Single cell epigenetic visualization assay

Abstract Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5′-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles.</jats:p

Increased S-nitrosylation and proteasomal degradation of caspase-3 during infection contribute to the persistence of adherent invasive escherichia coli (AIEC) in immune cells

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients

Molecular layer deposition of "magnesicone", a magnesium-based hybrid material

Molecular layer deposition (MLD) offers the deposition of ultrathin and conformal organic or hybrid films which have a wide range of applications. However, some critical potential applications require a very specific set of properties. For application as desiccant layers in water barrier films, for example, the films need to exhibit water uptake and swelling and be overcoatable. For application as a backbone for a solid composite electrolyte for lithium ions on the other hand, the films need to be stable against lithium and need to be transformable from a hybrid MLD film to a porous metal oxide film. Magnesium-based MLD films, called "magnesicone", are promising on both these aspects, and thus, an MLD process is developed using Mg(MeCp)(2) as a metal source and ethylene glycol (EG) or glycerol (GL) as organic reactants. Saturated growth could be achieved at 2 to 3 angstrom/cycle in a wide temperature window from 100 to 250 degrees C. The resulting magnesicone films react with ambient air and exhibit water uptake, which is in the case of the GL-based films associated with swelling (up to 10%) and in the case of EG-based magnesicone with Mg(CO)(3) formation, and are overcoatable with an ALD of Al2O3. Furthermore, by carefully tuning the annealing rate, the EG-grown films can be made porous at 350 degrees C. Hence, these functional tests demonstrate the potential of magnesicone films as reactive barrier layers and as the porous backbone of lithium ion composite solid electrolytes, making it a promising material for future applications

Enzyme replacement reverses abnormal cerebrovascular responses in Fabry disease

BACKGROUND: Fabry disease is a lysosomal X-linked enzyme deficiency of α-galactosidase A associated with an increased mortality and morbidity due to renal failure, cardiac disease and early onset stroke. METHODS: We examined the functional blood flow response of the brain after visual stimulation (reversing checkerboard pattern), and cerebral vasoreactivity following acetazolamide (15 mg/kg) with [(15)O]H(2)O and positron emission tomography (PET) in Fabry disease. Twenty-six hemizygous patients (age range 19–47 years) were enrolled in a randomized double-blind placebo-controlled 6-month trial of enzyme replacement therapy administered by intravenous infusion every two weeks. Regional cerebral blood flow (rCBF) was measured with PET at the beginning and end of the trial. RESULTS: Fabry patients had a significantly greater increase in rCBF following visual stimulation and acetazolamide challenge compared to controls. Visual reactivity was normal. The time for recovery of the cerebral vasculature following acetazolamide was prolonged in Fabry patients compared to controls. The abnormal rCBF response induced by visual stimulation and acetazolamide decreased significantly following enzyme replacement therapy, as did the prolonged recovery of the cerebral vasculature. CONCLUSIONS: Enzyme replacement therapy reverses the exaggerated cerebrovascular response in Fabry disease

Travel-related schistosomiasis, strongyloidiasis, filariasis, and toxocariasis: the risk of infection and the diagnostic relevance of blood eosinophilia

<p>Abstract</p> <p>Background</p> <p>This study prospectively assessed the occurrence of clinical and subclinical schistosomiasis, strongyloidiasis, filariasis, and toxocariasis, and the screening value of eosinophilia in adult short-term travelers to helminth-endemic countries.</p> <p>Methods</p> <p>Visitors of a pre-travel health advice centre donated blood samples for serology and blood cell count before and after travel. Samples were tested for eosinophilia, and for antibodies against schistosomiasis, strongyloidiasis, filariasis, and toxocariasis. Previous infection was defined as seropositivity in pre- and post-travel samples. Recent infection was defined as a seroconversion. Symptoms of parasitic disease were recorded in a structured diary.</p> <p>Results</p> <p>Previous infection was found in 112 of 1207 subjects: schistosomiasis in 2.7%, strongyloidiasis in 2.4%, filariasis in 3.4%, and toxocariasis in 1.8%. Recent schistosomiasis was found in 0.51% of susceptible subjects at risk, strongyloidiasis in 0.25%, filariasis in 0.09%, and toxocariasis in 0.08%. The incidence rate per 1000 person-months was 6.4, 3.2, 1.1, and 1.1, respectively. Recent infections were largely contracted in Asia. The positive predictive value of eosinophilia for diagnosis was 15% for previous infection and 0% for recent infection. None of the symptoms studied had any positive predictive value.</p> <p>Conclusion</p> <p>The chance of infection with schistosomiasis, strongyloidiasis, filariasis, and toxocariasis during one short-term journey to an endemic area is low. However, previous stay leads to a cumulative risk of infection. Testing for eosinophilia appeared to be of no value in routine screening of asymptomatic travelers for the four helminthic infections. Findings need to be replicated in larger prospective studies.</p

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales

Exercising 'soft closure' on lay health knowledge? Harnessing the declining power of the medical profession to improve online health information

This study aims to address the increasingly complex medical predicament of low quality online health information contributing to lay health knowledge and consequently to clinical outcomes. We situate the predicament within a social change paradigm of individualism, choice, diminishing medical power, and emergence of the legitimacy of lay health knowledge. We contend that the prominence of lay health knowledge has been facilitated by the internet, and is due to a surge in broadcasting of experiential knowledge coupled with increased access to and enactment of medical and non-medically sanctioned online information on health and illness. We draw on and further test the application of social closure theory to help conceive a potential solution to this enduring problem. We conduct a quality assessment of an indicative case study, Apicectomies, and test the application of our notion of soft closure on its findings, resulting in targeted, feasible and potentially beneficial solutions to increasing the medical quality of online health information. We further present the extant application of soft closure by Healthtalkonline.org, which collates a medically reliable set of experiential knowledge on a range of health issues. As such, we propose a constructive re-enactment of the traditional closure of the medical profession on medical knowledge