Identification of Naegleria fowleri in domestic water sources by nested PCR

The free-living amoeboflagellate Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system. In the United States, the disease is generally acquired while swimming and diving in freshwater lakes and ponds. In addition to swimming, exposure to N. fowleri and the associated disease can occur by total submersion in bathwater or small backyard wading pools. In the present study, swipe samples and residual pipe water from homes in Arizona were examined for N. fowleri by nested PCR due to the death of two previously healthy children from PAM. Since neither child had a history of swimming in a freshwater lake or pond prior to the onset of disease symptoms, the domestic water supply was the suspected source of infection. Of 19 samples collected from bathroom and kitchen pipes and sink traps, 17 samples were positive for N. fowleri by PCR. A sample from a Micro-Wynd II filter was obtained by passing water from bathtubs through the filter. Organisms attached to the filter also tested positive by PCR. The two samples that tested negative for N. fowleri were one that was obtained from a kitchen sink trap and a swipe sample from the garbage disposal of one home.     

Determination of Parameters for the Supercritical Extraction of Antioxidant Compounds from Green Propolis Using Carbon Dioxide and Ethanol as Co-Solvent

The aim of this study was to determine the best processing conditions to extract Brazilian green propolis using a supercritical extraction technology. For this purpose, the influence of different parameters was evaluated such as S/F (solvent mass in relation to solute mass), percentage of co-solvent (1 and 2% ethanol), temperature (40 and 50°C) and pressure (250, 350 and 400 bar) using supercritical carbon dioxide. The Global Yield Isotherms (GYIs) were obtained through the evaluation of the yield, and the chemical composition of the extracts was also obtained in relation to the total phenolic compounds, flavonoids, antioxidant activity and 3,5-diprenyl-4-hydroxicinnamic acid (Artepillin C) and acid 4-hydroxycinnamic (p-coumaric acid). The best results were identified at 50°C, 350 bar, 1% ethanol (co-solvent) and S/F of 110. These conditions, a content of 8.93±0.01 and 0.40±0.05 g/100 g of Artepillin C and p-coumaric acid, respectively, were identified indicating the efficiency of the extraction process. Despite of low yield of the process, the extracts obtained had high contents of relevant compounds, proving the viability of the process to obtain green propolis extracts with important biological applications due to the extracts composition.     

Combined Genetic and Genealogic Studies Uncover a Large BAP1 Cancer Syndrome Kindred Tracing Back Nine Generations to a Common Ancestor from the 1700s

We recently discovered an inherited cancer syndrome caused by BRCA1-Associated Protein 1 (BAP1) germline mutations, with high incidence of mesothelioma, uveal melanoma and other cancers and very high penetrance by age 55. To identify families with the BAP1 cancer syndrome, we screened patients with family histories of multiple mesotheliomas and melanomas and/or multiple cancers. We identified four families that shared an identical BAP1 mutation: they lived across the US and did not appear to be related. By combining family histories, molecular genetics, and genealogical approaches, we uncovered a BAP1 cancer syndrome kindred of ~80,000 descendants with a core of 106 individuals, whose members descend from a couple born in Germany in the early 1700s who immigrated to North America. Their descendants spread throughout the country with mutation carriers affected by multiple malignancies. Our data show that, once a proband is identified, extended analyses of these kindreds, using genomic and genealogical studies to identify the most recent common ancestor, allow investigators to uncover additional branches of the family that may carry BAP1 mutations. Using this knowledge, we have identified new branches of this family carrying BAP1 mutations. We have also implemented early-detection strategies that help identify cancers at early-stage, when they can be cured (melanomas) or are more susceptible to therapy (MM and other malignancies).     

Comparison of Leishmania killicki (syn. L. tropica) and Leishmania tropica Population Structure in Maghreb by Microsatellite Typing

Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica) (from Tunisia, Algeria and Libya) and 113 L. tropica specimens (all from Morocco) were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica) between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica) in space and time. Comparison of the L. tropica (exclusively from Morocco) and L. killicki (syn. L. tropica) population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.     

