Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo. (C) 2008 Elsevier B.V. All rights reserved.

Short-term effect of targeted placements of sheep excrement on grassland in inner mongolia on soil and plant parameters

Grazing animal excrement plays an important role in nutrient cycling and redistribution in grazing ecosystems, due to grazing in large areas and return in small areas. To elucidate the changes to the soil and pasture caused by sheep urine, fresh dung, and compost patches, a short- term field experiment using artificially placed pats was set up in the autumn of 2003 in the Inner Mongolian steppe. Urine application significantly increased soil pH during the first 32 days in soil layers at depths of both 0 - 5 cm and 5 - 15 cm. Rapid hydrolysis of urea gave large amounts of urine- nitrogen ( N) as ammonium ( NH4+) in soil extracts and was followed by apparent nitrification from day 2. Higher inorganic N content in the urine- treated soil was found throughout the experiment compared with the control. No significant effects of sheep excrement on soil microbial carbon ( C) and soil microbial N was found, but microbial activities significantly increased compared with the control after application of sheep excrement. Forty- six percent of dung- N and 27% of compost- N were transferred into vegetation after the experiment. The results from this study suggest that large amounts of nutrients have been lost from the returned excrement patches in the degraded grassland of Inner Mongolia, especially from sheep urine- N.

Framingham Heart Study 100K project: genome-wide associations for cardiovascular disease outcomes

BACKGROUND:Cardiovascular disease (CVD) and its most common manifestations - including coronary heart disease (CHD), stroke, heart failure (HF), and atrial fibrillation (AF) - are major causes of morbidity and mortality. In many industrialized countries, cardiovascular disease (CVD) claims more lives each year than any other disease. Heart disease and stroke are the first and third leading causes of death in the United States. Prior investigations have reported several single gene variants associated with CHD, stroke, HF, and AF. We report a community-based genome-wide association study of major CVD outcomes.METHODS:In 1345 Framingham Heart Study participants from the largest 310 pedigrees (54% women, mean age 33 years at entry), we analyzed associations of 70,987 qualifying SNPs (Affymetrix 100K GeneChip) to four major CVD outcomes: major atherosclerotic CVD (n = 142; myocardial infarction, stroke, CHD death), major CHD (n = 118; myocardial infarction, CHD death), AF (n = 151), and HF (n = 73). Participants free of the condition at entry were included in proportional hazards models. We analyzed model-based deviance residuals using generalized estimating equations to test associations between SNP genotypes and traits in additive genetic models restricted to autosomal SNPs with minor allele frequency [greater than or equal to]0.10, genotype call rate [greater than or equal to]0.80, and Hardy-Weinberg equilibrium p-value [greater than or equal to] 0.001.RESULTS:Six associations yielded p <10-5. The lowest p-values for each CVD trait were as follows: major CVD, rs499818, p = 6.6 x 10-6; major CHD, rs2549513, p = 9.7 x 10-6; AF, rs958546, p = 4.8 x 10-6; HF: rs740363, p = 8.8 x 10-6. Of note, we found associations of a 13 Kb region on chromosome 9p21 with major CVD (p 1.7 - 1.9 x 10-5) and major CHD (p 2.5 - 3.5 x 10-4) that confirm associations with CHD in two recently reported genome-wide association studies. Also, rs10501920 in CNTN5 was associated with AF (p = 9.4 x 10-6) and HF (p = 1.2 x 10-4). Complete results for these phenotypes can be found at the dbgap website http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.CONCLUSION:No association attained genome-wide significance, but several intriguing findings emerged. Notably, we replicated associations of chromosome 9p21 with major CVD. Additional studies are needed to validate these results. Finding genetic variants associated with CVD may point to novel disease pathways and identify potential targeted preventive therapies.

