Development and mapping of peach candidate genes involved in fruit quality and their transferability and potential use in other Rosaceae species

The goal of the present study was to identify candidate genes (CGs) involved in fruit quality in peach that can be transferred to other Rosaceae species. Two cDNA libraries from fruit of the “Fantasia” peach cultivar, constructed at two stages of development, were used to generate a set of expressed sequence tag sequences. A total of 1,730 peach unigenes were obtained after clustering. Sequences and corresponding annotations were stored in a relational database and are available through a web interface. Fifty-nine CGs involved in fruit growth and development or fruit quality at maturity, focusing on sweetness, acidity, and phenolic compound content, were selected according to their annotation. Fifty-five primer pairs, designed from peach CG sequences and giving PCR products in peach, were tested in strawberry and 36 gave amplified products. Eight CGs were mapped in peach, 14 in strawberry, four in both species and confirmed the pattern of synteny already proposed using comparative mapping. In peach, the CGs are located in three linkage groups (3, 5, 7), and in strawberry they are distributed in all seven Fragaria linkage groups. Colocalization between some of these CGs and quantitative trait loci for fruit quality traits were identified and are awaiting confirmation in further analyses.     

Loss of α1β1 Soluble Guanylate Cyclase,the Major Nitric Oxide Receptor,Leads to Moyamoya and Achalasia

Moyamoya is a cerebrovascular condition characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and the compensatory development of abnormal “moyamoya” vessels. The pathophysiological mechanisms of this condition, which leads to ischemic and hemorrhagic stroke, remain unknown. It can occur as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes). Here, we describe an autosomal-recessive disease leading to severe moyamoya and early-onset achalasia in three unrelated families. This syndrome is associated in all three families with homozygous mutations in GUCY1A3, which encodes the α1 subunit of soluble guanylate cyclase (sGC), the major receptor for nitric oxide (NO). Platelet analysis showed a complete loss of the soluble α1β1 guanylate cyclase and showed an unexpected stimulatory role of sGC within platelets. The NO-sGC-cGMP pathway is a major pathway controlling vascular smooth-muscle relaxation, vascular tone, and vascular remodeling. Our data suggest that alterations of this pathway might lead to an abnormal vascular-remodeling process in sensitive vascular areas such as ICA bifurcations. These data provide treatment options for affected individuals and strongly suggest that investigation of GUCY1A3 and other members of the NO-sGC-cGMP pathway is warranted in both isolated early-onset achalasia and nonsyndromic moyamoya.     

Randomized Controlled Ferret Study to Assess the Direct Impact of 2008–09 Trivalent Inactivated Influenza Vaccine on A(H1N1)pdm09 Disease Risk

During spring-summer 2009, several observational studies from Canada showed increased risk of medically-attended, laboratory-confirmed A(H1N1)pdm09 illness among prior recipients of 2008–09 trivalent inactivated influenza vaccine (TIV). Explanatory hypotheses included direct and indirect vaccine effects. In a randomized placebo-controlled ferret study, we tested whether prior receipt of 2008–09 TIV may have directly influenced A(H1N1)pdm09 illness. Thirty-two ferrets (16/group) received 0.5 mL intra-muscular injections of the Canadian-manufactured, commercially-available, non-adjuvanted, split 2008–09 Fluviral or PBS placebo on days 0 and 28. On day 49 all animals were challenged (Ch0) with A(H1N1)pdm09. Four ferrets per group were randomly selected for sacrifice at day 5 post-challenge (Ch+5) and the rest followed until Ch+14. Sera were tested for antibody to vaccine antigens and A(H1N1)pdm09 by hemagglutination inhibition (HI), microneutralization (MN), nucleoprotein-based ELISA and HA1-based microarray assays. Clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. Baseline characteristics were similar between the two groups of influenza-naïve animals. Antibody rise to vaccine antigens was evident by ELISA and HA1-based microarray but not by HI or MN assays; virus challenge raised antibody to A(H1N1)pdm09 by all assays in both groups. Beginning at Ch+2, vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at Ch+5 (7.4% vs. 5.2%; p = 0.01). At Ch+5 vaccinated animals had higher lung virus titers (log-mean 4.96 vs. 4.23pfu/mL, respectively; p = 0.01), lung inflammatory scores (5.8 vs. 2.1, respectively; p = 0.051) and cytokine levels (p>0.05). At Ch+14, both groups had recovered. Findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. While they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior 2008–09 TIV receipt on A(H1N1)pdm09 illness. As such, they warrant further in-depth investigation and search for possible mechanistic explanations.     

CRISPR Is an Optimal Target for the Design of Specific PCR Assays for Salmonella enterica Serotypes Typhi and Paratyphi A

Background

Serotype-specific PCR assays targeting Salmonella enterica serotypes Typhi and Paratyphi A, the causal agents of typhoid and paratyphoid fevers, are required to accelerate formal diagnosis and to overcome the lack of typing sera and, in some situations, the need for culture. However, the sensitivity and specificity of such assays must be demonstrated on large collections of strains representative of the targeted serotypes and all other bacterial populations producing similar clinical symptoms.

Methodology

Using a new family of repeated DNA sequences, CRISPR (clustered regularly interspaced short palindromic repeats), as a serotype-specific target, we developed a conventional multiplex PCR assay for the detection and differentiation of serotypes Typhi and Paratyphi A from cultured isolates. We also developed EvaGreen-based real-time singleplex PCR assays with the same two sets of primers.

Principal findings

We achieved 100% sensitivity and specificity for each protocol after validation of the assays on 188 serotype Typhi and 74 serotype Paratyphi A strains from diverse genetic groups, geographic origins and time periods and on 70 strains of bacteria frequently encountered in bloodstream infections, including 29 other Salmonella serotypes and 42 strains from 38 other bacterial species.

