New methods to analyse microarray data that partially lack a reference signal

BACKGROUND: Microarray-based Comparative Genomic Hybridisation (CGH) has been used to assess genetic variability between bacterial strains. Crucial for interpretation of microarray data is the availability of a reference to compare signal intensities to reliably determine presence or divergence each DNA fragment. However, the production of a good reference becomes unfeasible when microarrays are based on pan-genomes.When only a single strain is used as a reference for a multistrain array, the accessory gene pool will be partially represented by reference DNA, although these genes represent the genomic repertoire that can explain differences in virulence, pathogenicity or transmissibility between strains. The lack of a reference makes interpretation of the data for these genes difficult and, if the test signal is low, they are often deleted from the analysis. We aimed to develop novel methods to determine the presence or divergence of genes in a Staphylococcus aureus multistrain PCR product microarray-based CGH approach for which reference DNA was not available for some probes. RESULTS: In this study we have developed 6 new methods to predict divergence and presence of all genes spotted on a multistrain Staphylococcus aureus DNA microarray, published previously, including those gene spots that lack reference signals. When considering specificity and PPV (i.e. the false-positive rate) as the most important criteria for evaluating these methods, the method that defined gene presence based on a signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics. For this method specificity was 100% and 82% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively, and PPV were 100% and 76% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively. CONCLUSION: A definition of gene presence based on signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics, allowing the analysis of 6-17% more of the genes not present in the reference strain. This method is recommended to analyse microarray data that partially lack a reference signal

Effects of Partial and Complete Ablation of the Slow Pathway on Fast Pathway Properties in Patients with Atrioventricular Nodal Reentrant Tachycardia

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73520/1/j.1540-8167.1994.tb01187.x.pd

Docosahexaenoic acid reduces microglia phagocytic activity via miR-124 and induces neuroprotection in rodent models of spinal cord contusion injury

Microglia are activated after spinal cord injury (SCI), but their phagocytic mechanisms and link to neuroprotection remain incompletely characterized. Docosahexaenoic acid (DHA) has been shown to have significant neuroprotective effects after hemisection and compression SCI and can directly affect microglia in these injury models. In rodent contusion SCI, we demonstrate that DHA (500 nmol/kg) administered acutely post-injury confers neuroprotection and enhances locomotor recovery, and also exerts a complex modulation of the microglial response to injury. In rodents, at 7 days after SCI, the level of phagocytosed myelin within Iba1-positive or P2Y12-positive cells was significantly lower after DHA treatment, and this occurred in parallel with an increase in intracellular miR-124 expression. Furthermore, intraspinal administration of a miR-124 inhibitor significantly reduced the DHA-induced decrease in myelin phagocytosis in mice at 7 days post-SCI. In rat spinal primary microglia cultures, DHA reduced the phagocytic response to myelin, which was associated with an increase in miR-124, but not miR-155. A similar response was observed in a microglia cell line (BV2) treated with DHA, and the effect was blocked by a miR-124 inhibitor. Furthermore, the phagocytic response of BV2 cells to stressed neurones was also reduced in the presence of DHA. In peripheral monocyte-derived macrophages, the expression of the M1, but not the M0 or M2 phenotype, was reduced by DHA, but the phagocytic activation was not altered. These findings show that DHA induces neuroprotection in contusion injury. Furthermore, the improved outcome is via a miR-124-dependent reduction in the phagocytic response of microglia.US Department of Defence CDMRP/SCIRP award (W81XWH-10-1-1040 to P.K.Y., T.B. and A.M.T.); The Barts and London Charity (to P.K.Y. and A.M.T.); Rod Flower Vacation Scholarship (to A.L.B.); International Spinal Research Trust (to J.H. and P.G.P.); Ray W. Poppleton Endowment (to P.G.P.); Chang Gung Memorial Hospital, Taiwan (CMRPG3A1051–1054 to Z.-H.L.). M.A.B. is funded by the Spanish Ministry of Economy and Competitivity (Programa Ramón y Cajal: RYC-2017-21804)

Autoimmune and autoinflammatory mechanisms in uveitis

The eye, as currently viewed, is neither immunologically ignorant nor sequestered from the systemic environment. The eye utilises distinct immunoregulatory mechanisms to preserve tissue and cellular function in the face of immune-mediated insult; clinically, inflammation following such an insult is termed uveitis. The intra-ocular inflammation in uveitis may be clinically obvious as a result of infection (e.g. toxoplasma, herpes), but in the main infection, if any, remains covert. We now recognise that healthy tissues including the retina have regulatory mechanisms imparted by control of myeloid cells through receptors (e.g. CD200R) and soluble inhibitory factors (e.g. alpha-MSH), regulation of the blood retinal barrier, and active immune surveillance. Once homoeostasis has been disrupted and inflammation ensues, the mechanisms to regulate inflammation, including T cell apoptosis, generation of Treg cells, and myeloid cell suppression in situ, are less successful. Why inflammation becomes persistent remains unknown, but extrapolating from animal models, possibilities include differential trafficking of T cells from the retina, residency of CD8(+) T cells, and alterations of myeloid cell phenotype and function. Translating lessons learned from animal models to humans has been helped by system biology approaches and informatics, which suggest that diseased animals and people share similar changes in T cell phenotypes and monocyte function to date. Together the data infer a possible cryptic infectious drive in uveitis that unlocks and drives persistent autoimmune responses, or promotes further innate immune responses. Thus there may be many mechanisms in common with those observed in autoinflammatory disorders

