In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Recently, <it>Plasmodium falciparum </it>parasites bearing <it>Pfdhfr </it>I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves.</p> <p>Methods</p> <p>Thirty <it>Plasmodium falciparum </it>clinical isolates were collected in 2008 in the south-east of Madagascar. A part of <it>Pfdhfr </it>gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC_50sto pyrimethamine was performed on adapted isolates.</p> <p>Results</p> <p>Four different <it>Pfdhfr </it>alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of <it>P. falciparum </it>isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine <it>in vitro </it>susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC₅₀) < 10 nM. The geometric mean of IC₅₀of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC₅₀of the triple mutant parasite (13,804 nM).</p> <p>Conclusion</p> <p>The IC₅₀s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC₅₀s directly on parasites did not confirm what has been found on transgenic bacteria. The prevalence increase of the <it>Pfdhfr </it>I164L single mutant parasite since 2006 could be explained by the selective advantage of this allele under sulphadoxine-pyrimethamine pressure. The emergence of highly resistant alleles should be considered in the future, in particular because an unexpected double mutant-type allele S108N/I164L has been already detected.</p

The Knee Clinical Assessment Study – CAS(K). A prospective study of knee pain and knee osteoarthritis in the general population

BACKGROUND: Knee pain affects an estimated 25% of the adult population aged 50 years and over. Osteoarthritis is the most common diagnosis made in older adults consulting with knee pain in primary care. However, the relationship between this diagnosis and both the current disease-based definition of osteoarthritis and the regional pain syndrome of knee pain and disability is unclear. Expert consensus, based on current evidence, views the disease and the syndrome as distinct entities but the clinical usefulness of these two approaches to classifying knee pain in older adults has not been established. We plan to conduct a prospective, population-based, observational cohort study to investigate the relative merits of disease-based and regional pain syndrome-based approaches to classification and prognosis of knee pain in older adults. METHODS: All patients aged 50 years and over registered with three general practices in North Staffordshire will be invited to take part in a two-stage postal survey. Respondents to this survey phase who indicate that they have experienced knee pain within the previous 12 months will be invited to attend a research clinic for a detailed assessment. This will consist of clinical interview, physical examination, digital photography, plain x-rays, anthropometric measurement and a brief self-complete questionnaire. All consenting clinic attenders will be followed up by (i) general practice medical record review, (ii) repeat postal questionnaire at 18-months

Selective estrogen receptor modulators inhibit growth and progression of premalignant lesions in a mouse model of ductal carcinoma in situ

INTRODUCTION: Ductal carcinoma in situ (DCIS) is a noninvasive premalignant lesion and is considered a precursor to invasive carcinoma. DCIS accounts for nearly 20% of newly diagnosed breast cancer, but the lack of experimentally amenable in vivo DCIS models hinders the development of treatment strategies. Here, we demonstrate the utility of a mouse transplantation model of DCIS for chemoprevention studies using selective estrogen receptor modulators (SERMs). This model consists of a set of serially transplanted lines of genetically engineered mouse mammary intraepithelial neoplasia (MIN) outgrowth (MIN-O) tissue that have stable characteristics. We studied the ovarian-hormone-responsiveness of one of the lines with a particular focus on the effects of two related SERMs, tamoxifen and ospemifene. METHODS: The estrogen receptor (ER) status and ovarian-hormone-dependence of the mouse MIN outgrowth tissue were determined by immunohistochemistry and ovarian ablation. The effects of tamoxifen and ospemifene on the growth and tumorigenesis of MIN outgrowth were assessed at 3 and 10 weeks after transplantation. The effects on ER status, cell proliferation, and apoptosis were studied with immunohistochemistry. RESULTS: The MIN-O was ER-positive and ovarian ablation resulted in reduced MIN-O growth and tumor development. Likewise, tamoxifen and ospemifene treatments decreased the MIN growth and tumor incidence in comparison with the control (P < 0.01). Both SERMs significantly decreased cell proliferation. Between the two SERM treatment groups, there were no statistically significant differences in MIN-O size, tumor latency, or proliferation rate. In contrast, the ospemifene treatment significantly increased ER levels while tamoxifen significantly decreased them. CONCLUSION: Tamoxifen and ospemifene inhibit the growth of premalignant mammary lesions and the progression to invasive carcinoma in a transplantable mouse model of DCIS. The inhibitory effects of these two SERMs are similar except for their effects on ER modulation. These differences in ER modulation may suggest different mechanisms of action between the two related SERMs and may portend different long-term outcomes. These data demonstrate the value of this model system for preclinical testing of antiestrogen or other therapies designed to prevent or delay the malignant transformation of premalignant mammary lesions in chemoprevention

An evolutionary approach to international political economy: the case of corporate tax avoidance

Corporate tax avoidance is both widespread and diverse in its practical mechanics. The scope of the phenomenon often leads economists to conclude that in the jungle of economic competition, tax planning (or optimisation) is among the necessary tools to ensure the survival of the fittest. This theory is increasingly associated with a Darwinian theory of economic evolution. In this paper, I develop a contrasting framework of the evolutionary political economy of corporate tax avoidance. Analysing core concepts of Old Institutionalist Economics (OIE), I examine the core drivers of corporate tax avoidance in a globalised system of states. The major contrast, I find, is between that of the corporate and legal personality and the institutional environment in which it operates. Historically, each corporate entity has been considered a separate legal person, yet a series of ‘mutations’ of incorporations laws created a widening gap between theory and reality, and these, in turn, give rise to tax arbitrage. Narrowing this gap, however, impinges on another venerable historical institution, the institution of sovereignty and sovereign inequality

