Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe,Protective Vaccine Antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.     

KSHV MicroRNAs Mediate Cellular Transformation and Tumorigenesis by Redundantly Targeting Cell Growth and Survival Pathways

Kaposi''s sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi''s sarcoma, primary effusion lymphoma and multicentric Castleman''s disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.     

Identification of methylated flavonoid regioisomeric metabolites using enzymatic semisynthesis and liquid chromatography-tandem mass spectrometry

Discrimination of isomeric methylated metabolites is an important step toward identifying genes responsible for methylation, but presents substantial challenges because authentic standards are often unavailable and mass spectra of isomers have been considered indistinguishable. In this report, an approach is described for identifying methyl group positions in multiply methylated flavonoid metabolites using combinations of tandem mass spectrometry, liquid chromatography retention, and site-selective methylation by recombinant O-methyltransferases from Solanum habrochaites LA1777. The basis for observed fragment ions in tandem mass spectra of multiply methylated myricetin was further established using enzymatic incorporation of deuterium-labeled methyl groups using S-adenosylmethionine-d ₃ as precursor.     

Soluble Guanylate Cyclase α1–Deficient Mice: A Novel Murine Model for Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α₁ subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α₁–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α₁ and β₁ subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.     

Enduring Effects of Early Life Stress on Firing Patterns of Hippocampal and Thalamocortical Neurons in Rats: Implications for Limbic Epilepsy

Early life stress results in an enduring vulnerability to kindling-induced epileptogenesis in rats, but the underlying mechanisms are not well understood. Recent studies indicate the involvement of thalamocortical neuronal circuits in the progression of kindling epileptogenesis. Therefore, we sought to determine in vivo the effects of early life stress and amygdala kindling on the firing pattern of hippocampus as well as thalamic and cortical neurons. Eight week old male Wistar rats, previously exposed to maternal separation (MS) early life stress or early handling (EH), underwent amygdala kindling (or sham kindling). Once fully kindled, in vivo juxtacellular recordings in hippocampal, thalamic and cortical regions were performed under neuroleptic analgesia. In the thalamic reticular nucleus cells both kindling and MS independently lowered firing frequency and enhanced burst firing. Further, burst firing in the thalamic reticular nucleus was significantly increased in kindled MS rats compared to kindled EH rats (p<0.05). In addition, MS enhanced burst firing of hippocampal pyramidal neurons. Following a stimulation-induced seizure, somatosensory cortical neurons exhibited a more pronounced increase in burst firing in MS rats than in EH rats. These data demonstrate changes in firing patterns in thalamocortical and hippocampal regions resulting from both MS and amygdala kindling, which may reflect cellular changes underlying the enhanced vulnerability to kindling in rats that have been exposed to early life stress.     

Volunteer Bias in Recruitment,Retention, and Blood Sample Donation in a Randomised Controlled Trial Involving Mothers and Their Children at Six Months and Two Years: A Longitudinal Analysis

Background

The vulnerability of clinical trials to volunteer bias is under-reported. Volunteer bias is systematic error due to differences between those who choose to participate in studies and those who do not.

Methods and Results

This paper extends the applications of the concept of volunteer bias by using data from a trial of probiotic supplementation for childhood atopy in healthy dyads to explore 1) differences between a) trial participants and aggregated data from publicly available databases b) participants and non-participants as the trial progressed 2) impact on trial findings of weighting data according to deprivation (Townsend) fifths in the sample and target populations. 1) a) Recruits (n = 454) were less deprived than the target population, matched for area of residence and delivery dates (n = 6,893) (mean [SD] deprivation scores 0.09[4.21] and 0.79[4.08], t = 3.44, df = 511, p<0.001). b) i)As the trial progressed, representation of the most deprived decreased. These participants and smokers were less likely to be retained at 6 months (n = 430[95%]) (OR 0.29,0.13–0.67 and 0.20,0.09–0.46), and 2 years (n = 380[84%]) (aOR 0.68,0.50–0.93 and 0.55,0.28–1.09), and consent to infant blood sample donation (n = 220[48%]) (aOR 0.72,0.57–0.92 and 0.43,0.22–0.83). ii)Mothers interested in probiotics or research or reporting infants’ adverse events or rashes were more likely to attend research clinics and consent to skin-prick testing. Mothers participating to help children were more likely to consent to infant blood sample donation. 2) In one trial outcome, atopic eczema, the intervention had a positive effect only in the over-represented, least deprived group. Here, data weighting attenuated risk reduction from 6.9%(0.9–13.1%) to 4.6%(−1.4–+10.5%), and OR from 0.40(0.18–0.91) to 0.56(0.26–1.21). Other findings were unchanged.