Patient-centric trials for therapeutic development in precision oncology

An enhanced understanding of the molecular pathology of disease gained from genomic studies is facilitating the development of treatments that target discrete molecular subclasses of tumours. Considerable associated challenges include how to advance and implement targeted drug-development strategies. Precision medicine centres on delivering the most appropriate therapy to a patient on the basis of clinical and molecular features of their disease. The development of therapeutic agents that target molecular mechanisms is driving innovation in clinical-trial strategies. Although progress has been made, modifications to existing core paradigms in oncology drug development will be required to realize fully the promise of precision medicine

Demographic, criminal and psychiatric factors related to inmate suicide

A review of 19 studies suggests that it may be feasible to identify prisoners with suicide risk on the basis of demographic, psychiatric, and criminal characteristics. The present study aimed to identify combinations of characteristics that are capable of identifying potential suicide victims. Characteristics of 95 suicide victims in the Dutch prison system were compared with those of a random sample of 247 inmates in ten jails. Combinations of indicators for suicide risk were also tested for their capability of identifying 209 suicides in U.S. jails and 279 prison suicides in England and Wales. A combination of six characteristics (age 40+, homelessness, history of psychiatric care, history of drug abuse, one prior incarceration, violent offence) was capable of correctly classifying 82% of the Dutch suicide victims (82% specificity). Less powerful combinations correctly classified 53% of the U.S. suicides and 47% of the U.K. suicides. It is concluded that a set of demographic and criminal characteristics and indicators of psychiatric problems is useful for the identification of suicide risk in jails and prisons

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient

Comparison of two modes of vitamin B12 supplementation on neuroconduction and cognitive function among older people living in Santiago, Chile: a cluster randomized controlled trial. a study protocol [ISRCTN 02694183]

BACKGROUND: Older people have a high risk of vitamin B12 deficiency; this can lead to varying degrees of cognitive and neurological impairment. CBL deficiency may present as macrocytic anemia, subacute combined degeneration of the spinal cord, or as neuropathy, but is often asymptomatic in older people. Less is known about subclinical vitamin B12 deficiency and concurrent neuroconduction and cognitive impairment. A Programme of Complementary Feeding for the Older Population (PACAM) in Chile delivers 2 complementary fortified foods that provide approximately 1.4 μg/day of vitamin B12 (2.4 μg/day elderly RDA). The aim of the present study is to assess whether supplementation with vitamin B12 will improve neuroconduction and cognitive function in older people who have biochemical evidence of vitamin B12 insufficiency in the absence of clinical deficiency. METHODS: We designed a cluster double-blind placebo-controlled trial involving community dwelling people aged 70-79 living in Santiago, Chile. We randomized 15 clusters (health centers) involving 300 people (20 per cluster). Each cluster will be randomly assigned to one of three arms: a) a 1 mg vitamin B12 pill taken daily and a routine PACAM food; b) a placebo pill and the milk-PACAM food fortified to provide 1 mg of vitamin B12; c) the routine PACAM food and a placebo pill.The study has been designed as an 18 month follow up period. The primary outcomes assessed at baseline, 4, 9 and 18 months will be: serum levels of vitamin B12, neuroconduction and cognitive function. CONCLUSIONS: In view of the high prevalence of vitamin B12 deficiency in later life, the present study has potential public health interest because since it will measure the impact of the existing program of complementary feeding as compared to two options that provide higher vitamin B12 intakes that might potentially may contribute in preserving neurophysiologic and cognitive function and thus improve quality of life for older people in Chile. TRIAL REGISTRATION: ISRCTN: ISRCTN02694183

Simvastatin activates single skeletal RyR1 channels but exerts more complex regulation of the cardiac RyR2 isoform

Background and Purpose: Statins are amongst the most widely prescribed drugs for those at risk of cardiovascular disease, lowering cholesterol levels by inhibiting 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA reductase. Although effective at preventing cardiovascular disease, statin use is associated with muscle weakness, myopathies and, occasionally, fatal rhabdomyolysis. As simvastatin, a commonly prescribed statin, promotes Ca²⁺ release from sarcoplasmic reticulum (SR) vesicles, we investigated if simvastatin directly activates skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. Experimental Approach: RyR1 and RyR2 single‐channel behaviour was investigated after incorporation of sheep cardiac or mouse skeletal SR into planar phospholipid bilayers under voltage‐clamp conditions. LC‐MS was used to monitor the kinetics of interconversion of simvastatin between hydroxy‐acid and lactone forms during these experiments. Cardiac and skeletal myocytes were permeabilised to examine simvastatin modulation of SR Ca²⁺ release. Key Results: Hydroxy acid simvastatin (active at HMG‐CoA reductase) significantly and reversibly increased RyR1 open probability (Po) and shifted the distribution of Ca²⁺ spark frequency towards higher values in skeletal fibres. In contrast, simvastatin reduced RyR2 Po and shifted the distribution of spark frequency towards lower values in ventricular cardiomyocytes. The lactone pro‐drug form of simvastatin (inactive at HMG‐CoA reductase) also activated RyR1, suggesting that the HMG‐CoA inhibitor pharmacophore was not responsible for RyR1 activation. Conclusion and Implications: Simvastatin interacts with RyR1 to increase SR Ca²⁺ release and thus may contribute to its reported adverse effects on skeletal muscle. The ability of low concentrations of simvastatin to reduce RyR2 Po may also protect against Ca²⁺‐dependent arrhythmias and sudden cardiac death