On the Action of Methotrexate and 6-Mercaptopurine on M. avium Subspecies paratuberculosis

BACKGROUND: Clinical improvement in inflammatory bowel disease (IBD) treated with methotrexate and 6-mercaptopurine (6-MP) is associated with a decrease in pro-inflammatory cytokines. This has been presumed to indicate the mechanism of action of methotrexate and 6-MP. Although controversial, there are increasingly compelling data that Mycobacterium avium subspecies paratuberculosis (MAP) may be an etiological agent in some or all of IBD. We hypothesized that the clinical efficacy of methotrexate and 6-MP in IBD may be to simply inhibit the growth of MAP. METHODOLOGY: The effect on MAP growth kinetics by methotrexate and 6-MP were evaluated in cell culture of two strains each of MAP and M. avium using a radiometric ((14)CO(2) BACTEC®) detection system that quantifies mycobacterial growth as arbitrary “growth index units” (GI). Efficacy data are presented as “percent decrease in cumulative GI” (% −ΔcGI). PRINCIPAL FINDINGS: The positive control antibiotic (clarithromycin) has ≥85% −ΔcGI at a concentration of 0.5 µg/ml. The negative control (ampicillin) has minimal inhibition at 64 µg/ml. MAP ATCC 19698 shows ≥80% −ΔcGI for both agents by 4 µg/ml. With the other three isolates, although more effective than ampicillin, 6-MP is consistently less effective than methotrexate. CONCLUSIONS: We show that methotrexate and 6-MP inhibit MAP growth in vitro. Each of the four isolates manifests different % −ΔcGI. These data are compatible with the hypothesis that the clinical improvement in patients with IBD treated with methotrexate and 6-MP could be due to treating a MAP infection. The decrease in pro-inflammatory cytokines, thought to be the primary mechanism of action, may simply be a normal, secondary, physiological response. We conclude that henceforth, in clinical studies that evaluate the effect of anti-MAP agents in IBD, the use of methotrexate and 6-MP should be excluded from any control groups

miRNA-Dependent Translational Repression in the Drosophila Ovary

Background: The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary. Methodology/Principal Findings: Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed. Conclusions/Significance: Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.This work was supported in part by NIH grant GM54409 and in part by Research Grant No. 1-FY08-445. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Cellular and Molecular Biolog

Forecasting Non-Stationary Diarrhea, Acute Respiratory Infection, and Malaria Time-Series in Niono, Mali

BACKGROUND: Much of the developing world, particularly sub-Saharan Africa, exhibits high levels of morbidity and mortality associated with diarrhea, acute respiratory infection, and malaria. With the increasing awareness that the aforementioned infectious diseases impose an enormous burden on developing countries, public health programs therein could benefit from parsimonious general-purpose forecasting methods to enhance infectious disease intervention. Unfortunately, these disease time-series often i) suffer from non-stationarity; ii) exhibit large inter-annual plus seasonal fluctuations; and, iii) require disease-specific tailoring of forecasting methods. METHODOLOGY/PRINCIPAL FINDINGS: In this longitudinal retrospective (01/1996-06/2004) investigation, diarrhea, acute respiratory infection of the lower tract, and malaria consultation time-series are fitted with a general-purpose econometric method, namely the multiplicative Holt-Winters, to produce contemporaneous on-line forecasts for the district of Niono, Mali. This method accommodates seasonal, as well as inter-annual, fluctuations and produces reasonably accurate median 2- and 3-month horizon forecasts for these non-stationary time-series, i.e., 92% of the 24 time-series forecasts generated (2 forecast horizons, 3 diseases, and 4 age categories = 24 time-series forecasts) have mean absolute percentage errors circa 25%. CONCLUSIONS/SIGNIFICANCE: The multiplicative Holt-Winters forecasting method: i) performs well across diseases with dramatically distinct transmission modes and hence it is a strong general-purpose forecasting method candidate for non-stationary epidemiological time-series; ii) obliquely captures prior non-linear interactions between climate and the aforementioned disease dynamics thus, obviating the need for more complex disease-specific climate-based parametric forecasting methods in the district of Niono; furthermore, iii) readily decomposes time-series into seasonal components thereby potentially assisting with programming of public health interventions, as well as monitoring of disease dynamics modification. Therefore, these forecasts could improve infectious diseases management in the district of Niono, Mali, and elsewhere in the Sahel

Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by Laser Capture Microdissection

<p>Abstract</p> <p>Background</p> <p>Successful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult.</p> <p>The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA.</p> <p>Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis.</p> <p>Results</p> <p>We developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (<it>SOHLH2</it>, <it>MAEL</it>, <it>MATER</it>, <it>VASA</it>, <it>GDF9</it>, <it>BMP15</it>) and three granulosa cell-specific genes (<it>KL</it>, <it>GATA4</it>, <it>AMH</it>).</p> <p>A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte.</p> <p>Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA.</p> <p>Conclusions</p> <p>The ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.</p

Marine, Estuarine, and Diadromous Fish Stocks at Risk of Extinction in North America (Exclusive of Pacific Salmonids)

The problem of extinction in the sea has been recognized only recently by the scientific community (Roberts and Hawkins 1999). For the last decade, the American Fisheries Society (AFS) has pursued an initiative to identify marine fish stocks that may be at risk of extinction in North America (Musick 1998; Coleman et al. 2000; Musick et al. 2000a; Musick et al. 2000b; Parker et al. 2000). The present paper presents the first recognized AFS list of marine, estuarine, and diadromous fishes that may be at risk of extinction in North America (exclusive of Pacific salmonids). The geographic scope of this review includes the coastal waters of Mexico, the continental United States, Alaska, and Canada. The objectives in preparing this list have been to identify stocks at risk at a sufficiently early stage of decline to avoid listing as threatened or endangered, minimize the probability of under- or overestimating the risk of extinction, use the best existing knowledge of stock dynamics at low population levels, and allow experts on stocks to bring to bear all the available knowledge about life history and conservation status in order to categorize risk of extinction

Valorisation of Biowastes for the Production of Green Materials Using Chemical Methods

With crude oil reserves dwindling, the hunt for a sustainable alternative feedstock for fuels and materials for our society continues to expand. The biorefinery concept has enjoyed both a surge in popularity and also vocal opposition to the idea of diverting food-grade land and crops for this purpose. The idea of using the inevitable wastes arising from biomass processing, particularly farming and food production, is, therefore, gaining more attention as the feedstock for the biorefinery. For the three main components of biomass—carbohydrates, lipids, and proteins—there are long-established processes for using some of these by-products. However, the recent advances in chemical technologies are expanding both the feedstocks available for processing and the products that be obtained. Herein, this review presents some of the more recent developments in processing these molecules for green materials, as well as case studies that bring these technologies and materials together into final products for applied usage

Inhibitory effects of (±)-propranolol on excitation-contraction coupling in isolated soleus muscles of the rat

1. The effect of a β-adrenoceptor antagonist, propranolol, was investigated on excitation-contraction coupling in small, intact bundles of soleus muscle fibres from the rat. 2. (±)-Propranolol significantly inhibited twitch and tetanic tension with IC(50) values of 6.7 μM and 3.5 μM, respectively. 3. (+)-Propranolol (which has 100 times less β-blocking activity than the (±) form) was approximately one third as effective as the (±) form at inhibiting isometric tension. 4. (±)-Propranolol (20 μM) had no significant effect on the amplitude of caffeine contractures, suggesting that it did not directly inhibit Ca(2+) release from the sarcoplasmic reticulum. 5. The resting membrane potential measured after 15 min perfusion with 20 μM (±)-propranolol was not significantly different from control. However, this concentration of (±)-propranolol significantly reduced both the peak amplitude and the maximum rate of rise of the action potential. Both effects were only partially reversible after extensive washing. 6. (±)-Propranolol perfusion caused a modest reduction in the amplitude of sub-maximal K(+) contractures at concentrations (5  μM) that markedly depressed tetanic tension. 7. The results indicate that (±)-propranolol can decrease isometric tension independently of β-receptor occupation by (i) reducing the amplitude and rate of rise of the action potential and (ii) by directly inhibiting excitation-contraction coupling. The relatively low IC(50) for the ‘membrane-stabilizing' action of propranolol on tetanic tension (3.5 μM), combined with the ability of the drug to accumulate gradually in biological membranes, may contribute to a peripheral component of the tremorolytic and fatigue-inducing actions of propranolol on skeletal muscle