Molecular basis of a new ovine model for human 3M syndrome-2.

BACKGROUND: Brachygnathia, cardiomegaly and renal hypoplasia syndrome (BCRHS, OMIA 001595-9940 ) is a previously reported recessively inherited disorder in Australian Poll Merino/Merino sheep. Affected lambs are stillborn with various congenital defects as reflected in the name of the disease, as well as short stature, a short and broad cranium, a small thoracic cavity, thin ribs and brachysternum. The BCRHS phenotype shows similarity to certain human short stature syndromes, in particular the human 3M syndrome-2. Here we report the identification of a likely disease-causing variant and propose an ovine model for human 3M syndrome-2. RESULTS: Eight positional candidate genes were identified among the 39 genes in the approximately 1 Mb interval to which the disease was mapped previously. Obscurin like cytoskeletal adaptor 1 (OBSL1) was selected as a strong positional candidate gene based on gene function and the resulting phenotypes observed in humans with mutations in this gene. Whole genome sequencing of an affected lamb (BCRHS3) identified a likely causal variant ENSOARG00000020239:g.220472248delC within OBSL1. Sanger sequencing of seven affected, six obligate carrier, two phenotypically unaffected animals from the original flock and one unrelated control animal validated the variant. A genotyping assay was developed to genotype 583 animals from the original flock, giving an estimated allele frequency of 5%. CONCLUSIONS: The identification of a likely disease-causing variant resulting in a frameshift (p.(Val573Trpfs*119)) in the OBSL1 protein has enabled improved breeding management of the implicated flock. The opportunity for an ovine model for human 3M syndrome and ensuing therapeutic research is promising given the availability of carrier ram semen for BCRHS.

Mapping of host-parasite-microbiome interactions reveals metabolic determinants of tropism and tolerance in Chagas disease.

Chagas disease (CD) is a parasitic disease caused by Trypanosoma cruzi protozoa, presenting with cardiomyopathy, megaesophagus, and/or megacolon. To determine the mechanisms of gastrointestinal (GI) CD tissue tropism, we systematically characterized the spatial localization of infection-induced metabolic and microbiome alterations, in a mouse model of CD. Notably, the impact of the transition between acute and persistent infection differed between tissue sites, with sustained large-scale effects of infection in the esophagus and large intestine, providing a potential mechanism for the tropism of CD within the GI tract. Infection affected acylcarnitine metabolism; carnitine supplementation prevented acute-stage CD mortality without affecting parasite burden by mitigating infection-induced metabolic disturbances and reducing cardiac strain. Overall, results identified a previously-unknown mechanism of disease tolerance in CD, with potential for new therapeutic regimen development. More broadly, results highlight the potential of spatially resolved metabolomics to provide insight into disease pathogenesis and infectious disease drug development.

Size effects of aromatic substitution in the ortho position on the photodimerization kinetics of alpha-trans cinnamic acid derivatives. A solid-state NMR study.

The photoreaction of two alpha-cinnamic acid derivatives, alpha-o-methoxy and alpha-o-ethoxy cinnamic acid, was studied by (13)C CPMAS solid-state NMR spectroscopy in order to elucidate effects of aromatic substitution and substituent size on the kinetics of the [2+2] photodimerization. The reactants and products can be clearly differentiated and a detailed spectroscopic characterization was carried out, including 2D PASS spectra, at a low spinning frequency to determine the principal values of the chemical shift tensor. Density functional theory (DFT) calculations of chemical shifts and chemical shift anisotropy tensors were found to be in good agreement with the experimental results and helped in the individual assignments of reactant and photoproduct carbon atoms. The photoreaction kinetics show no systematic variation with substituent size, in that the alpha-o-methoxy cinnamic acid progresses at a slower rate than unsubstituted alpha-cinnamic acid, but alpha-o-ethoxy cinnamic acid at a faster one. Interestingly, the distance between reacting double bonds is not a good indicator of photoreaction rate. The observed trend is in part due to a larger degree of reorientation of the aromatic ring for the o-methoxy cinnamic acid, and a more dominant interaction appears to be the p-orbital overlap between two reacting double bonds in determining the reaction kinetics.

Temperature stability and photodimerization kinetics of beta-cinnamic acid and comparison to its alpha-polymorph as studied by solid-state NMR spectroscopy techniques and DFT calculations.

Photoreactions of the alpha- and beta-polymorphs of trans-cinnamic acid were studied by (13)C CPMAS solid-state nuclear magnetic resonance spectroscopy, and the reactants and products were spectroscopically characterized in detail. Chemical shifts and chemical shift anisotropy tensors calculated using density functional theory (DFT) were found to be in good agreement with the experimental results and helped to identify the polymorphs and the individual assignments of reactant and photoproduct carbon atoms. The beta-polymorph is metastable. Its transformation into the alpha-cinnamic acid polymorph is monitored by temperature-dependent (13)C NMR spectroscopy. The transformation occurs at a very slow rate at room temperature but is highly accelerated at elevated temperatures. Analysis of the kinetics of the photoreaction shows that the beta-polymorph progresses at a slower rate compared to that of alpha-cinnamic acid. Based on chemical shift tensor values of reactants and products as obtained from 2D PASS spectra, the difference in reaction rates is suggested to be due to the higher amount of molecular reorientation of functional groups upon photoreaction and the larger distance between the reacting double bonds.

Characterization of a mutant pancreatic eIF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells.

Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

Electrofractionation: a technique for detecting and recovering biomolecules.

Electrofractionation (EF) is a technique that allows electrophoretic materials to be detected and recovered following electrophoresis. The EF apparatus utilizes the resolving power of electrophoresis and the mobile phase of liquid chromatography to create a continuous elution system. Our data indicate that EF is able to detect and recover oligonucleotides, as DNA fragments, proteins, and presumably other material that can be analyzed by electrophoresis. EF shares functional similarities with high-performance electrophoresis chromatography (HPEC) but operates by a different strategy and at a fraction of the cost. Moreover, EF can be constructed largely from standard laboratory equipment. The simplicity, rapid analysis times, low cost, and high recovery yields of EF make this system a practical alternative to the conventional detection and purification methods used for biomolecules.