Norwalk Virus–specific Binding to Oyster Digestive Tissues

Specific binding of virus to oysters can selectively concentrate a human pathogen

Histo-Blood Group Antigens Act as Attachment Factors of Rabbit Hemorrhagic Disease Virus Infection in a Virus Strain-Dependent Manner

Rabbit Hemorrhagic disease virus (RHDV), a calicivirus of the Lagovirus genus, and responsible for rabbit hemorrhagic disease (RHD), kills rabbits between 48 to 72 hours post infection with mortality rates as high as 50–90%. Caliciviruses, including noroviruses and RHDV, have been shown to bind histo-blood group antigens (HBGA) and human non-secretor individuals lacking ABH antigens in epithelia have been found to be resistant to norovirus infection. RHDV virus-like particles have previously been shown to bind the H type 2 and A antigens. In this study we present a comprehensive assessment of the strain-specific binding patterns of different RHDV isolates to HBGAs. We characterized the HBGA expression in the duodenum of wild and domestic rabbits by mass spectrometry and relative quantification of A, B and H type 2 expression. A detailed binding analysis of a range of RHDV strains, to synthetic sugars and human red blood cells, as well as to rabbit duodenum, a likely gastrointestinal site for viral entrance was performed. Enzymatic cleavage of HBGA epitopes confirmed binding specificity. Binding was observed to blood group B, A and H type 2 epitopes in a strain-dependent manner with slight differences in specificity for A, B or H epitopes allowing RHDV strains to preferentially recognize different subgroups of animals. Strains related to the earliest described RHDV outbreak were not able to bind A, whereas all other genotypes have acquired A binding. In an experimental infection study, rabbits lacking the correct HBGA ligands were resistant to lethal RHDV infection at low challenge doses. Similarly, survivors of outbreaks in wild populations showed increased frequency of weak binding phenotypes, indicating selection for host resistance depending on the strain circulating in the population. HBGAs thus act as attachment factors facilitating infection, while their polymorphism of expression could contribute to generate genetic resistance to RHDV at the population level

Disorders of sex development : insights from targeted gene sequencing of a large international patient cohort

Background: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. Results: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46, XY DSD and 48 with 46, XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46, XY DSD. In patients with 46, XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. Conclusions: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes

Early Energy Deficit in Huntington Disease: Identification of a Plasma Biomarker Traceable during Disease Progression

Huntington disease (HD) is a fatal neurodegenerative disorder, with no effective treatment. The pathogenic mechanisms underlying HD have not been elucidated, but weight loss, associated with chorea and cognitive decline, is a characteristic feature of the disease that is accessible to investigation. We, therefore, performed a multiparametric study exploring body weight and the mechanisms of its loss in 32 presymptomatic carriers and HD patients in the early stages of the disease, compared to 21 controls. We combined this study with a multivariate statistical analysis of plasma components quantified by proton nuclear magnetic resonance (1H NMR) spectroscopy. We report evidence of an early hypermetabolic state in HD. Weight loss was observed in the HD group even in presymptomatic carriers, although their caloric intake was higher than that of controls. Inflammatory processes and primary hormonal dysfunction were excluded. 1H NMR spectroscopy on plasma did, however, distinguish HD patients at different stages of the disease and presymptomatic carriers from controls. This distinction was attributable to low levels of the branched chain amino acids (BCAA), valine, leucine and isoleucine. BCAA levels were correlated with weight loss and, importantly, with disease progression and abnormal triplet repeat expansion size in the HD1 gene. Levels of IGF1, which is regulated by BCAA, were also significantly lower in the HD group. Therefore, early weight loss in HD is associated with a systemic metabolic defect, and BCAA levels may be used as a biomarker, indicative of disease onset and early progression. The decreased plasma levels of BCAA may correspond to a critical need for Krebs cycle energy substrates in the brain that increased metabolism in the periphery is trying to provide

Long-term treatment of acromegaly with pegvisomant, a growth hormone receptor antagonist