The biosynthesis of acetovanillone in tobacco cell-suspension cultures

A soluble enzyme, extracted from tobacco cell-suspension cultures 24 h after treatment with 100 μM methyl jasmonate, has been shown to synthesize acetovanillone (apocynin) from feruloyl-CoA in the presence of NAD. The enzyme displayed Michaelis-Menten kinetics with apparent K_m values of 5.6 μM for feruloyl-CoA and 260 μM for NAD and exhibited very high specificity for its substrates. The increase in acetovanillone synthase activity was followed by an increase in the concentration of both acetovanillone and acetosyringone in the culture medium. No intermediate could be detected when analysing the reaction medium by HPLC during the formation of acetovanillone in cell-free extracts. The apparent molecular mass estimated by gel permeation on an FPLC column was ca. 79 kDa. To our knowledge, this is the first report of an enzymic system catalysing the synthesis of an acetophenone. This work demonstrates that the biosynthesis of acetophenones in tobacco proceeds from hydroxycinnamic acids through a CoA-dependent β-oxidation pathway. Interestingly in methyl jasmonate-treated cells, which synthesize very large amounts of hydroxycinnamoylputrescines, inhibition of the synthesis of these conjugates increased the concentration of acetovanillone and acetosyringone in the culture medium, suggesting that the two metabolic pathways can compete for their common precursors, i.e. hydroxycinnamoyl-CoA thioesters.     

Structure of Pinus sylvestris L. populations in Bulgaria revealed by chloroplast microsatellites and terpenes analysis: Provenance tests

In the present study we investigated the genetic structure and genetic diversity of Pinus sylvestris populations in Bulgaria using chloroplast microsatellite markers and terpene analysis. We were interested in addressing the following questions: (1) can population structure in Scots pine be detected via chloroplast microsatellites markers and terpenes; (2) are there differences in population differentiation between the two analyses; and (3) how are the patterns related to geographic distances. Twelve provenances were chosen throughout the species' range in Bulgaria. Following DNA extraction, chloroplast microsatellite (cpSSR) loci were surveyed using six primer pairs. Between 4 to 8 size variants were identified at each locus. A total of 35 size variants at the six loci were identified, 11 occurring at low frequencies (<1%). They were combined in 134 different haplotypes, of which seven represent 1/3 of the genetic structure. AMOVA analysis revealed that 10.99% of the variation was found among populations, while 89.01% was expressed within populations. The cpSSR analysis divided Scots pine populations into two groups, the first represented by populations located in the south-western part of the Rhodopes and Pirin mountains, while the second group is located in the northeast of Rhodopes and Rila mountains. Terpene analysis revealed that on average, 53% of the monoterpene pool in P. sylvestris was accounted for by -pinene (range 47–59%) followed by β-pinene (range 6–12%). The presence of two distinct groups is weekly consistent with physical distances between populations, similar significant correlation between genetic distance determined by chloroplast microsatellites analysis and chemotype distance (determined by terpenes) was observed. Our results suggest that the structural pattern of genetic diversity of cpDNA in Scots pine populations is the consequence of historical biogeographic processes.     

Insulin release: Reconciliation of the receptor and metabolic hypotheses

Summary Nutrients which stimulate insulin secretion are currently thought to initiate the series of cellular events eventually leading to insulin release either by interacting with a stereospecific receptor system (the regulatory site hypothesis) or by acting as a fuel (the substrate site hypothesis) in the pancreaticB-cell. The latter hypothesis is supported by a number of observations indicating that the capacity of nutrients to stimulate insulin release is indeed highly dependent on their capacity to increase catabolic fluxes in isolated pancreatic islets. However, these observations do not rule out the existence of nutrient receptors in islet cells. For instance, a nonmetabolized analog of L-leucine stimulates insulin release by causing allosteric activation of glutamate dehydrogenase, which should be considered, therefore, as a receptor for certain amino acids. Likewise, the increase in glycolytic flux, which is associated with the process of glucose-stimulated insulin release, is attributable not solely to a mass action phenomenon but also to the activation of phosphofructokinase by fructose 2.6-bisphosphate. The biosynthesis of this activator may involve a glucose receptor system. The fact that certain nutrient secretagogues (e.g D-glucose and L-leucine) act in the B-cell both as substrates and enzyme activators permits reconciliation of the substrate site and regulatory site hypotheses for insulin release.