High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP‐IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP‐IV is highly specific for the male mouse pheromone 2‐sec‐butyl‐4,5‐dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X‐ray crystal structure of MUP‐IV bound to three mouse pheromones: SBT, 2,5‐dimethylpyrazine, and 2‐heptanone. We also obtained the structure of MUP‐IV with 2‐ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water‐mediated hydrogen bond network in the MUP‐I and MUP‐II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP‐IV.     

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer''s and Parkinson''s, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications.     

Genomics through the lens of next-generation sequencing

A report on the 23rd annual meeting on 'The Biology of Genomes', 11-15 May 2010, Cold Spring Harbor, USA.     

MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels

Three-dimensional (3D) cultures are increasing in use because of their ability to represent in vivo human physiology when compared to monolayer two-dimensional (2D) cultures. When grown in 3D using scaffold-free agarose hydrogels, MCF-7 human breast cancer cells self-organize to form directionally-oriented microtissues that contain a luminal space, reminiscent of the in vivo structure of the mammary gland. When compared to MCF-7 cells cultured in 2D monolayer culture, MCF-7 microtissues exhibit increased mRNA expression of luminal epithelial markers keratin 8 and keratin 19 and decreased expression of basal marker keratin 14 and the mesenchymal marker vimentin. These 3D MCF-7 microtissues remain responsive to estrogens, as demonstrated by induction of known estrogen target mRNAs following exposure to 17β-estradiol. Culture of MCF-7 cells in scaffold-free conditions allows for the formation of more differentiated, estrogen-responsive structures that are a more relevant system for evaluation of estrogenic compounds than traditional 2D models.     

Differentiating Coeliac Disease from Irritable Bowel Syndrome by Urinary Volatile Organic Compound Analysis – A Pilot Study

Coeliac disease (CD), a T-cell-mediated gluten sensitive enteropathy, affects ∼1% of the UK population and can present with wide ranging clinical features, often being mistaken for Irritable Bowel Syndrome (IBS). Heightened clinical awareness and serological screening identifies those with potential coeliac disease; the diagnosis is confirmed with duodenal biopsies, and symptom improvement with a gluten-free diet. Limitations to diagnosis are false negative serology and reluctance to undergo biopsy. The gut microbiome is altered in several gastrointestinal disorders, causing altered gut fermentation patterns recognisable by volatile organic compounds (VOC) analysis in urine, breath and faeces. We aimed to determine if CD alters the urinary VOC pattern, distinguishing it from IBS. 47 patients were recruited, 27 with established CD, on gluten free diets, and 20 with diarrhoea-predominant IBS (D-IBS). Collected urine was stored frozen in 10 ml aliquots. For assay, the specimens were heated to 40±0.1°C and the headspace analysed by Field Asymmetric Ion Mobility Spectrometry (FAIMS). Machine learning algorithms were used for statistical evaluation. Samples were also analysed using Gas chromatography and mass spectroscopy (GC-MS). Sparse logistic regression showed that FAIMS distinguishes VOCs in CD vs D-IBS with ROC curve AUC of 0.91 (0.83–0.99), sensitivity and specificity of 85% respectively. GCMS showed a unique peak at 4′67 found only in CD, not D-IBS, which correlated with the compound 1,3,5,7 cyclooctatetraene. This study suggests that FAIMS offers a novel, non-invasive approach to identify those with possible CD, and distinguishes from D-IBS. It offers the potential for monitoring compliance with a gluten-free diet at home. The presence of cyclooctatetraene in CD specimens will need further validation.     

Advances in the Conceptualization and Measurement of Health Care Empowerment: Development and Validation of the Health Care Empowerment Inventory

The Health Care Empowerment Model offers direction for the investigation of patient-controlled engagement and involvement in health care. At the core of the model is the construct of Health Care Empowerment (HCE), for which there exist no validated measures. A set of 27 candidate self-report survey items was constructed to capture five hypothesized inter-related facets of HCE (informed, engaged, committed, collaborative, and tolerant of uncertainty). The full item set was administered to 644 HIV-infected persons enrolled in three ongoing research studies. Exploratory and confirmatory factor analyses resulted in a two factor solution comprising four items each on two subscales: (1) HCE: Informed, Committed, Collaborative, and Engaged HCE ICCE) and (2) HCE Tolerance of Uncertainty (HCE TU). Subscale scores were evaluated for relationships with relevant constructs measured in the three studies, including depression, provider relationships, medication adherence, and HIV-1 viral load. Findings suggest the utility of this 8-item Health Care Empowerment Inventory (HCEI) in efforts to measure, understand, and track changes in the ways in which individuals engage in health care.     

