Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer

The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (~400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (~1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1LRR. In contrast, neither MBP alone nor MBP fused to CWP1CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase. Author SummaryWhile the walls of plants and fungi contain numerous sugar homopolymers (cellulose, chitin, and β-1,3-glucans) and dozens of proteins, the cyst wall of Giardia is relatively simple. The Giardia wall contains a unique homopolymer of β-1,3-linked N-acetylgalactosamine (GalNAc) and at least three cyst wall proteins (CWPs), each of which is composed of Leu-rich repeats and a C-terminal Cys-rich region. The three major discoveries here are: 1) Fibrils of the GalNAc homopolymer are curled and form a lattice that is compressed into a narrow plane by bound protein in intact cyst walls. 2) Leu-rich repeats of CWP1 form a novel lectin domain that is specific for fibrils of the GalNAc homopolymer, which can be isolated by methods used to deproteinate fungal walls. 3) A cyst-specific glycohydrolase is able to degrade deproteinated fibrils of the GalNAc homopolymer. We incorporate these findings into a new curled fiber and lectin model of the intact Giardia cyst wall and a protease and glycohydrolase model of excystation.National Institutes of Health (AI048082, AI44070, GM31318, RR1088

Cholesterol dependence of HTLV-I infection

Cholesterol-rich plasma membrane microdomains are important for entry of many viruses, including retro-viruses. Depletion of cholesterol with 2-hydroxypropyl-β-cyclodextrin inhibits entry of human T cell leukemia virus type I (HTLV-1) and HTLV-I envelope pseudotyped lentivirus particles. Using a soluble fusion protein of the HTLV-I surface envelope protein with the immunoglobulin Fc domain, the HTLV-I receptor was found to colocalize with a raft-associated marker and to cluster in specific plasma membrane microdomains. Depletion of cholesterol did not alter receptor binding activity, suggesting a requirement for cholesterol in a postbinding virus entry step

Oligosaccharide and Glycoprotein Microarrays as Tools in HIV Glycobiology Glycan-Dependent gp120/Protein Interactions

AbstractDefining HIV envelope glycoprotein interactions with host factors or binding partners advances our understanding of the infectious process and provides a basis for the design of vaccines and agents that interfere with HIV entry. Here we employ carbohydrate and glycoprotein microarrays to analyze glycan-dependent gp120-protein interactions. In concert with new linking chemistries and synthetic methods, the carbohydrate arrays combine the advantages of microarray technology with the flexibility and precision afforded by organic synthesis. With these microarrays, we individually and competitively determined the binding profiles of five gp120 binding proteins, established the carbohydrate structural requirements for these interactions, and identified a potential strategy for HIV vaccine development

Femtosecond photodissociation dynamics of 1,4-diiodobenzene by gas-phase X-ray scattering and photoelectron spectroscopy

We present a multifaceted investigation into the initial photodissociation dynamics of 1,4-diiodobenzene (DIB) following absorption of 267 nm radiation. We combine ultrafast time-resolved photoelectron spectroscopy and X-ray scattering experiments performed at the Linac Coherent Light Source (LCLS) to study the initial electronic excitation and subsequent rotational alignment, and interpret the experiments in light of Complete Active Space Self-Consistent Field (CASSCF) calculations of the excited electronic landscape. The initially excited state is found to be a bound 1B1 surface, which undergoes ultrafast population transfer to a nearby state in 35 ± 10 fs. The internal conversion most likely leads to one or more singlet repulsive surfaces that initiate the dissociation. This initial study is an essential and prerequisite component of a comprehensive study of the complete photodissociation pathway(s) of DIB at 267 nm. Assignment of the initially excited electronic state as a bound state identifies the mechanism as predissociative, and measurement of its lifetime establishes the time between excitation and initiation of dissociation, which is crucial for direct comparison of photoelectron and scattering experiments.</p

Single-Cell Analysis of Experience-Dependent Transcriptomic States in Mouse Visual Cortex

Activity-dependent transcriptional responses shape cortical function. However, we lack a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease. Here we applied high-throughput single-cell RNA-sequencing to investigate the breadth of transcriptional changes that occur across cell types in mouse visual cortex following exposure to light. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibit inter- and intra-laminar heterogeneity in the induction of stimulus responsive genes. Non-neuronal cells demonstrated clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of stimulus-dependent transcriptional changes that occur across cell types in visual cortex, which are likely critical for cortical function and may be sites of de-regulation in developmental brain disorders

An empirical approach to the nucleation of sulfuric acid droplets in the atmosphere

We use quantum mechanical evaluations of the Gibbs free energy of the hydrates of sulfuric acid, H2SO4. nH2O and (H2SO4)2 . nH2O to evaluate an empirical surface tension for sulfuric acid-water clusters containing few molecules. We use this surface tension to evaluate nucleation rates using classical heteromolecular theory. At low temperatures (T 213 K) the nucleation rates obtained with the empirical surface tensions are signifi cantly greater than those using bulk values of the surface tension. At higher temperatures the difference disappears

The Evaluation of a Rapid In Situ HIV Confirmation Test in a Programme with a High Failure Rate of the WHO HIV Two-Test Diagnostic Algorithm