Measuring Bottleneck Bandwidth of Targeted Path Segments

Accurate measurement of network bandwidth is crucial for flexible Internet applications and protocols which actively manage and dynamically adapt to changing utilization of network resources. These applications must do so to perform tasks such as distributing and delivering high-bandwidth media, scheduling service requests and performing admission control. Extensive work has focused on two approaches to measuring bandwidth: measuring it hop-by-hop, and measuring it end-to-end along a path. Unfortunately, best-practice techniques for the former are inefficient and techniques for the latter are only able to observe bottlenecks visible at end-to-end scope. In this paper, we develop and simulate end-to-end probing methods which can measure bottleneck bandwidth along arbitrary, targeted subpaths of a path in the network, including subpaths shared by a set of flows. As another important contribution, we describe a number of practical applications which we foresee as standing to benefit from solutions to this problem, especially in emerging, flexible network architectures such as overlay networks, ad-hoc networks, peer-to-peer architectures and massively accessed content servers.

FLT3-driven redox signalling in acute myeloid leukaemia

Acute myeloid leukaemia refers to cancer of the blood and bone marrow characterised by the rapid expansion of immature blasts of the myeloid lineage. The aberrant proliferation of these blasts interferes with normal haematopoiesis, resulting in symptoms such as anaemia, poor coagulation and infections. The molecular mechanisms underpinning acute myeloid leukaemia are multi-faceted and complex, with a range of diverse genetic and cytogenetic abnormalities giving rise to the acute myeloid leukaemia phenotype. Amongst the most common causative factors are mutations of the FLT3 gene, which codes for a growth factor receptor tyrosine kinase required by developing haematopoietic cells. Disruptions to this gene can result in constitutively active FLT3, driving the de-regulated proliferation of undifferentiated precursor blasts. FLT3-targeted drugs provide the opportunity to inhibit this oncogenic receptor, but over time can give rise to resistance within the blast population. The identification of targetable components of the FLT3 signalling pathway may allow for combination therapies to be used to impede the emergence of resistance. However, the intracellular signal transduction pathway of FLT3 is relatively obscure. The objective of this study is to further elucidate this pathway, with particular focus on the redox signalling element which is thought to be involved. Signalling via reactive oxygen species is becoming increasingly recognised as a crucial aspect of physiological and pathological processes within the cell. The first part of this study examined the effects of NADPH oxidase-derived reactive oxygen species on the tyrosine phosphorylation levels of acute myeloid leukaemia cell lines. Using two-dimensional phosphotyrosine immunoblotting, a range of proteins were identified as undergoing tyrosine phosphorylation in response to NADPH oxidase activity. Ezrin, a cytoskeletal regulatory protein and substrate of Src kinase, was selected for further study. The next part of this study established that NADPH oxidase is subject to regulation by FLT3. Both wild type and oncogenic FLT3 signalling were shown to affect the expression of a key NADPH oxidase subunit, p22phox, and FLT3 was also demonstrated to drive intracellular reactive oxygen species production. The NADPH oxidase target protein, Ezrin, undergoes phosphorylation on two tyrosine residues downstream of FLT3 signalling, an effect which was shown to be p22phox-dependent and which was attributed to the redox regulation of Src. The cytoskeletal associations of Ezrin and its established role in metastasis prompted the investigation of the effects of FLT3 and NADPH oxidase activity on the migration of acute myeloid leukaemia cell lines. It was found that inhibition of either FLT3 or NADPH oxidase negatively impacted on the motility of acute myeloid leukaemia cells. The final part of this study focused on the relationship between FLT3 signalling and phosphatase activity. It was determined, using phosphatase expression profiling and real-time PCR, that several phosphatases are subject to regulation at the levels of transcription and post-translational modification downstream of oncogenic FLT3 activity. In summary, this study demonstrates that FLT3 signal transduction utilises a NADPH oxidase-dependent redox element, which affects Src kinase, and modulates leukaemic cell migration through Ezrin. Furthermore, the expression and activity of several phosphatases is tightly linked to FLT3 signalling. This work reveals novel components of the FLT3 signalling cascade and indicates a range of potential therapeutic targets.