Conclusions

The performance and convenience of our serotype-specific PCR assays should facilitate the rapid and accurate identification of these two major serotypes in a large range of clinical and public health laboratories with access to PCR technology. These assays were developed for use with DNA from cultured isolates, but with modifications to the assay, the CRISPR targets could be used in the development of assays for use with clinical and other samples.     

Characterization of the interaction between protein Snu13p/15.5K and the Rsa1p/NUFIP factor and demonstration of its functional importance for snoRNP assembly

The yeast Snu13p protein and its 15.5K human homolog both bind U4 snRNA and box C/D snoRNAs. They also bind the Rsa1p/NUFIP assembly factor, proposed to scaffold immature snoRNPs and to recruit the Hsp90-R2TP chaperone complex. However, the nature of the Snu13p/15.5K–Rsa1p/NUFIP interaction and its exact role in snoRNP assembly remained to be elucidated. By using biophysical, molecular and imaging approaches, here, we identify residues needed for Snu13p/15.5K–Rsa1p/NUFIP interaction. By NMR structure determination and docking approaches, we built a 3D model of the Snup13p–Rsa1p interface, suggesting that residues R₂₄₉, R₂₄₆ and K₂₅₀ in Rsa1p and E₇₂ and D₇₃ in Snu13p form a network of electrostatic interactions shielded from the solvent by hydrophobic residues from both proteins and that residue W₂₅₃ of Rsa1p is inserted in a hydrophobic cavity of Snu13p. Individual mutations of residues in yeast demonstrate the functional importance of the predicted interactions for both cell growth and snoRNP formation. Using archaeal box C/D sRNP 3D structures as templates, the association of Snu13p with Rsa1p is predicted to be exclusive of interactions in active snoRNPs. Rsa1p and NUFIP may thus prevent premature activity of pre-snoRNPs, and their removal may be a key step for active snoRNP production.     

Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1

One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.     

Selecting the quality of mule duck fatty liver based on near-infrared spectroscopy

Background

“Foie gras” is produced predominantly in France and about 90% of the commercialized product is obtained from male mule ducks. The melting rate (percentage of fat released during cooking) is the main criterion used to determine the quality of “foie gras”. However, up to now the melting rate could not be predicted without causing liver damage, which means that selection programs could not use this criterion.

Methods

Fatty liver phenotypes were obtained for a population of over 1400 overfed male mule ducks. The phenotypes were based on two types of near-infrared spectra (on the liver surface and on ground liver) in order to predict the melting rate and liver composition (ash, dry matter, lipid and protein contents). Genetic parameters were computed in multiple traits with a “sire-dam” model and using a Gibbs sampling approach.

Results

The estimates for the genetic parameters show that the measured melting rate and the predicted melting rate obtained with two near-infrared spectrometer devices are genetically the same trait: genetic correlations are very high (ranging from +0.89 to +0.97 depending on the mule duck parental line and the spectrometer) and heritabilities are comparable. The predictions based on the spectra of ground liver samples using a laboratory spectrometer correlate with those based on the surface spectra using a portable spectrometer (from +0.83 to +0.95 for dry matter, lipid and protein content) and are particularly high for the melting rate (higher than +0.95). Although less accurate than the predictions obtained using the spectra of ground liver samples, the phenotypic prediction of the melting rate based on surface spectra is sufficiently accurate to be used by “foie gras” processors.

Conclusions

Near-infrared spectrometry is an efficient tool to select liver quality in breeding programs because animals can be ranked according to their liver melting rate without damaging their livers. Thus, these original results will help breeders to select ducks based on the liver melting rate, a crucial criterion that defines the quality of the liver and for which there was previously no accurate predictor.     

Sedation Agents Differentially Modulate Cortical and Subcortical Blood Oxygenation: Evidence from Ultra-High Field MRI at 17.2 T

Background

Sedation agents affect brain hemodynamic and metabolism leading to specific modifications of the cerebral blood oxygenation level. We previously demonstrated that ultra-high field (UHF) MRI detects changes in cortical blood oxygenation following the administration of sedation drugs commonly used in animal research. Here we applied the UHF-MRI method to study clinically relevant sedation drugs for their effects on cortical and subcortical (thalamus, striatum) oxygenation levels.

Methods

We acquired T2*-weighted images of Sprague-Dawley rat brains at 17.2T in vivo. During each MRI session, rats were first anesthetized with isoflurane, then with a second sedative agent (sevoflurane, propofol, midazolam, medetomidine or ketamine-xylazine) after stopping isoflurane. We computed a T2*-oxygenation-ratio that aimed at estimating cerebral blood oxygenation level for each sedative agent in each region of interest: cortex, hippocampus, thalamus and striatum.

Results

The T2*-oxygenation-ratio was consistent across scan sessions. This ratio was higher with inhalational agents than with intravenous agents. Under sevoflurane and medetomidine, T2*-oxygenation-ratio was homogenous across the brain regions. Intravenous agents (except medetomidine) induced a T2*-oxygenation-ratio imbalance between cortex and subcortical regions: T2*-oxygenation-ratio was higher in the cortex than the subcortical areas under ketamine-xylazine; T2*-oxygenation-ratio was higher in subcortical regions than in the cortex under propofol or midazolam.

Conclusion

Preclinical UHF MRI is a powerful method to monitor the changes in cerebral blood oxygenation level induced by sedative agents across brain structures. This approach also allows for a classification of sedative agents based on their differential effects on cerebral blood oxygenation level.     

Identification and Characterization of Novel Classes of Macrophage Migration Inhibitory Factor (MIF) Inhibitors with Distinct Mechanisms of Action

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC₅₀ values in the range of 0.2–15.5 μm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro¹; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.