Giardia Colonizes and Encysts in High-Density Foci in the Murine Small Intestine.

Giardia lamblia is a highly prevalent yet understudied protistan parasite causing significant diarrheal disease worldwide. Hosts ingest Giardia cysts from contaminated sources. In the gastrointestinal tract, cysts excyst to become motile trophozoites, colonizing and attaching to the gut epithelium. Trophozoites later differentiate into infectious cysts that are excreted and contaminate the environment. Due to the limited accessibility of the gut, the temporospatial dynamics of giardiasis in the host are largely inferred from laboratory culture and thus may not mirror Giardia physiology in the host. Here, we have developed bioluminescent imaging (BLI) to directly interrogate and quantify the in vivo temporospatial dynamics of Giardia infection, thereby providing an improved murine model to evaluate anti-Giardia drugs. Using BLI, we determined that parasites primarily colonize the proximal small intestine nonuniformly in high-density foci. By imaging encystation-specific bioreporters, we show that encystation initiates shortly after inoculation and continues throughout the duration of infection. Encystation also initiates in high-density foci in the proximal small intestine, and high density contributes to the initiation of encystation in laboratory culture. We suggest that these high-density in vivo foci of colonizing and encysting Giardia likely result in localized disruption to the epithelium. This more accurate visualization of giardiasis redefines the dynamics of the in vivo Giardia life cycle, paving the way for future mechanistic studies of density-dependent parasitic processes in the host. IMPORTANCEGiardia is a single-celled parasite causing significant diarrheal disease in several hundred million people worldwide. Due to limited access to the site of infection in the gastrointestinal tract, our understanding of the dynamics of Giardia infections in the host has remained limited and largely inferred from laboratory culture. To better understand Giardia physiology and colonization in the host, we developed imaging methods to quantify Giardia expressing bioluminescent physiological reporters in two relevant animal models. We discovered that parasites primarily colonize and encyst in the proximal small intestine in discrete, high-density foci. We also show that high parasite density contributes to encystation initiation

Uncultivated Microbial Eukaryotic Diversity: A Method to Link ssu rRNA Gene Sequences with Morphology

Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA “phylotypes” from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages, identified in diverse environments

Subaru FOCAS Spectroscopic Observations of High-Redshift Supernovae

We present spectra of high-redshift supernovae (SNe) that were taken with the Subaru low resolution optical spectrograph, FOCAS. These SNe were found in SN surveys with Suprime-Cam on Subaru, the CFH12k camera on the Canada-France-Hawaii Telescope (CFHT), and the Advanced Camera for Surveys (ACS) on the Hubble Space Telescope (HST). These SN surveys specifically targeted z>1 Type Ia supernovae (SNe Ia). From the spectra of 39 candidates, we obtain redshifts for 32 candidates and spectroscopically identify 7 active candidates as probable SNe Ia, including one at z=1.35, which is the most distant SN Ia to be spectroscopically confirmed with a ground-based telescope. An additional 4 candidates are identified as likely SNe Ia from the spectrophotometric properties of their host galaxies. Seven candidates are not SNe Ia, either being SNe of another type or active galactic nuclei. When SNe Ia are observed within a week of maximum light, we find that we can spectroscopically identify most of them up to z=1.1. Beyond this redshift, very few candidates were spectroscopically identified as SNe Ia. The current generation of super red-sensitive, fringe-free CCDs will push this redshift limit higher.Comment: 19 pages, 26 figures. PASJ in press. see http://www.supernova.lbl.gov/2009ClusterSurvey/ for additional information pertaining to the HST Cluster SN Surve

Protocol: does sodium nitrite administration reduce ischaemia-reperfusion injury in patients presenting with acute ST segment elevation myocardial infarction? Nitrites in acute myocardial infarction (NIAMI)