Thuricin CD: a potential therapeutic targeted against Clostridium difficile-associated diarrhea (CDAD)

Clostridium difficile is mainly a nosocomial pathogen and is a significant cause of antibioticassociated diarrhea. It is also implicated in the majority of cases of pseudomembranous colitis. The main etiological agent of C. difficile-associated diarrhea (CDAD) is perturbations to the gut microbiota by broad-spectrum antibiotics. Recently, thuricin CD, a two-peptide narrow spectrum sactibiotic bacteriocin with potent activity against C. difficile has been discovered. It is produced by Bacillus thuringiensis DPC6431. The efficacy of thuricin CD against a range of C. difficile clinical isolates has been determined in the form of minimum inhibitory concentration (MIC) values and compared to metronidazole, vancomycin, ramoplanin and actagardine in this thesis. Furthermore, by assessing paired combinations of the above-mentioned antimicrobials, it was determined that ramoplanin and actagardine function in a synergistic manner against the majority of C. difficile isolates. The functions of the genes in the thuricin CD gene cluster have also been elucidated by cloning the cluster and expressing thuricin CD in a heterologous Bacillus subtilis host and are described herein. In addition, the immunity mechanisms employed by the B. thuringiensis DPC6431 producer to protect itself from the antimicrobial actions of thuricin CD have also been elucidated. It has been shown that a small immunity peptide, TrnI, is involved in thuricin CD immunity, most likely by intercepting the thuricin CD peptides and/or blocking their access to the thuricin CD receptor. This immunity peptide and also the ABC-transporter system TrnFG serve to protect the B. thuringiensis host against thuricin CD.

Breast cancer therapies in development. A review of their pharmacology and clinical potential.

Although the management of breast cancer has improved over the past few decades, it remains an important challenge for the clinician. Cytotoxic chemotherapy and hormonotherapy, when given in the adjuvant setting, have a definitive though modest impact on the outcome of early-stage breast cancer. In metastatic disease, these therapies help to provide substantial palliation of symptoms but have a limited impact on survival. The discovery of vinorelbine and the taxanes, paclitaxel and docetaxel, certainly represented the most encouraging clinical development of the 1980s in breast cancer therapy. Several other new cytotoxic agents have been recognised for their potential in the treatment of this disorder. Many of them are only in a very early phase of their clinical development and it remains to be proven that they will have a major role in daily practice in the near future.

Prostate cancer: an evolving paradigm.

Since at least the early 1990s, stage and risk migration have been seen in patients with prostate cancer, likely corresponding to the institution of prostate specific antigen (PSA) screening in health systems. Preoperative risk factors, including PSA level and clinical stage, have decreased significantly. These improved prognostic variables have led to a larger portion of men being stratified with low-risk disease, as per the classification of D'Amico and associates. This, in turn, has corresponded with more favorable postoperative variables, including decreased extraprostatic tumor extension and prolonged biochemical-free recurrence rates. The advent of focal therapy is bolstered by findings of increased unilateral disease with decreased tumor volume. Increasingly, targeted or delayed therapies may be possible within the current era of lower risk disease.

Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors.

Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa. Carbohydrate binding agents (CBAs) demonstrate the ability to act as entry inhibitors due to their ability to bind to glycans and prevent gp120 binding to CD4+ cells. However, as proteins they present significant challenges in regard to economical production and formulation for resource-poor environments. We have synthesized water-soluble polymer CBAs that contain multiple benzoboroxole moieties. A benzoboroxole-functionalized monomer was synthesized and incorporated into linear oligomers with 2-hydroxypropylmethacrylamide (HPMAm) at different feed ratios using free radical polymerization. The benzoboroxole small molecule analogue demonstrated weak affinity for HIV-1BaL gp120 by SPR; however, the 25 mol % functionalized benzoboroxole oligomer demonstrated a 10-fold decrease in the K(D) for gp120, suggesting an increased avidity for the multivalent polymer construct. High molecular weight polymers functionalized with 25, 50, and 75 mol % benzoboroxole were synthesized and tested for their ability to neutralize HIV-1 entry for two HIV-1 clades and both R5 and X4 coreceptor tropism. All three polymers demonstrated activity against all viral strains tested with EC(50)s that decrease from 15000 nM (1500 microg mL(-1)) for the 25 mol % functionalized polymers to 11 nM (1 microg mL(-1)) for the 75 mol % benzoboroxole-functionalized polymers. These polymers exhibited minimal cytotoxicity after 24 h exposure to a human vaginal cell line.

MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

BACKGROUND: Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). METHODS: We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. RESULTS: Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). CONCLUSIONS: Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.

Targeting A20 decreases glioma stem cell survival and tumor growth.

Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-kappaB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFalpha-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFalpha-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.

Beta-arrestin-2 regulates the development of allergic asthma.

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking beta-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the beta-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking beta-arrestin-2. beta-arrestin-2-deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine-mediated CD4+ T cell migration to the lung. This report provides the first evidence that beta-arrestin-2 is required for the manifestation of allergic asthma. Because beta-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

Accessibility, availability and affordability of anti-malarials in a rural district in Kenya after implementation of a national subsidy scheme.

BACKGROUND: Poor access to prompt and effective treatment for malaria contributes to high mortality and severe morbidity. In Kenya, it is estimated that only 12% of children receive anti-malarials for their fever within 24 hours. The first point of care for many fevers is a local medicine retailer, such as a pharmacy or chemist. The role of the medicine retailer as an important distribution point for malaria medicines has been recognized and several different strategies have been used to improve the services that these retailers provide. Despite these efforts, many mothers still purchase ineffective drugs because they are less expensive than effective artemisinin combination therapy (ACT). One strategy that is being piloted in several countries is an international subsidy targeted at anti-malarials supplied through the retail sector. The goal of this strategy is to make ACT as affordable as ineffective alternatives. The programme, called the Affordable Medicines Facility - malaria was rolled out in Kenya in August 2010. METHODS: In December 2010, the affordability and accessibility of malaria medicines in a rural district in Kenya were evaluated using a complete census of all public and private facilities, chemists, pharmacists, and other malaria medicine retailers within the Webuye Demographic Surveillance Area. Availability, types, and prices of anti-malarials were assessed. There are 13 public or mission facilities and 97 medicine retailers (registered and unregistered). RESULTS: The average distance from a home to the nearest public health facility is 2 km, but the average distance to the nearest medicine retailer is half that. Quinine is the most frequently stocked anti-malarial (61% of retailers). More medicine retailers stocked sulphadoxine-pyramethamine (SP; 57%) than ACT (44%). Eleven percent of retailers stocked AMFm subsidized artemether-lumefantrine (AL). No retailers had chloroquine in stock and only five were selling artemisinin monotherapy. The mean price of any brand of AL, the recommended first-line drug in Kenya, was $2.7 USD. Brands purchased under the AMFm programme cost 40% less than non-AMFm brands. Artemisinin monotherapies cost on average more than twice as much as AMFm-brand AL. SP cost only $0.5, a fraction of the price of ACT. CONCLUSIONS: AMFm-subsidized anti-malarials are considerably less expensive than unsubsidized AL, but the price difference between effective and ineffective therapies is still large.

β-Arrestin-2 regulates the development of allergic asthma

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking β-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the β-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking β-arrestin-2. β-arrestin-2-deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine-mediated CD4+ T cell migration to the lung. This report provides the first evidence that β-arrestin-2 is required for the manifestation of allergic asthma. Because β-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice.

Heart failure is accompanied by severely impaired beta-adrenergic receptor (betaAR) function, which includes loss of betaAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of betaAR function is agonist-stimulated receptor phosphorylation by the betaAR kinase (betaARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in betaAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of betaARK1 or the beta2AR were mated into a genetic model of murine heart failure (MLP-/-). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP-/- and MLP-/-/beta2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP-/-/betaARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP-/-/betaARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP-/- mice but less than controls. Importantly, heightened betaAR desensitization in the MLP-/- mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the betaARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal betaAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit betaARK1 as a novel mode of therapy.