Detection of Infectious Cryptosporidium Oocysts by Cell Culture Immunofluorescence Assay: Applicability to Environmental Samples

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.     

Resting energy expenditure and delayed-onset muscle soreness after full-body resistance training with an eccentric concentration

The purpose of this investigation was to determine the effect of an acute bout of high-volume, full-body resistance training with an eccentric concentration on resting energy expenditure (REE) and indicators of delayed-onset muscle soreness (DOMS). Eight resistance trained (RT) and eight untrained (UT) participants (mean: age = 23.5 years; height = 180.76 cm; weight = 87.58 kg; body fat = 19.34%; lean mass = 68.71 kg) were measured on four consecutive mornings for REE and indicators of DOMS: creatine kinase (CK) and rating of perceived muscle soreness (RPMS). Delayed-onset muscle soreness was induced by performing eight exercises, eight sets, and six repetitions using a 1-second concentric and 3-second eccentric muscle action duration. A two-factor repeated-measures analysis of variance revealed that REE was significantly (p < 0.05) elevated at 24, 48, and 72 hours post compared with baseline measures for both UT and RT groups. Ratings of perceived muscle soreness were significantly elevated within groups for UT and RT at 24 and 48 hours post and for UT only at 72 hours post compared with baseline (p < 0.05). Nonparametric analyses revealed that CK was significantly increased at 24 hours post for both UT and RT and at 48 and 72 hours post for UT only compared with baseline (p < 0.05). Resting energy expenditure and indicators of DOMS were higher in UT compared with RT on all measures, but no significant differences were determined. The main finding of this investigation is that full-body resistance training with an eccentric concentration significantly increased REE up to 72 hours postexercise in UT and RT participants.     

Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production

Metabolic engineering in microbes could be used to produce large amounts of valuable metabolites that are difficult to extract from their natural sources and too expensive or complex to produce by chemical synthesis. As a step towards the production of Taxol in the yeast Saccharomyces cerevisiae, we introduced heterologous genes encoding biosynthetic enzymes from the early part of the taxoid biosynthetic pathway, isoprenoid pathway, as well as a regulatory factor to inhibit competitive pathways, and studied their impact on taxadiene synthesis. Expression of Taxus chinensis taxadiene synthase alone did not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of T. chinensis taxadiene synthase and geranylgeranyl diphosphate synthase failed to increase levels, probably due to steroid-based negative feedback, so we also expressed a truncated version of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMG-CoA reductase) isoenzyme 1 that is not subject to feedback inhibition and a mutant regulatory protein, UPC2-1, to allow steroid uptake under aerobic conditions, resulting in a 50% increase in taxadiene. Finally, we replaced the T. chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the T. chinensis taxadiene synthase gene to ensure high-level expression, resulting in a 40-fold increase in taxadiene to 8.7±0.85 mg/l as well as significant amounts of geranylgeraniol (33.1±5.6 mg/l), suggesting taxadiene levels could be increased even further. This is the first demonstration of such enhanced taxadiene levels in yeast and offers the prospect for Taxol production in recombinant microbes.     

Mecardonia tenella (Plantaginaceae) Attracts Oil-, Perfume-, and Pollen-Gathering Bees in Southern Brazil

Floral characteristics often indicate the pollinators' functional group visiting the plant and the pollination syndromes associated with them. This idea has been challenged in the past decades due to increasing evidence that most plants, including those exhibiting floral syndromes, are visited by large arrays of species that differ in their effectiveness as pollinators. Our study focuses on Mecardonia tenella (Plantaginaceae) from the Araucaria forest of southern Brazil, which exhibits characteristics of the oil flower pollination syndrome. However, it is visited by three types of functional groups of bees: male orchid bees, oil-collecting bees, and pollen-seeking bees. The relative contribution of each functional group to the plant's reproductive success was estimated based on their pollen load, visitation frequency, and morphology. We assessed resources, phenology, and breeding system of M. tenella . Our results indicate that flowers lack nectar, but volatiles, lipids, and pollen are resources that can be gathered by visitors. This combination of floral traits and visitors' assemblage makes M. tenella a challenge to the concept of pollination syndromes. Our findings indicate that the current interactions may not reflect the circumstances under which some floral traits of this plant were selected.     

Synthesis and biological evaluation of asymmetric gramicidin S analogues containing modified d-phenylalanine residues

The synthesis of new analogues of the cationic antimicrobial peptide gramicidin S, having a modified d-phenylalanine residue, their antibacterial properties against several Gram positive and negative strains, as well as their hemolytic activity is reported.     

A four-year surveillance program for detection of Plasmodium falciparum chloroquine resistance in Honduras

Countries could use the monitoring of drug resistance in malaria parasites as an effective early warning system to develop the timely response mechanisms that are required to avert the further spread of malaria. Drug resistance surveillance is essential in areas where no drug resistance has been reported, especially if neighbouring countries have previously reported resistance. Here, we present the results of a four-year surveillance program based on the sequencing of the pfcrt gene of Plasmodium falciparum populations from endemic areas of Honduras. All isolates were susceptible to chloroquine, as revealed by the pfcrt “CVMNK” genotype in codons 72-76.     

Changes in fossil chironomid remains along a depth gradient: evidence for common faunal thresholds within lakes

Many environmental variables that are important for the development of chironomid larvae (such as water temperature, oxygen availability, and food quantity) are related to water depth, and a statistically strong relationship between chironomid distribution and water depth is therefore expected. This study focuses on the distribution of fossil chironomids in seven shallow lakes and one deep lake from the Plymouth Aquifer (Massachusetts, USA) and aims to assess the influence of water depth on chironomid assemblages within a lake. Multiple samples were taken per lake in order to study the distribution of fossil chironomid head capsules within a lake. Within each lake, the chironomid assemblages are diverse and the changes that are seen in the assemblages are strongly related to changes in water depth. Several thresholds (i.e., where species turnover abruptly changes) are identified in the assemblages, and most lakes show abrupt changes at about 1–2 and 5–7 m water depth. In the deep lake, changes also occur at 9.6 and 15 m depth. The distribution of many individual taxa is significantly correlated to water depth, and we show that the identification of different taxa within the genus Tanytarsus is important because different morphotypes show different responses to water depth. We conclude that the chironomid fauna is sensitive to changes in lake level, indicating that fossil chironomid assemblages can be used as a tool for quantitative reconstruction of lake level changes.