"Background Pegvisomant is a new growth hormone receptor antagonist that improves symptoms and normalises insulin-like growth factor-1 (IGF-1) in a high proportion of patients with acromegaly treated for up to 12 weeks. We assessed the effects of pegvisomant in 160 patients with acromegaly treated for an average of 425 days. Methods Treatment efficacy was assessed by measuring changes in tumour volume by magnetic resonance imaging, and serum growth hormone and IGF-1 concentrations in 152 patients who received pegvisomant by daily subcutaneous injection for up to 18 months. The safety analysis included 160 patients some of whom received weekly injections and are excluded from the efficacy analysis. Findings Mean serum IGF-1 concentrations fell by at least 50%: 467 μg/L (SE 24), 526 μg/L (29), and 523 μg/L (40) in patients treated for 6, 12 and 18 months, respectively (p less than 0·001), whereas growth hormone increased by 12·5 μg/L (2·1), 12·5 μg/L (3·0), and 14·2 μg/L (5·7) (p less than 0·001). Of the patients treated for 12 months or more, 87 of 90 (97%) achieved a normal serum IGF-1 concentration. In patients withdrawn from pegvisomant (n=45), serum growth hormone concentrations were 8·0 μg/L (2·5) at baseline, rose to 15·2 μg/L (2·4) on drug, and fell back within 30 days of withdrawal to 8·3 μg/L (2·7). Antibodies to growth hormone were detected in 27 (16·9%) of patients, but no tachyphylaxis was seen. Serum insulin and glucose concentrations were significantly decreased (p less than 0·05). Two patients experienced progressive growth of their pituitary tumours, and two other patients had increased alanine and asparate aminotransferase concentrations requiring withdrawal from treatment. Mean pituitary tumour volume in 131 patients followed for a mean of 11·46 months (0·70) decreased by 0·033 cm3(0·057; p=0·353). Interpretation Pegvisomant is an effective medical treatment for acromegaly.

Evolutionary Trace Residues in Noroviruses: Importance in Receptor Binding, Antigenicity, Virion Assembly, and Strain Diversity

Noroviruses cause major epidemic gastroenteritis in humans. A large number of strains of these single-stranded RNA viruses have been reported. Due to the absence of infectious clones of noroviruses and the high sequence variability in their capsids, it has not been possible to identify functionally important residues in these capsids. Consequently, norovirus strain diversity is not understood on the basis of capsid functions, and the development of therapeutic compounds has been hampered. To determine functionally important residues in noroviruses, we have analyzed a number of norovirus capsid sequences in the context of the Norwalk virus capsid crystal structure by using the evolutionary trace method. This analysis has identified capsid protein residues that uniquely characterize different norovirus strains and provide new insights into capsid assembly and disassembly pathways and the strain diversity of these viruses. Such residues form specific three-dimensional clusters that may be of functional importance in noroviruses. One of these clusters includes residues known to participate in the proteolytic cleavage of these viruses at high pH. Other clusters are formed in capsid regions known to be important in the binding of antibodies to noroviruses, thereby indicating residues that may be important in the antigenicity of these viruses. The highly variable region of the capsid shows a distinct cluster whose residues may participate in norovirus-receptor interactions

Norwalk Virus-Like Particle Hemagglutination by Binding to H Histo-Blood Group Antigens