BACKGROUND: Concerns about false-positive HIV results led to a review of testing procedures used in a Médecins Sans Frontières (MSF) HIV programme in Bukavu, eastern Democratic Republic of Congo. In addition to the WHO HIV rapid diagnostic test algorithm (RDT) (two positive RDTs alone for HIV diagnosis) used in voluntary counselling and testing (VCT) sites we evaluated in situ a practical field-based confirmation test against western blot WB. In addition, we aimed to determine the false-positive rate of the WHO two-test algorithm compared with our adapted protocol including confirmation testing, and whether weakly reactive compared with strongly reactive rapid test results were more likely to be false positives. METHODOLOGY/PRINCIPAL FINDINGS: 2864 clients presenting to MSF VCT centres in Bukavu during January to May 2006 were tested using Determine HIV-1/2 and UniGold HIV rapid tests in parallel by nurse counsellors. Plasma samples on 229 clients confirmed as double RDT positive by laboratory retesting were further tested using both WB and the Orgenics Immunocomb Combfirm HIV confirmation test (OIC-HIV). Of these, 24 samples were negative or indeterminate by WB representing a false-positive rate of the WHO two-test algorithm of 10.5% (95%CI 6.6-15.2). 17 of the 229 samples were weakly positive on rapid testing and all were negative or indeterminate by WB. The false-positive rate fell to 3.3% (95%CI 1.3-6.7) when only strong-positive rapid test results were considered. Agreement between OIC-HIV and WB was 99.1% (95%CI 96.9-99.9%) with no false OIC-HIV positives if stringent criteria for positive OIC-HIV diagnoses were used. CONCLUSIONS: The WHO HIV two-test diagnostic algorithm produced an unacceptably high level of false-positive diagnoses in our setting, especially if results were weakly positive. The most probable causes of the false-positive results were serological cross-reactivity or non-specific immune reactivity. Our findings show that the OIC-HIV confirmation test is practical and effective in field contexts. We propose that all double-positive HIV RDT samples should undergo further testing to confirm HIV seropositivity until the accuracy of the RDT testing algorithm has been established at programme level

Infection of Cultured Human Endothelial Cells by Legionella pneumophila

Legionella pneumophila is a gram-negative pathogen that causes a severe pneumonia known as Legionnaires' disease. Here, we demonstrate for the first time that L. pneumophila infects and grows within cultured human endothelial cells. Endothelial infection may contribute to lung damage observed during Legionnaires' disease and to systemic spread of this organism

Culture-Independent Microbiological Analysis of Foley Urinary Catheter Biofilms

Background: Prevention of catheter-associated urinary tract infection (CAUTI), a leading cause of nosocomial disease, is complicated by the propensity of bacteria to form biofilms on indwelling medical devices [1,2,3,4,5]. Methodology/Principal Findings: To better understand the microbial diversity of these communities, we report the results of a culture-independent bacterial survey of Foley urinary catheters obtained from patients following total prostatectomy. Two patient subsets were analyzed, based on treatment or no treatment with systemic fluoroquinolone antibiotics during convalescence. Results indicate the presence of diverse polymicrobial assemblages that were most commonly observed in patients who did not receive systemic antibiotics. The communities typically contained both Gram-positive and Gramnegative microorganisms that included multiple potential pathogens. Conclusion/Significance: Prevention and treatment of CAUTI must take into consideration the possible polymicrobial nature of any particular infection

Cardioprotective effects of lixisenatide in rat myocardial ischemia-reperfusion injury studies

BACKGROUND: Lixisenatide is a glucagon-like peptide-1 analog which stimulates insulin secretion and inhibits glucagon secretion and gastric emptying. We investigated cardioprotective effects of lixisenatide in rodent models reflecting the clinical situation. METHODS: The acute cardiac effects of lixisenatide were investigated in isolated rat hearts subjected to brief ischemia and reperfusion. Effects of chronic treatment with lixisenatide on cardiac function were assessed in a modified rat heart failure model after only transient coronary occlusion followed by long-term reperfusion. Freshly isolated cardiomyocytes were used to investigate cell-type specific mechanisms of lixisenatide action. RESULTS: In the acute setting of ischemia-reperfusion, lixisenatide reduced the infarct-size/area at risk by 36% ratio without changes on coronary flow, left-ventricular pressure and heart rate. Treatment with lixisenatide for 10 weeks, starting after cardiac ischemia and reperfusion, improved left ventricular end-diastolic pressure and relaxation time and prevented lung congestion in comparison to placebo. No anti-fibrotic effect was observed. Gene expression analysis revealed a change in remodeling genes comparable to the ACE inhibitor ramipril. In isolated cardiomyocytes lixisenatide reduced apoptosis and increased fractional shortening. Glucagon-like peptide-1 receptor (GLP1R) mRNA expression could not be detected in rat heart samples or isolated cardiomyocytes. Surprisingly, cardiomyocytes isolated from GLP-1 receptor knockout mice still responded to lixisenatide. CONCLUSIONS: In rodent models, lixisenatide reduced in an acute setting infarct-size and improved cardiac function when administered long-term after ischemia-reperfusion injury. GLP-1 receptor independent mechanisms contribute to the described cardioprotective effect of lixisenatide. Based in part on these preclinical findings patients with cardiac dysfunction are currently being recruited for a randomized, double-blind, placebo-controlled, multicenter study with lixisenatide. TRIAL REGISTRATION: (ELIXA, ClinicalTrials.gov Identifier: NCT01147250