BACKGROUND: Whilst advances in reperfusion therapies have reduced early mortality from acute myocardial infarction, heart failure remains a common complication, and may develop very early or long after the acute event. Reperfusion itself leads to further tissue damage, a process described as ischaemia-reperfusion-injury (IRI), which contributes up to 50% of the final infarct size. In experimental models nitrite administration potently protects against IRI in several organs, including the heart. In the current study we investigate whether intravenous sodium nitrite administration immediately prior to percutaneous coronary intervention (PCI) in patients with acute ST segment elevation myocardial infarction will reduce myocardial infarct size. This is a phase II, randomised, placebo-controlled, double-blinded and multicentre trial. METHODS AND OUTCOMES: The aim of this trial is to determine whether a 5 minute systemic injection of sodium nitrite, administered immediately before opening of the infarct related artery, results in significant reduction of IRI in patients with first acute ST elevation myocardial infarction (MI). The primary clinical end point is the difference in infarct size between sodium nitrite and placebo groups measured using cardiovascular magnetic resonance imaging (CMR) performed at 6-8 days following the AMI and corrected for area at risk (AAR) using the endocardial surface area technique. Secondary end points include (i) plasma creatine kinase and Troponin I measured in blood samples taken pre-injection of the study medication and over the following 72 hours; (ii) infarct size at six months; (iii) Infarct size corrected for AAR measured at 6-8 days using T2 weighted triple inversion recovery (T2-W SPAIR or STIR) CMR imaging; (iv) Left ventricular (LV) ejection fraction measured by CMR at 6-8 days and six months following injection of the study medication; and (v) LV end systolic volume index at 6-8 days and six months. FUNDING,ETHICS AND REGULATORY APPROVALS: This study is funded by a grant from the UK Medical Research Council. This protocol is approved by the Scotland A Research Ethics Committee and has also received clinical trial authorisation from the Medicines and Healthcare products Regulatory Agency (MHRA) (EudraCT number: 2010-023571-26)

Giardia Flagellar Motility Is Not Directly Required to Maintain Attachment to Surfaces

Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined “suction-based” mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing “hydrodynamic model” of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is important for positioning and orienting trophozoites prior to attachment. Drugs affecting flagellar motility may result in lower levels of attachment by indirectly limiting the number of parasites that can position the ventral disc properly against a surface and against peristaltic flow

Novel Structural Components of the Ventral Disc and Lateral Crest in Giardia intestinalis

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment

Groups without cultured representatives dominate eukaryotic picophytoplankton in the oligotrophic South East Pacific Ocean

Background: Photosynthetic picoeukaryotes (PPE) with a cell size less than 3 µm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone. Methodology/Principal Findings: In order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX), clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales) dominated. Conclusions/Significance: Our data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics, and also to obtain into culture strains representative of these novel groups

Updated fracture incidence rates for the US version of FRAX®

# The Author(s) 2009. This article is published with open access at Springerlink.com Summary On the basis of updated fracture and mortality data, we recommend that the base population values used in the US version of FRAX ® be revised. The impact of suggested changes is likely to be a lowering of 10-year fracture probabilities. Introduction Evaluation of results produced by the US version of FRAX ® indicates that this tool overestimates the likelihood of major osteoporotic fracture. In an attempt to correct this, we updated underlying fracture and mortality rates for the model. Methods We used US hospital discharge data from 2006 t

A multiplex marker set for microsatellite typing and sexing of sooty terns Onychoprion fuscatus

OBJECTIVES: Seabirds have suffered dramatic population declines in recent decades with one such species being the sooty tern Onychoprion fuscatus. An urgent call to re-assess their conservation status has been made given that some populations, such as the one on Ascension Island, South Atlantic, have declined by over 80% in three generations. Little is known about their population genetics, which would aid conservation management through understanding ecological processes and vulnerability to environmental change. We developed a multiplex microsatellite marker set for sooty terns including sex-typing markers to assist population genetics studies. RESULTS: Fifty microsatellite loci were isolated and tested in 23 individuals from Ascension Island. Thirty-one were polymorphic and displayed between 4 and 20 alleles. Three loci were Z-linked and two autosomal loci deviated from Hardy-Weinberg equilibrium. The remaining 26 autosomal loci together with three sex-typing makers were optimised in seven polymerase chain reaction plexes. These 26 highly polymorphic markers will be useful for understanding genetic structure of the Ascension Island population and the species as a whole. Combining these with recently developed microsatellite markers isolated from Indian Ocean birds will allow for assessment of global population structure and genetic diversity