The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate.

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumours (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11-13 and EXT3 on 19p (refs 6-9). Most HME cases have been attributed to missense or frameshift mutations in these tumour-supressor genes, whose functions have remained obscure. Here, we show that EXT1 is an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing aetiologic missense mutations failed to alter cell-surface glycosaminoglycans, despite retaining their ER-localization.

Survival of calicivirus in foods and on surfaces: experiments with feline calicivirus as a surrogate for norovirus.

Although there is a large body of evidence incriminating foods as vehicles in the transmission of norovirus, little is known about virus survival in foods and on surfaces. Feline calicivirus was used as a surrogate for norovirus to investigate its survival in representative foods of plant and animal origin and on metal surfaces. Known concentrations of feline calicivirus in a natural fecal suspension were deposited onto lettuce, strawberries, ham, or stainless steel and incubated for 7 days at refrigeration or room temperatures. Virus was recovered at 1-day intervals, and the titers of the virus were determined by plaque assay. Infectious virus was recoverable until day 7 from lettuce, ham, and stainless steel. Statistically higher titers of feline calicivirus (P < 0.05) were recovered from ham under all conditions than from lettuce, strawberries, or stainless steel. These data provide valuable information for epidemiological and monitoring purposes as well as for the development of food processing practices and appropriate strategies to inactivate norovirus and control its transmission via foods and surfaces.

Evaluation of murine norovirus persistence in environments relevant to food production and processing.

Human norovirus (NoV) causes outbreaks of acute gastroenteritis associated with many ready-to-eat foods, including fresh produce. Effective inactivation procedures must consider virus survival under conditions of produce production and processing. This study aimed to investigate the persistence of NoV in a variety of environments, using murine NoV (MNV) as a surrogate for NoV. MNV was incubated for up to 42 days at room temperature on stainless steel disks, on lettuce, on soil, and in potable water and titers determined by plaque assay. A 1-log reduction of MNV infectivity was observed after 29 days in water, 4 days on lettuce, 12 days on soil, and 15 days on stainless steel disks. MNV survived longer in water than in any of the other environments, indicating that drying may contribute to NoV inactivation. MNV genomes were not significantly reduced for up to 42 days, suggesting that genomic detection is not a reliable indicator of viability. Overall, our findings provide valuable information regarding the potential for NoV transmission in the food supply.

Review: norovirus prevalence in Belgian, Canadian and French fresh produce: a threat to human health?

Foodborne viruses, especially noroviruses (NoV), are increasingly reported as the cause of foodborne outbreaks. NoV outbreaks have been reported linked to fresh soft red fruits and leafy greens. Belgium, Canada and France were the first countries to provide data about the prevalence of NoV on fresh produce. In total, 867 samples of leafy greens, 180 samples of fresh soft red fruits and 57 samples of other types of fresh produce (tomatoes, cucumber and fruit salads) were analyzed. Firstly, the NoV detection methodology, including virus and RNA extraction, real-time RT-PCR and quality controls were compared among the three countries. In addition, confirmation and genotyping of the NoV strains was attempted for a subset of NoV positive samples using conventional RT-PCR targeting an alternative region followed by sequencing. Analysis of the process control showed that 653, 179 and 18 samples of the leafy greens, soft red fruits and other fresh produce types were valid for analysis based on the recovery of the process control. NoV was detected by real-time RT-PCR in 28.2% (N=641), 33.3% (N=6) and 50% (N=6) of leafy greens tested in Canada, Belgium and France, respectively. Soft red fruits were found positive by real-time RT-PCR in 34.5% (N=29) and 6.7% (N=150) of the samples tested in Belgium and France, respectively. 55.5% (N=18) of the other fresh produce types, analyzed in Belgium, were found NoV positive by real-time RT-PCR. Conventional RT-PCR resulted in an amplicon of the expected size in 19.5% (52/266) of the NoV positive samples where this assay was attempted. Subsequent sequencing was only successful in 34.6% (18/52) of the suspected amplicons obtained by conventional RT-PCR. From this study, using the described methodology, NoV genomes were frequently detected in fresh produce however sequence confirmation was not successful for the majority of the samples tested. Infection or outbreaks were rarely or not known to be related to the NoV positive samples. With the increase in sensitivity of the detection methodology, there is an increasing concern about the interpretation of positive NoV results by real-time amplification. Strategies to confirm the results by real-time RT-PCR should be developed in analogy with the detection of microbial pathogens in foods. Detection might indicate contact with NoV in the fresh produce chain. Consequently, a potential risk for infection cannot be excluded but the actual risk from RT-PCR NoV positive produce is still unknown. Studies should be designed determining the probability of infection related to the presence or levels of NoV genomic copies.

Identification by integrated computer modeling and light scattering studies of an electrostatic serum albumin-hyaluronic acid binding site.

Dynamic light scattering and turbidimetry, carried out on solutions of hyaluronic acid (HA) and bovine or human serum albumin (SA) at fixed ionic strength (I), revealed a critical pH corresponding to the onset of HA-SA soluble complex formation. Subsequent reduction of pH below pH(c), corresponding to an increase in protein net positive charge, results in phase separation of the complex. The sensitivity of pH(c) to I indicated the primacy of electrostatic interactions in this process. Since pH(c) was always above the pK(a) of HA, these effects could be attributed to the influence of protein charge. The electrostatic potential around HSA was modeled using DelPhi (MSI) under pH, I conditions corresponding to incipient binding, phase separation, and noninteraction. At all incipient binding conditions (i.e., pH(c), at varying I), an identical region of positive potential 5 A from the protein van der Waals surface appeared. This unique domain intensified with a decrease in pH or I (corresponding to stronger binding), and diminished with an increase in pH or I (i.e., at noninteracting conditions). The size and low curvature of this domain could readily accommodate a 12 nm (decamer) sequence of HA. Simple electrostatic considerations indicate an electrostatic binding energy for the formation of this complex of ca. 1 kT, consistent with the condition of incipient complex formation. We suggest that such weak electrostatic binding may characterize nonspecific interactions for other protein-gylcosaminoglycan pairs.

The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration.

Human cytomegalovirus (HCMV) infection of smooth muscle cells (SMCs) in vivo has been linked to a viral etiology of vascular disease. In this report, we demonstrate that HCMV infection of primary arterial SMCs results in significant cellular migration. Ablation of the chemokine receptor, US28, abrogates SMC migration, which is rescued only by expression of the viral homolog and not a cellular G protein-coupled receptor (GPCR). Expression of US28 in the presence of CC chemokines including RANTES or MCP-1 was sufficient to promote SMC migration by both chemokinesis and chemotaxis, which was inhibited by protein tyrosine kinase inhibitors. US28-mediated SMC migration provides a molecular basis for the correlative evidence that links HCMV to the acceleration of vascular disease.