A novel sialic acid-binding adhesin present in multiple species contributes to the pathogenesis of Infective endocarditis

Bacterial binding to platelets is a key step in the development of infective endocarditis (IE). Sialic acid, a common terminal carbohydrate on host glycans, is the major receptor for streptococci on platelets. So far, all defined interactions between streptococci and sialic acid on platelets are mediated by serine-rich repeat proteins (SRRPs). However, we identified Streptococcus oralis subsp. oralis IE-isolates that bind sialic acid but lack SRRPs. In addition to binding sialic acid, some SRRP^- isolates also bind the cryptic receptor β-1,4-linked galactose through a yet unknown mechanism. Using comparative genomics, we identified a novel sialic acid-binding adhesin, here named AsaA (associated with sialic acid adhesion A), present in IE-isolates lacking SRRPs. We demonstrated that S. oralis subsp. oralis AsaA is required for binding to platelets in a sialic acid-dependent manner. AsaA comprises a non-repeat region (NRR), consisting of a FIVAR/CBM and two Siglec-like and Unique domains, followed by 31 DUF1542 domains. When recombinantly expressed, Siglec-like and Unique domains competitively inhibited binding of S. oralis subsp. oralis and directly interacted with sialic acid on platelets. We further demonstrated that AsaA impacts the pathogenesis of S. oralis subsp. oralis in a rabbit model of IE. Additionally, we found AsaA orthologues in other IE-causing species and demonstrated that the NRR of AsaA from Gemella haemolysans blocked binding of S. oralis subsp. oralis, suggesting that AsaA contributes to the pathogenesis of multiple IE-causing species. Finally, our findings provide evidence that sialic acid is a key factor for bacterial-platelets interactions in a broader range of species than previously appreciated, highlighting its potential as a therapeutic target.     

A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis

BackgroundThe C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis.ResultsWe found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1.ConclusionsPTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.     

Feather isotope analysis discriminates age-classes of Western, Least, and Semipalmated sandpipers when plumage methods are unreliable

ABSTRACT Avian age‐class discrimination is typically based on the completeness of the first prebasic molt. In several calidrid sandpiper species, juvenal flight feathers grown on Arctic breeding grounds are retained through the first three migrations. Thereafter, flight feathers are grown annually at temperate migratory stopover sites during the fall or on the subtropical wintering grounds. Standard methods for distinguishing age classes of sandpipers rely on a combination of traits, including body plumage, coloration of protected inner median covert edges, and extent of flight feather wear. We tested the ability of stable hydrogen isotope ratios in flight feathers (δD_f) to distinguish young birds in their first winter through second fall from older adults in three calidrid sandpiper species, Western (Calidris mauri), Least (C. minutilla), and Semipalmated (C. pusilla) sandpipers. We compared the apparent reliability of the isotope approach to that of plumage‐based aging. The large expected differences in δD_f values of flight feathers grown at Arctic versus non‐Arctic latitudes enabled use of this technique to discriminate between age‐classes. We determined δD_f values of known Arctic‐grown feathers from juveniles that grew their flight feathers on the breeding grounds. Flight feather δD_f values of southward‐migrating adults showed bimodal distributions for all three species. Negative values overlapped with species‐specific juvenile values, identifying putative second fall birds with high‐latitude grown juvenal feathers retained from the previous year. The more positive values identified older adults who grew their feathers at mid‐ and low latitudes. Importantly, δD_f analysis successfully identified first‐winter and second‐fall birds not detected by plumage‐based aging. Flight feather wear alone was a poor basis for age classification because scores overlapped extensively between putative second fall birds and older adults. Flight feather hydrogen isotope analysis enables more definitive assignment of age classes when standard plumage methods are unreliable.