACU nurses were trained in skin assessment and in pressure ulcer staging. Patients were not assessed again after their discharge from the PACU. RESULTS: Seven patients (1.3%) had existing pressure injuries (ulcers) and a further 6 (1.3%) developed a surgery-related pressure ulcer. Risk factors associated with surgery-related pressure injuries were similar to non-surgically related risks and included older age, skin condition, and being admitted from a location other than one's own home. Length of surgery was not associated with pressure ulcer development in this cohort. CONCLUSION: Perioperative nurses play an important role in identifying existing or new pressure injuries. However, many of these nurses are unfamiliar with pressure ulcer classification, so education in this area is essential. Although the incidence of surgically acquired pressure ulcers was low in this cohort, careful skin inspection before and after surgery provides an opportunity for early treatment and may prevent existing lesions progressing to higher stages.

Transgenerational inheritance of non-genetically determined phenotypes.

Inheritance of non-genetic factors permits ancestral environmental history to inform the development of subsequent generations. This form of soft inheritance has been shown in mammals, yet the molecular underpinnings of this phenomenon are poorly understood. In the present article, we focus on gametic inheritance of non-genetic factors, utilizing examples of paternal transmission to explore the core issues that need to be addressed in order to gain greater insight into the molecular mechanisms. Three essential processes are identified: (i) how the environment affects the germline to establish an altered molecular milieu, (ii) the molecular nature of the inherited mark, and (iii) how this affects genome function in the developing embryo to elicit an alternative developmental outcome.

Inactive or moderately active human promoters are enriched for inter-individual epialleles.

BACKGROUND: Inter-individual epigenetic variation, due to genetic, environmental or random influences, is observed in many eukaryotic species. In mammals, however, the molecular nature of epiallelic variation has been poorly defined, partly due to the restricted focus on DNA methylation. Here we report the first genome-scale investigation of mammalian epialleles that integrates genomic, methylomic, transcriptomic and histone state information. RESULTS: First, in a small sample set, we demonstrate that non-genetically determined inter-individual differentially methylated regions (iiDMRs) can be temporally stable over at least 2 years. Then, we show that iiDMRs are associated with changes in chromatin state as measured by inter-individual differences in histone variant H2A.Z levels. However, the correlation of promoter iiDMRs with gene expression is negligible and not improved by integrating H2A.Z information. We find that most promoter epialleles, whether genetically or non-genetically determined, are associated with low levels of transcriptional activity, depleted for housekeeping genes, and either depleted for H3K4me3/enriched for H3K27me3 or lacking both these marks in human embryonic stem cells. The preferential enrichment of iiDMRs at regions of relative transcriptional inactivity validates in a larger independent cohort, and is reminiscent of observations previously made for promoters that undergo hypermethylation in various cancers, in vitro cell culture and ageing. CONCLUSIONS: Our work identifies potential key features of epiallelic variation in humans, including temporal stability of non-genetically determined epialleles, and concomitant perturbations of chromatin state. Furthermore, our work suggests a novel mechanistic link among inter-individual epialleles observed in the context of normal variation, cancer and ageing.