Conservation and divergence of known apicomplexan transcriptional regulons

<p>Abstract</p> <p>Background</p> <p>The apicomplexans are a diverse phylum of parasites causing an assortment of diseases including malaria in a wide variety of animals and lymphoproliferation in cattle. Little is known about how these varied parasites regulate their transcriptional regulons. Even less is known about how regulon systems, consisting of transcription factors and target genes together with their associated biological process, evolve in these diverse parasites.</p> <p>Results</p> <p>In order to obtain insights into the differences in transcriptional regulation between these parasites we compared the orthology profiles of putative malaria transcription factors across species and examined the enrichment patterns of four binding sites across eleven apicomplexans.</p> <p>About three-fifths of the factors are broadly conserved in several phylogenetic orders of sequenced apicomplexans. This observation suggests the existence of regulons whose regulation is conserved across this ancient phylum. Transcription factors not broadly conserved across the phylum are possibly involved in regulon systems that have diverged between species. Examining binding site enrichment patterns in light of transcription factor conservation patterns suggests a second mode via which regulon systems may diverge - rewiring of existing transcription factors and their associated binding sites in specific ways. Integrating binding sites with transcription factor conservation patterns also facilitated prediction of putative regulators for one of the binding sites.</p> <p>Conclusions</p> <p>Even though transcription factors are underrepresented in apicomplexans, the distribution of these factors and their associated regulons reflect common and family-specific transcriptional regulatory processes.</p

Orally Active Multi-Functional Antioxidants Delay Cataract Formation in Streptozotocin (Type 1) Diabetic and Gamma-Irradiated Rats

Age-related cataract is a worldwide health care problem whose progression has been linked to oxidative stress and the accumulation of redox-active metals. Since there is no specific animal model for human age-related cataract, multiple animal models must be used to evaluate potential therapies that may delay and/or prevent cataract formation.Proof of concept studies were conducted to evaluate 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8), multi-functional antioxidants that can independently chelate redox metals and quench free radicals, on their ability to delay the progression of diabetic "sugar" cataracts and gamma radiation-induced cataracts. Prior to 15 Gy of whole head irradiation, select groups of Long Evans rats received either diet containing compound 4 or 8, or a single i.p. injection of panthethine, a radioprotective agent. Compared to untreated, irradiated rats, treatment with pantethine, 4 and 8 delayed initial lens changes by 4, 47, and 38 days, respectively, and the average formation of posterior subcapsular opacities by 23, 53 and 58 days, respectively. In the second study, select groups of diabetic Sprague Dawley rats were administered chow containing compounds 4, 8 or the aldose reductase inhibitor AL1576. As anticipated, treatment with AL1576 prevented cataract by inhibiting sorbitol formation in the lens. However, compared to untreated rats, compounds 4 and 8 delayed vacuole formation by 20 days and 12 days, respectively, and cortical cataract formation by 8 and 3 days, respectively, without reducing lenticular sorbitol. Using in vitro lens culture in 30 mM xylose to model diabetic "sugar" cataract formation, western blots confirmed that multi-functional antioxidants reduced endoplasmic reticulum stress.Multi-functional antioxidants delayed cataract formation in two diverse rat models. These studies provide a proof of concept that a general cataract treatment focused on reducing oxidative stress instead of a specific mechanism of cataractogenesis can be developed

Food site residence time and female competitive relationships in wild gray-cheeked mangabeys (Lophocebus albigena)

Authors of socioecological models propose that food distribution affects female social relationships in that clumped food resources, such as fruit, result in strong dominance hierarchies and favor coalition formation with female relatives. A number of Old World monkey species have been used to test predictions of the socioecological models. However, arboreal forest-living Old World monkeys have been understudied in this regard, and it is legitimate to ask whether predominantly arboreal primates living in tropical forests exhibit similar or different patterns of behavior. Therefore, the goal of our study was to investigate female dominance relationships in relation to food in gray-cheeked mangabeys (Lophocebus albigena). Since gray-cheeked mangabeys are largely frugivorous, we predicted that females would have linear dominance hierarchies and form coalitions. In addition, recent studies suggest that long food site residence time is another important factor in eliciting competitive interactions. Therefore, we also predicted that when foods had long site residence times, higher-ranking females would be able to spend longer at the resource than lower-ranking females. Analyses showed that coalitions were rare relative to some other Old World primate species, but females had linear dominance hierarchies. We found that, contrary to expectation, fruit was not associated with more agonism and did not involve long site residence times. However, bark, a food with a long site residence time and potentially high resource value, was associated with more agonism, and higher-ranking females were able to spend more time feeding on it than lower-ranking females. These results suggest that higher-ranking females may benefit from higher food and energy intake rates when food site residence times are long. These findings also add to accumulating evidence that food site residence time is a behavioral contributor to female dominance hierarchies in group-living species

Interactions Between Laminin Receptor and the Cytoskeleton During Translation and Cell Motility

Human laminin receptor acts as both a component of the 40S ribosomal subunit to mediate cellular translation and as a cell surface receptor that interacts with components of the extracellular matrix. Due to its role as the cell surface receptor for several viruses and its overexpression in several types of cancer, laminin receptor is a pathologically significant protein. Previous studies have determined that ribosomes are associated with components of the cytoskeleton, however the specific ribosomal component(s) responsible has not been determined. Our studies show that laminin receptor binds directly to tubulin. Through the use of siRNA and cytoskeletal inhibitors we demonstrate that laminin receptor acts as a tethering protein, holding the ribosome to tubulin, which is integral to cellular translation. Our studies also show that laminin receptor is capable of binding directly to actin. Through the use of siRNA and cytoskeletal inhibitors we have shown that this laminin receptor-actin interaction is critical for cell migration. These data indicate that interactions between laminin receptor and the cytoskeleton are vital in mediating two processes that are intimately linked to cancer, cellular translation and migration

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved