Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue.

BACKGROUND: Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR) which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG) densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes. RESULTS: Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression). Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ-line and the somatic DMRs. CONCLUSIONS: Our validated pyrosequencing methylation assays can be widely used as a tool to investigate DNA methylation levels of imprinted genes in clinical samples. This first comprehensive analysis of normal methylation levels in adult somatic tissues at human imprinted regions confirm that, despite intra-individual variability and tissue specific expression, imprinted genes faithfully maintain their DNA methylation in healthy adult tissue. DNA methylation levels of a selection of imprinted genes are, therefore, a valuable indicator for epigenetic stability.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Development of the Bristol Rabbit Pain Scale (BRPS):A multidimensional composite pain scale specific to rabbits (Oryctolagus cuniculus)

A species-specific composite pain scale is a prerequisite for adequate pain assessment. The aim of this study was to develop a multidimensional pain scale specific to rabbits (Oryctolagus cuniculus) called the Bristol Rabbit Pain Scale (BRPS). The scale was developed over five phases using a unique combination of methods: focus groups and behavioural observation. The first two phases aimed at identifying descriptors to describe a rabbit in pain, and then reducing their number, both using focus groups. A total of 72 pain descriptors were grouped under six categories (Demeanour, Posture, Facial expression, Attention to the painful area, Audible and Other) and ‘No pain’ descriptors were added. The third phase aimed to confirm, through video observation of rabbits, the categories and descriptors previously described, to reject those terms that were ambiguous, and identify any new descriptors that had not been included in the previous list of descriptors. This led to the rejection of the categories Audible and Attention to the painful area and of 34 descriptors. Seven new descriptors were identified. The last two phases constructed the final format of the BRPS by refining the categories, ranking the descriptors on an ordinal scale and testing the internal reliability of the scale using Cronbach’s alpha test. This led to a composite pain scale of six categories (Demeanour, Posture, Locomotion, Ears, Eyes and Grooming) with four intensities of pain (0, 1, 2, and 3), a total score of 0–18, and a high Cronbach’s alpha coefficient (alpha = 0.843). This BRPS fills an important gap in the field of rabbit medicine and has the potential to improve the assessment and management of pain in rabbits providing veterinary professionals with a novel multidimensional pain assessment tool. Further studies will investigate the clinical utility, validity and reliability of the BRPS

Long-term and trans-generational effects of neonatal experience on sheep behaviour

Early life experiences can have profound long-term, and sometimes trans-generational, effects on individual phenotypes. However, there is a relative paucity of knowledge about effects on pain sensitivity, even though these may impact on an individual's health and welfare, particularly in farm animals exposed to painful husbandry procedures. Here, we tested in sheep whether neonatal painful and non-painful challenges can alter pain sensitivity in adult life, and also in the next generation. Ewes exposed to tail-docking or a simulated mild infection (lipopolysaccharide (LPS)) on days 3–4 of life showed higher levels of pain-related behaviour when giving birth as adults compared with control animals. LPS-treated ewes also gave birth to lambs who showed decreased pain sensitivity in standardized tests during days 2–3 of life. Our results demonstrate long-term and trans-generational effects of neonatal experience on pain responses in a commercially important species and suggest that variations in early life management can have important implications for animal health and welfare

Comparative Genomics and Mutational Analysis Reveals a Novel XoxF-Utilizing Methylotroph in the Roseobacter Group Isolated From the Marine Environment

The Roseobacter group comprises a significant group of marine bacteria which are involved in global carbon and sulfur cycles. Some members are methylotrophs, using one-carbon compounds as a carbon and energy source. It has recently been shown that methylotrophs generally require a rare earth element when using the methanol dehydrogenase enzyme XoxF for growth on methanol. Addition of lanthanum to methanol enrichments of coastal seawater facilitated the isolation of a novel methylotroph in the Roseobacter group: Marinibacterium anthonyi strain La 6. Mutation of xoxF5 revealed the essential nature of this gene during growth on methanol and ethanol. Physiological characterization demonstrated the metabolic versatility of this strain. Genome sequencing revealed that strain La 6 has the largest genome of all Roseobacter group members sequenced to date, at 7.18 Mbp. Multilocus sequence analysis (MLSA) showed that whilst it displays the highest core gene sequence similarity with subgroup 1 of the Roseobacter group, it shares very little of its pangenome, suggesting unique genetic adaptations. This research revealed that the addition of lanthanides to isolation procedures was key to cultivating novel XoxF-utilizing methylotrophs from the marine environment, whilst genome sequencing and MLSA provided insights into their potential genetic adaptations and relationship to the wider community

Clinical measurements performed during alfaxalone total intravenous anaesthesia for radiography and neurophysiological investigations in dogs

Objective: To describe clinically relevant, physiological measurements collected during a 3- hour duration alfaxalone total intravenous anaesthesia.Study design: Case series.Animals: A total of 112 client-owned middle aged or older dogs.Methods: Dogs were premedicated with intramuscular acepromazine (0.03 mg kg-1). Anaesthesia was induced and subsequently maintained for up to 3 hours with alfaxalone administered intravenously. Dogs breathed 100% oxygen via an endotracheal tube. Heart rate, respiratory rate, and blood pressure were evaluated 30 minutes after administration of acepromazine and used as baseline values for comparisons of intra-anaesthetic data. Blood glucose was measured one week prior to anaesthesia and every hour during alfaxalone anaesthesia. Quality and duration of recovery were recorded. Mean data for physiological variables were compared over three time points; before induction of anaesthesia, forthe first hour of anaesthesia and from 60 minutes to discontinuation of anaesthesia.Results: Mean induction dose of alfaxalone was 1.4 (95% CI 1.3 - 1.5) mg kg-1. Post induction apnoea for greater than 60 seconds occurred in 13 (11.6%) dogs. Mean alfaxalone infusion rate during the first 60 minutes of anaesthesia was 0.099 mg kg-1 minute-1; from 60 minutes until discontinuation of anaesthesia, mean infusion rate was 0.092 mg kg-1 minute-1. Heart rate was well maintained; hypotension (mean arterial blood pressure less than 60 mmHg) was encountered in 23 (21%) of dogs. Blood glucose levels did not alter during anaesthesia. Median time between discontinuation of alfaxalone infusion and extubation was 17 (7 – 35 minutes), time to assuming sternal recumbency was 75 (58 - 110 minutes), and time to standing was 109 (88 - 140 minutes).Conclusions and clinical relevance: Alfaxalone infusion provided effective anaesthesia in this population. In a minority of cases respiratory and haemodynamic support of the patient was required

Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms, in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined DNA-stable isotope probing with metagenomics and metaproteomics to characterize an as yet uncultivated marine methylotroph that actively incorporated carbon from 13C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which theculture-independent techniques of DNA- and protein-stable isotope probing have been used to characterize the metabolism of a naturally-ocurring Methylophaga-like bacterium in the marine environment (i.e. M. thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment