The Interconversion between a Flexible β-Sheet and a Fibril β-Arrangement Constitutes the Main Conformational Event during Misfolding of PSD95-PDZ3 Domain

The temperature-induced misfolding pathway of PDZ3, the third PDZ domain of the PSD95 neuronal protein, is populated by a trimeric β-sheet-rich intermediate state that leads to a stepwise and reversible formation of supramacromolecular structures. Using FTIR, we have found that misfolding of this pathway is not due to different ensembles of a variety of precursors, but comes mainly from the interconversion of a flexible β-sheet of the domain to wormlike fibrils. The appearance of the wormlike fibril FTIR component is also accompanied by a slight decrease of the band that corresponds to loops in the native state, whereas the rest of the regular elements of secondary structure are fairly well maintained upon misfolding. Transmission electron microscope micrographs have confirmed the presence of wormlike fibrils upon heating at 60°C, where the trimeric intermediate is maximally populated. Toxicity assays in the human neuroblastoma cell line SH-SY5Y show that cytotoxicity increases as the aggregation pathway proceeds. NMR analysis of chemical shifts as a function of temperature has revealed, as one of the main conformational aspects of such an interconversion at the residue level, that the β-sheet arrangement around strand β3 promotes the change that drives misfolding of the PDZ3 domain.     

Determinants of species evenness in a neotropical bat ensemble

Evenness is an important property of communities. Species richness alone does not capture the fact that one or a few species may dominate total abundance and biomass of a community. This in turn has important consequences for ecosystem functioning and species interactions. Evenness has been observed to vary systematically along environmental and productivity gradients. However, a truly general theory about which factors control evenness in a community has yet to emerge. Prior research on evenness has suggested that high richness, biomass and abundance should lead to lower community evenness in our study system of bats in Panama. However, only few empirical studies examine the simultaneous effects of species richness, biomass or abundance on evenness. For the first time, we applied path analysis in the study of evenness to tease apart the relative importance and direction (positive or negative) of causality among these three factors. As predicted, we found that evenness decreases with increasing species richness, abundance and biomass. The negative effect of abundance was mediated by the positive joint effect of biomass and richness. The selected models varied in the strength of the correlation between the three variables with evenness but their direction was consistent. Overall, we argue that rarity, high mobility and differences in resource availability at sites with lower environmental stress can explain the negative effects of richness on evenness.     

Anandamide induces sperm release from oviductal epithelia through nitric oxide pathway in bovines

Mammalian spermatozoa are not able to fertilize an egg immediately upon ejaculation. They acquire this ability during their transit through the female genital tract in a process known as capacitation. The mammalian oviduct acts as a functional sperm reservoir providing a suitable environment that allows the maintenance of sperm fertilization competence until ovulation occurs. After ovulation, spermatozoa are gradually released from the oviductal reservoir in the caudal isthmus and ascend to the site of fertilization. Capacitating-related changes in sperm plasma membrane seem to be responsible for sperm release from oviductal epithelium. Anandamide is a lipid mediator that participates in the regulation of several female and male reproductive functions. Previously we have demonstrated that anandamide was capable to release spermatozoa from oviductal epithelia by induction of sperm capacitation in bovines. In the present work we studied whether anandamide might exert its effect by activating the nitric oxide (NO) pathway since this molecule has been described as a capacitating agent in spermatozoa from different species. First, we demonstrated that 1 μM NOC-18, a NO donor, and 10 mM L-Arginine, NO synthase substrate, induced the release of spermatozoa from the oviductal epithelia. Then, we observed that the anandamide effect on sperm oviduct interaction was reversed by the addition of 1 μM L-NAME, a NO synthase inhibitor, or 30 μg/ml Hemoglobin, a NO scavenger. We also demonstrated that the induction of bull sperm capacitation by nanomolar concentrations of R(+)-methanandamide or anandamide was inhibited by adding L-NAME or Hemoglobin. To study whether anandamide is able to produce NO, we measured this compound in both sperm and oviductal cells. We observed that anandamide increased the levels of NO in spermatozoa, but not in oviductal cells. These findings suggest that anandamide regulates the sperm release from oviductal epithelia probably by activating the NO pathway during sperm capacitation.     

Administration of triclabendazole is safe and effective in controlling fascioliasis in an endemic community of the bolivian altiplano

Background

The Bolivian northern Altiplano is characterized by a high prevalence of Fasciola hepatica infection. In order to assess the feasibility, safety and efficacy of large-scale administration of triclabendazole as an appropriate public health measure to control morbidity associated with fascioliasis, a pilot intervention was implemented in 2008.

Materials and Methods

Schoolchildren from an endemic community were screened for fascioliasis and treated with a single administration of triclabendazole (10 mg/kg). Interviews to assess the occurrence of adverse events were conducted on treatment day, one week later, and one month after treatment. Further parasitological screenings were performed three months after treatment and again two months later (following a further treatment) in order to evaluate the efficacy of the intervention.

Results

Ninety infected children were administered triclabendazole. Adverse events were infrequent and mild. No serious adverse events were reported. Observed cure rates were 77.8% after one treatment and 97.8% after two treatments, while egg reduction rates ranged between 74% and 90.3% after one treatment, and between 84.2% and 99.9% after two treatments. The proportion of high-intensity infections (≥400 epg) decreased from 7.8% to 1.1% after one treatment and to 0% after two treatments.

Conclusion

Administration of triclabendazole is a feasible, safe and efficacious public health intervention in an endemic community in the Bolivian Altiplano, suggesting that preventive chemotherapy can be applied to control of fascioliasis. Further investigations are needed to define the most appropriate frequency of treatment.     

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies ¹². The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used^1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocystsand Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously^{1-3,5-8,10,11}. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters⁴. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices^9,13 .This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.     

Polyphosphate Degradation in Stationary Phase Triggers Biofilm Formation via LuxS Quorum Sensing System in Escherichia coli

In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk⁻ppx⁻ or ppk⁻ppx⁻/ppk⁺ strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.     

Acute podocyte vascular endothelial growth factor (VEGF-A) knockdown disrupts alphaVbeta3 integrin signaling in the glomerulus

Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGF(KD)) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ~20% of non-induced controls and urine VEGF-A to ~30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alpha(V)beta(3) integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta(3) integrin and neuropilin-1 in the kidney in vivo and in VEGF(KD) podocytes. Podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGF(KD) podocytes downregulates fibronectin and disrupts alpha(V)beta(3) integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alpha(V)beta(3) integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure.     

Novel phospholipolytic activities associated with electronegative low-density lipoprotein are involved in increased self-aggregation

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction present in plasma with increased platelet-activating factor acetylhydrolase (PAF-AH) activity. This activity could be involved in the proinflammatory effects of LDL(-). Our aim was to study the presence of additional phospholipolytic activities in LDL(-). Total LDL was fractionated into electropositive (LDL(+)) and LDL(-) by anion-exchange chromatography, and phospholipolytic activities were measured by fluorometric methods. Phospholipolytic activity was absent in LDL(+) whereas LDL(-) presented activity against lysophosphatidylcholine (LPC, 82.4 +/- 34.9 milliunits/mg of apoB), sphingomyelin (SM, 53.3 +/- 22.5 milliunits/mg of apoB), and phosphatidylcholine (PC, 25.7 +/- 4.3 milliunits/mg of apoB). LDL(-), but not LDL(+), presented spontaneous self-aggregation at 37 degrees C in parallel to phospholipid degradation. This was observed in the absence of lipid peroxidation and suggests the involvement of phospholipolytic activity in self-aggregation of LDL(-). Phospholipolytic activity was not due to PAF-AH, apoE, or apoC-III and was not increased in LDL(+) modified by Cu (2+) oxidation, acetylation, or secretory phospholipase A 2 (PLA 2). However, LDL(-) efficiently degraded phospholipids of lipoproteins enriched in LPC, such as oxidized LDL or PLA 2-LDL, but not native or acetylated LDL. This finding supports that LPC is the best substrate for LDL(-)-associated phospholipolytic activity. These results reveal novel properties of LDL(-) that could play a significant role in its atherogenic properties.     

Airborne fungi monitoring in Santiago, Chile

Fungal airborne spores were studied from September 1996throughout August 1997 in Santiago, Chile. Total concentrationsfluctuated between 308 and 10,334 spores/m³/day withan annual mean of 2,154 per m³, the highest dispersion beingduring April and May. Forty-five percent of total fungal content wasfound in autumn. Thirteen genera and 3 other spore types wereidentified. Cladosporium, the most abundant genera in ouratmosphere, contributed with 70.9% of the total fungi counts andreached an annual mean of 1,527 spores/m³/day, itshighest frequency being in autumn. Alternaria appeared as thesecond most frequent genera, with an annual mean of 40spores/m³/day, representing a 1.9% of theannual fungal catch. Altogether, Stemphylium, Torula, Epicoccum,Ganoderma, Helminthosporium, Chaetomiun, Pleospora and othersreached relative frequencies of 0.5% or less. It is concludedthat fungi are present in Santiago's atmosphere all year round, some ofthem with a clear seasonality.     

Weak acid accumulation in the serosal extracellular compartment of the frog gastric mucosa.

The dimethyloxazolidine dione distribution in the extracellular compartments of the frog gastric mucosa was analyzed by washout kinetics. The volumes of the two extracellular compartments, serosal and mucosal, were estimated by inulin washout as 0.435 +/- 0.019 and 0.176 +/- 0.018 microliter/microliter tissue water, respectively. In the serosal extracellular space, significant dimethyloxazolidine dione accumulations of 2.63 +/- 0.25, 2.28 +/- 0.16, and 1.86 +/- 0.08 times that of the bathing media were found for bathing solutions with pH values of 6.9, 7.4, and 7.9 respectively. A high pH of the serosal extracellular fluid by itself could not account for the high values of dimethyloxazolidine dione accumulation. A difference in the total dimethyloxazolidine dione accumulation requires: (a) the existence of differences in the pH values and also the existence of a difference in the diffusion coefficient of the two forms of dimethyloxazolidine dione; or (b), a binding of one of the two forms, i.e., binding of dimethyloxazolidine dione form by fixed charges.