Chromosomal instability by mutations in the novel minor spliceosome component CENATAC

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12‐dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT‐AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT‐AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.     

Sensitivity of PCR assays for murine gammaretroviruses and mouse contamination in human blood samples

Gammaretroviruses related to murine leukemia virus (MLV) have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs) or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.     

2-O-sulfotransferase regulates Wnt signaling, cell adhesion and cell cycle during zebrafish epiboly

O-sulfotransferases modify heparan sulfate proteoglycans (HSPGs) by catalyzing the transfer of a sulfate to a specific position on heparan sulfate glycosaminoglycan (GAG) chains. Although the roles of specific HSPG modifications have been described in cell culture and invertebrates, little is known about their functions or abilities to modulate specific cell signaling pathways in vertebrate development. Here, we report that 2-O-sulfotransferase (2-OST) is an essential component of canonical Wnt signaling in zebrafish development. 2-OST-deficient embryos have reduced GAG chain sulfation and are refractory to exogenous Wnt8 overexpression. Embryos in which maternally encoded 2-OST is knocked down have normal activation of several zygotic mesoderm, endoderm and ectoderm patterning genes, but have decreased deep cell adhesion and fail to initiate epiboly, which can be rescued by re-expression of 2-OST protein. Reduced cell adhesion and altered cell cycle regulation in 2-OST-deficient embryos are associated with decreased β-catenin and E-cadherin protein levels at cell junctions, and these defects can be rescued by reactivation of the intracellular Wnt pathway, utilizing stabilized β-catenin or dominant-negative Gsk3, but not by overexpression of Wnt8 ligand. Together, these results indicate that 2-OST functions within the Wnt pathway, downstream of Wnt ligand signaling and upstream of Gsk3β and β-catenin intracellular localization and function.     

Low brain penetrant CB1 receptor agonists for the treatment of neuropathic pain

Novel, low brain penetrant, orally bioavailable CB1 receptor agonists were designed starting from a mature lead series of potent brain penetrant CB1 receptor agonists. Increasing the calculated polar surface area was found to be a good strategy for reducing brain penetration whilst retaining drug-like properties. This in silico approach led to the discovery of LBP1, an orally bioavailable, low brain penetrant CB1 receptor agonist with robust activity in rodent models of neuropathic pain and a good preclinical therapeutic profile, which was selected for clinical development.     

Thienopyridine ureas as dual inhibitors of the VEGF and Aurora kinase families

In an effort to identify multi-targeted kinase inhibitors with a novel spectrum of kinase activity, a screen of Abbott proprietary KDR inhibitors against a broad panel of kinases was conducted and revealed a series of thienopyridine ureas with promising activity against the Aurora kinases. Modification of the diphenyl urea and C7 moiety of these compounds provided potent inhibitors with good pharmacokinetic profiles that were efficacious in mouse tumor models after oral dosing. Compound 2 (ABT-348) of this series is currently undergoing Phase I clinical trials in solid and hematological cancer populations.     

Supportive breeding boosts natural population abundance with minimal negative impacts on fitness of a wild population of Chinook salmon

While supportive breeding programmes strive to minimize negative genetic impacts to populations, case studies have found evidence for reduced fitness of artificially produced individuals when they reproduce in the wild. Pedigrees of two complete generations were tracked with molecular markers to investigate differences in reproductive success (RS) of wild and hatchery‐reared Chinook salmon spawning in the natural environment to address questions regarding the demographic and genetic impacts of supplementation to a natural population. Results show a demographic boost to the population from supplementation. On average, fish taken into the hatchery produced 4.7 times more adult offspring, and 1.3 times more adult grand‐offspring than naturally reproducing fish. Of the wild and hatchery fish that successfully reproduced, we found no significant differences in RS between any comparisons, but hatchery‐reared males typically had lower RS values than wild males. Mean relative reproductive success (RRS) for hatchery F₁ females and males was 1.11 (P = 0.84) and 0.89 (P = 0.56), respectively. RRS of hatchery‐reared fish (H) that mated in the wild with either hatchery or wild‐origin (W) fish was generally equivalent to W × W matings. Mean RRS of H × W and H × H matings was 1.07 (P = 0.92) and 0.94 (P = 0.95), respectively. We conclude that fish chosen for hatchery rearing did not have a detectable negative impact on the fitness of wild fish by mating with them for a single generation. Results suggest that supplementation following similar management practices (e.g. 100% local, wild‐origin brood stock) can successfully boost population size with minimal impacts on the fitness of salmon in the wild.     

Environmental Contamination as a Risk Factor for Intra-Household Staphylococcus aureus Transmission

Background

The household is a recognized community reservoir for Staphylococcus aureus. This study investigated potential risk factors for intra-household S. aureus transmission, including the contribution of environmental contamination.

Methods

We investigated intra-household S. aureus transmission using a sample of multiple member households from a community-based case-control study examining risk factors for CA-MRSA infection conducted in Northern Manhattan. During a home visit, index subjects completed a questionnaire. All consenting household members were swabbed, as were standardized environmental household items. Swabs were cultured for S. aureus. Positive isolates underwent further molecular characterization. Intra-household transmission was defined as having identical strains among two or more household members. Multiple logistic regression was used to identify independent risk factors for transmission.