Noroviruses are a major cause of epidemic acute nonbacterial gastroenteritis worldwide. Here we report our discovery that recombinant Norwalk virus virus-like particles (rNV VLPs) agglutinate red blood cells (RBCs). Since histo-blood group antigens are expressed on gut mucosa as well as RBCs, we used rNV VLP hemagglutination (HA) as a model system for studying NV attachment to cells in order to help identify a potential NV receptor(s). rNV VLP HA is dependent on low temperature (4°C) and acidic pH. Of the 13 species of RBCs tested, rNV VLPs hemagglutinated only chimpanzee and human RBCs. The rNV VLPs hemagglutinated all human type O (11 of 11), A (9 of 9), and AB (4 of 4) RBCs; however, few human type B RBC samples (4 of 14) were hemagglutinated. HA with periodate- and neuraminidase-treated RBCs indicated that rNV VLP binding was carbohydrate dependent and did not require sialic acid. The rNV VLPs did not hemagglutinate Bombay RBCs (zero of seven) that lack H type 2 antigen, and an anti-H type 2 antibody inhibited rNV VLP HA of human type O RBCs. These data indicated that the H type 2 antigen functions as the rNV VLP HA receptor on human type O RBCs. The rNV VLP HA was also inhibited by rNV VLP-specific monoclonal antibody 8812, an antibody that inhibits VLP binding to Caco-2 cells. Convalescent-phase sera from NV-infected individuals showed increased rNV VLP HA inhibition titers compared to prechallenge sera. In carbohydrate binding assays, the rNV VLPs bound to synthetic Lewis d (Le(d)), Le(b), H type 2, and Le(y) antigens, and these antigens also inhibited rNV VLP HA of human type O RBCs. Overall, our results indicate that carbohydrate antigens in the gut are a previously unrecognized factor in NV pathogenesis

Characterization of an epoxy filler for piezocomposites compatible with microfabrication processes

Miniature ultrasound transducer arrays that can operate at frequencies above 30 MHz are needed for high-resolution medical imaging. One way to achieve this is with a kerfless structure based on 1-3 connectivity piezocomposite with the electrodes defined by photolithography. To achieve this, not only does the composite need planar, parallel, and smooth surfaces, but it must also be made with an epoxy filler compatible with the chemicals, heat, and vacuum required for photolithography. This paper reports full characterization of an epoxy suitable for fine-scale kerfless array fabrication, including photolithographic processing. Material properties have been investigated as a function of cure temperature and for compatibility with solvents. By increasing the cure temperature, the crosslinking between the epoxy and the hardener in- creases, resulting in a higher glass transition temperature. The cured epoxy consequently has better resistance to both heat and solvents. An elevated cure temperature, near 100°C, is required to optimize material properties for photolithography on 1-3 piezocomposites. The acoustic properties of the epoxy have also been studied. These are similar to other epoxies used in piezocomposite fabrication and no significant changes have been observed for the different cure temperatures

The 3′ End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3′ untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3′ terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3′ elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3′UTR (ORF2+3+3′UTR). In contrast, expression of VP1 from constructs that lacked the 3′UTR (ORF2+3), ORF3 (ORF2+3′UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3′UTR revealed that the 3′UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells

Modular Laboratory Exercises to Analyze the Development of Zebrafish Motor Behavior

The embryonic zebrafish is an excellent research model to examine the neural networks that coordinate locomotive behavior. It demonstrates robust locomotive behavior early in development, its nervous system is relatively simple and accessible compared to mammalian systems, and there are mutants available with specific molecular and motor deficits. We have developed a series of four exercises that provide students with a basic understanding of locomotive behavior development, nervous system organization, development of neurotransmitter responsiveness, and genetics. The first two exercises can be performed in one 3-h laboratory period, and the third and fourth exercises, which build on the first two, can be completed in one or two subsequent periods. In the first exercise, students observe and quantify two distinct behaviors that characterize different developmental stages, spontaneous movement, and touch-evoked tail coiling. In the second, the students use a pharmacological approach to determine if the neurotransmitter glycine is required for the embryo to perform each behavior. In the third, they use simple lesions to assess whether the brain is required for each type of behavior. In the fourth, the students examine bandoneon, a zebrafish motility mutant that has a glycine receptor defect, by observing its behavior during spontaneous movement and touch-evoked tail coiling, performing lesions, and applying pharmacological drugs. These exercises are readily adaptable, such that portions can be omitted or expanded to examine other neurotransmitter systems or later stages of locomotive behavior development