Efficient depletion of host DNA contamination in malaria clinical sequencing.

The cost of whole-genome sequencing (WGS) is decreasing rapidly as next-generation sequencing technology continues to advance, and the prospect of making WGS available for public health applications is becoming a reality. So far, a number of studies have demonstrated the use of WGS as an epidemiological tool for typing and controlling outbreaks of microbial pathogens. Success of these applications is hugely dependent on efficient generation of clean genetic material that is free from host DNA contamination for rapid preparation of sequencing libraries. The presence of large amounts of host DNA severely affects the efficiency of characterizing pathogens using WGS and is therefore a serious impediment to clinical and epidemiological sequencing for health care and public health applications. We have developed a simple enzymatic treatment method that takes advantage of the methylation of human DNA to selectively deplete host contamination from clinical samples prior to sequencing. Using malaria clinical samples with over 80% human host DNA contamination, we show that the enzymatic treatment enriches Plasmodium falciparum DNA up to ∼9-fold and generates high-quality, nonbiased sequence reads covering >98% of 86,158 catalogued typeable single-nucleotide polymorphism loci

Genome-Wide Identification of Transcriptional Start Sites in the Plant Pathogen Pseudomonas syringae pv. tomato str. DC3000

RNA-Seq has provided valuable insights into global gene expression in a wide variety of organisms. Using a modified RNA-Seq approach and Illumina's high-throughput sequencing technology, we globally identified 5′-ends of transcripts for the plant pathogen Pseudomonas syringae pv. tomato str. DC3000. A substantial fraction of 5′-ends obtained by this method were consistent with results obtained using global RNA-Seq and 5′RACE. As expected, many 5′-ends were positioned a short distance upstream of annotated genes. We also captured 5′-ends within intergenic regions, providing evidence for the expression of un-annotated genes and non-coding RNAs, and detected numerous examples of antisense transcription, suggesting additional levels of complexity in gene regulation in DC3000. Importantly, targeted searches for sequence patterns in the vicinity of 5′-ends revealed over 1200 putative promoters and other regulatory motifs, establishing a broad foundation for future investigations of regulation at the genomic and single gene levels

Statistical analysis of reduction in tensile strength of cotton strips as a measure of soil microbial activity

The cotton strip assay (CSA) is an established technique for measuring soil microbial activity. The technique involves burying cotton strips and measuring their tensile strength after a certain time. This gives a measure of the rotting rate, R, of the cotton strips. R is then a measure of soil microbial activity. This paper examines properties of the technique and indicates how the assay can be optimised. Humidity conditioning of the cotton strips before measuring their tensile strength reduced the within and between day variance and enabled the distribution of the tensile strength measurements to approximate normality. The test data came from a three-way factorial experiment (two soils, two temperatures, three moisture levels). The cotton strips were buried in the soil for intervals of time ranging up to 6 weeks. This enabled the rate of loss of cotton tensile strength with time to be studied under a range of conditions. An inverse cubic model accounted for greater than 90% of the total variation within each treatment combination. This offers support for summarising the decomposition process by a single parameter R. The approximate variance of the decomposition rate was estimated from a function incorporating the variance of tensile strength and the differential of the function for the rate of decomposition, R, with respect to tensile strength. This variance function has a minimum when the measured strength is approximately 2/3 that of the original strength. The estimates of R are almost unbiased and relatively robust against the cotton strips being left in the soil for more or less than the optimal time. We conclude that the rotting rate X should be measured using the inverse cubic equation, and that the cotton strips should be left in the soil until their strength has been reduced to about 2/3

A helminth cathelicidin-like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase

We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin-like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM-1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM-1 is rapidly processed by lysosomal cathepsin L to release a short C-terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4+ T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host

Long term reversal of diabetes in non obese diabetic mice by liver-directed gene therapy