Conclusions

Potential for volunteer bias intensified during the trial, due to non-participation of the most deprived and smokers. However, these were not the only predictors of non-participation. Data weighting quantified volunteer bias and modified one important trial outcome.

Trial Registration

This randomised, double blind, parallel group, placebo controlled trial is registered with the International Standard Randomised Controlled Trials Register, Number (ISRCTN) 26287422. Registered title: Probiotics in the prevention of atopy in infants and children.     

Adaptive Evolution of the Myo6 Gene in Old World Fruit Bats (Family: Pteropodidae)

Myosin VI (encoded by the Myo6 gene) is highly expressed in the inner and outer hair cells of the ear, retina, and polarized epithelial cells such as kidney proximal tubule cells and intestinal enterocytes. The Myo6 gene is thought to be involved in a wide range of physiological functions such as hearing, vision, and clathrin-mediated endocytosis. Bats (Chiroptera) represent one of the most fascinating mammal groups for molecular evolutionary studies of the Myo6 gene. A diversity of specialized adaptations occur among different bat lineages, such as echolocation and associated high-frequency hearing in laryngeal echolocating bats, large eyes and a strong dependence on vision in Old World fruit bats (Pteropodidae), and specialized high-carbohydrate but low-nitrogen diets in both Old World and New World fruit bats (Phyllostomidae). To investigate what role(s) the Myo6 gene might fulfill in bats, we sequenced the coding region of the Myo6 gene in 15 bat species and used molecular evolutionary analyses to detect evidence of positive selection in different bat lineages. We also conducted real-time PCR assays to explore the expression levels of Myo6 in a range of tissues from three representative bat species. Molecular evolutionary analyses revealed that the Myo6 gene, which was widely considered as a hearing gene, has undergone adaptive evolution in the Old World fruit bats which lack laryngeal echolocation and associated high-frequency hearing. Real-time PCR showed the highest expression level of the Myo6 gene in the kidney among ten tissues examined in three bat species, indicating an important role for this gene in kidney function. We suggest that Myo6 has undergone adaptive evolution in Old World fruit bats in relation to receptor-mediated endocytosis for the preservation of protein and essential nutrients.     

Mechanism of Silver Nanoparticles Action on Insect Pigmentation Reveals Intervention of Copper Homeostasis

Silver nanoparticles (AgNPs), like almost all nanoparticles, are potentially toxic beyond a certain concentration because the survival of the organism is compromised due to scores of pathophysiological abnormalities past that concentration. However, the mechanism of AgNP toxicity remains undetermined. Instead of applying a toxic dose, we attempted to monitor the effects of AgNPs at a nonlethal concentration on wild type Drosophila melanogaster by exposing them throughout their development. All adult flies raised in AgNP doped food showed that up to 50 mg/L concentration AgNP has no negative influence on median survival; however, these flies appeared uniformly lighter in body color due to the loss of melanin pigments in their cuticle. Additionally, fertility and vertical movement ability were compromised due to AgNP feeding. Determination of the amount of free ionic silver (Ag⁺) led us to claim that the observed biological effects have resulted from the AgNPs and not from Ag⁺. Biochemical analysis suggests that the activity of copper dependent enzymes, namely tyrosinase and Cu-Zn superoxide dismutase, are decreased significantly following the consumption of AgNPs, despite the constant level of copper present in the tissue. Consequently, we propose a mechanism whereby consumption of excess AgNPs in association with membrane bound copper transporter proteins cause sequestration of copper, thus creating a condition that resembles copper starvation. This model also explains the cuticular demelanization effect resulting from AgNP since tyrosinase activity is essential for melanin biosynthesis. Finally, we claim that Drosophila, an established genetic model system, can be well utilized for further understanding of the biological effects of nanoparticles.