Results

We enrolled 291 households: 146 index cases, 145 index controls and 687 of their household contacts. The majority of indexes were Hispanic (85%), low income (74%), and female (67%), with a mean age of 31 (range 1–79). The average size of case and control households was 4 people. S. aureus colonized individuals in 62% of households and contaminated the environment in 54% of households. USA300 was the predominant clinical infection, colonizing and environmental strain. Eighty-one households had evidence of intra-household transmission: 55 (38%) case and 26 (18%) control households (P<.01). Environmental contamination with a colonizing or clinical infection strain (aOR: 5.4 [2.9–10.3] P<.01) and the presence of a child under 5 (aOR: 2.3 [1.2–4.5] P = .02) were independently associated with transmission. In separate multivariable models, environmental contamination was associated with transmission among case (aOR 3.3, p<.01) and control households (aOR 27.2, p<.01).

Conclusions

Environmental contamination with a colonizing or clinical infection strain was significantly and independently associated with transmission in a large community-based sample. Environmental contamination should be considered when treating S. aureus infections, particularly among households with multiple infected members.     

Subdomain location of mutations in cardiac actin correlate with type of functional change

Determining the molecular mechanisms that lead to the development of heart failure will help us gain better insight into the most costly health problem in the Western world. To understand the roles that the actin protein plays in the development of heart failure, we have taken a systematic approach toward characterizing human cardiac actin mutants that have been associated with either hypertrophic or dilated cardiomyopathy. Seven known cardiac actin mutants were expressed in a baculovirus system, and their intrinsic properties were studied. In general, the changes to the properties of the actin proteins themselves were subtle. The R312H variant exhibited reduced stability, with a T(m) of 53.6 °C compared to 56.8 °C for WT actin, accompanied with increased polymerization critical concentration and Pi release rate, and a marked increase in nucleotide release rates. Substitution of methionine for leucine at amino acid 305 showed no impact on the stability, nucleotide release rates, or DNase-I inhibition ability of the actin monomer; however, during polymerization, a 2-fold increase in Pi release was observed. Increases to both the T(m) and DNase-I inhibition activity suggested interactions between E99K actin molecules under monomer-promoting conditions. Y166C actin had a higher critical concentration resulting in a lower Pi release rate due to reduced filament-forming potential. The locations of mutations on the ACTC protein correlated with the molecular effects; in general, mutations in subdomain 3 affected the stability of the ACTC protein or affect the polymerization of actin filaments, while mutations in subdomains 1 and 4 more likely affect protein-protein interactions.     

How to blast osteoblasts? Novel dicarba analogues of amylin-(1–8) to treat osteoporosis

When administered in vivo, amylin (1–8) stimulates osteoblast proliferation increasing bone volume and bone strength. The native cyclic octapeptide amylin (1–8) is unstable, however, it provides an attractive framework for the creation of more stable, orally active synthetic analogues using various peptidomimetic techniques. On-resin ring closing metathesis (RCM) on the olefinic side chains of allylglycine residues and lysine moieties functionalized with an allyloxycarbonyl (Alloc) group, was used to prepare novel carba-bridged surrogates of the disulfide bridge between Cys/2 and Cys/7 in amylin-(1–8). Commercially available N^α-Fmoc N^ε-Alloc protected lysine was used as a convenient substrate for Grubbs’ ring closing metathesis. Analogues of amylin-(1–8) prepared by cyclization of allylglycine residues that also contained proline residues at either position 4 or 6, or both, were also prepared to investigate the effect of proline as a ‘kink-inducing’ residue on the efficiency of the RCM reaction. Of the nine novel alkene-bridged analogues prepared, five showed promising biological activity in a proliferation study in primary foetal rat osteoblasts at physiological concentrations. Two of these analogues were chosen for further in vivo evaluation.     

Redox potentials, laccase oxidation, and antilarval activities of substituted phenols

Laccases are copper-containing oxidases that are involved in sclerotization of the cuticle of mosquitoes and other insects. Oxidation of exogenous compounds by insect laccases may have the potential to produce reactive species toxic to insects. We investigated two classes of substituted phenolic compounds, halogenated di- and trihydroxybenzenes and substituted di-tert-butylphenols, on redox potential, oxidation by laccase and effects on mosquito larval growth. An inverse correlation between the oxidation potentials and laccase activity of halogenated hydroxybenzenes was found. Substituted di-tert-butylphenols however were found to impact mosquito larval growth and survival. In particular, 2,4-di-tert-butyl-6-(3-methyl-2-butenyl)phenol (15) caused greater than 98% mortality of Anophelesgambiae larvae in a concentration of 180nM, whereas 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-methylpropanal oxime (13) and 6,8-di-tert-butyl-2,2-dimethyl-3,4-dihydro-2H-chromene (33) caused 93% and 92% mortalities in concentrations of 3.4 and 3.7μM, respectively. Larvae treated with di-tert-butylphenolic compounds died just before pupation.