BACKGROUND: Type 1 diabetes (T1D) results from an autoimmune attack against the insulin-producing beta (β)-cells of the pancreas. The aim of this study was to reverse T1D by gene therapy. METHODS: We used a novel surgical technique, which involves isolating the liver from the circulation before delivery of a lentiviral vector carrying furin-cleavable human insulin (INS-FUR), or empty vector to the livers of diabetic non-obese diabetic mice (NOD). This was compared to the direct injection of the vector into the portal circulation. Mice were monitored for body weight and blood glucose. Intravenous glucose tolerance tests (IVGTT) were performed. Expression of insulin and pancreatic transcription factors was determined by reverse transcriptase PCR (RT-PCR) and immunohistochemistry and immunoelectron microscopy was used to localise insulin. RESULTS: Using the novel surgical technique, we achieved long-term transduction (42% efficiency) of hepatocytes, restored normoglycaemia for 150 days (experimental endpoint) and re-established normal glucose tolerance. We showed expression of β-cell transcription factors, murine insulin, glucagon and somatostatin, hepatic storage of insulin in granules. Expression of hepatic markers, C/EBP-β, G6PC, AAT, GLUI, were downregulated in INS-FUR-treated livers. Liver function tests remained normal with no evidence of intrahepatic inflammation or autoimmune destruction of the insulin-secreting liver tissue. By comparison, direct injection of INS-FUR reduced blood glucose levels, no pancreatic transdifferentiation or normal glucose tolerance was observed. CONCLUSIONS: This gene therapy protocol has for the first time permanently reversed T1D with normal glucose tolerance in NOD mice and, as such, opens a novel therapeutic strategy for the cure of T1D. Copyright © 2013 John Wiley & Sons, Ltd.status: publishe

FhES treatment modulates the phenotype of immune cells in the pancreatic draining lymph nodes of NOD mice.

<p>Four-week old female NOD mice were treated with 10 µg of FhES or PBS intraperitoneally on alternate days for a total of six injections. The cellular composition within the PLNs was examined by flow cytometry 24 h after the final injection (n = 6; data representative of 5 independent experiments). (A) Absolute cell numbers and percentages of B220⁺ B cells and CD3⁺ T cells in the PLNs; (B) representative plots of the proportions of CD3⁺ T cells; (C) subsets of CD3⁺ T cells; (D) proportion and absolute numbers of CD4⁺CD25⁺Foxp3⁺ CD3⁺ T cells; (E) B220⁺ B cells; (F) representative dot plots of proportions (left panel), and absolute numbers (right panel) of IL-10 secreting CD19⁺ B cells within the CD19⁺ gate; (G) expression of Ym1, Retlna, Ear 11 and Arg-1 by quantitative realtime RT-PCR presented as fold change in expression, calculated compared to the average expression of the PBS cohort (each data point represents a single mouse; n = 6; data representative of at least 2 independent experiments).</p

Treatment of NOD mice co-incident with the initiation of autoimmunity prevents T1D.

<p>Four-week old female NOD mice were injected intraperitoneally with FhES (10 µg in 100 µl sterile PBS) or vehicle (PBS), on alternate days, for a total of six injections. (A) Blood glucose levels were monitored and animals were sacrificed when they became diabetic (as defined by two consecutive blood glucose concentrations ≥14 mmol/L). The graphs represent an analysis of the age at which each animal was sacrificed and was performed using survival analysis. Data shown is from one of three independent experiments, all of which produced the same outcome. (B–E) Pancreas isolated from mice at 10 (n = 13), 15 (n = 15), 23 (n = 16) and 30 (n = 16) weeks of age were graded for insulitis on a scale of 0–4; whereby 0 = healthy islet or mild peri-insular mononuclear cell infiltration, 1 = infiltration up to 25% of islet mass, 2 = infiltration up to 50% of islet mass, 3 = infiltration from 50% up to 75% of islet mass, and 4 = less than 25% of islet mass present. The proportion of islets with each grade of insulitis is shown.</p

Peritoneal FhES-induced regulatory M2 macrophages expand Foxp3⁺ regulatory T cells <i>ex vivo</i>.

<p>(A) Four-week old female NOD mice were treated with 10 µg of FhES, SEA or PBS intraperitoneally on alternate days for a total of six injections. Peritoneal macrophages were harvested 24 h after the final injection and co-cultured with splenocytes isolated from age matched naive NOD mice in the presence of anti-CD3 (2 µg/ml) for 72 h. (B) Splenocytes were co-incubated with FhES (20 µg/ml), SEA (50 µg/ml), or PBS in the presence of anti-CD3 antibody (2 µg/ml) for 72 h. Representative flow cytometry dot plots are shown with the numbers representing the percentage of cells expressing both CD25⁺ and Foxp3. Histograms show the means of triplicate samples ± SEM, and are representative of two independent experiments. (C) Pancreata were isolated from female NOD mice (n = 3) 24 h after the final (sixth) FhES or PBS treatment and the expression levels of Arg1, Ym1, Retlna and Ear 11 were determined by quantitative realtime RT-PCR. All fold changes in expression levels were calculated compared to the average expression levels of the PBS cohort. Data shown is representative of at least two repeat experiments.</p