Chemical analysis of atherosclerotic plaque cholesterol combined with histology of the same tissue

Sensitive method for chemical analysis of free cholesterol (FC) and cholesterol esters (CE) was developed. Mouse arteries were dissected and placed in chloroform-methanol without tissue grinding. Extracts underwent hydrolysis of cholesteryl esters and derivatization of cholesterol followed by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. We demonstrated that FC and CE could be quantitatively extracted without tissue grinding and that lipid extraction simultaneously worked for tissue fixation. Delipidated tissues can be embedded in paraffin, sectioned, and stained. Microscopic images obtained from delipidated arteries have not revealed any structural alterations. Delipidation was associated with excellent antigen preservation compatible with traditional immunohistochemical procedures. In ApoE(-/-) mice, LC/MS/MS revealed early antiatherosclerotic effects of dual PPARalpha,gamma agonist LY465606 in brachiocephalic arteries of mice treated for 4 weeks and in ligated carotid arteries of animals treated for 2 weeks. Reduction in CE and FC accumulation in atherosclerotic lesions was associated with the reduction of lesion size. Thus, a combination of LC/MS/MS measurements of CE and FC followed by histology and immunohistochemistry of the same tissue provides novel methodology for sensitive and comprehensive analysis of experimental atherosclerotic lesions.     

Estimation in Markov models from aggregate data

In this paper, situations in which individuals move through a finite set of states according to a continuous-time Markov process are considered. Only aggregate data are available: these consist of the number of individuals in each state at specified observation times. We develop conditional least squares and approximate maximum-likelihood-estimation procedures for time-homogeneous models, and extend the methods so that they can handle immigration of individuals into the system during observation. Asymptotic covariance estimates are presented, and some problems for future study are noted.     

Potential of antimicrobial treatment of linear low-density polyethylene with poly((tert-butyl-amino)-methyl-styrene) to reduce biofilm formation in the food industry

Antimicrobial surfaces are one approach to prevent biofilms in the food industry. The aim of this study was to investigate the effect of poly((tert-butyl-amino)-methyl-styrene) (poly(TBAMS)) incorporated into linear low-density polyethylene (LLDPE) on the formation of mono- and mixed-species biofilms. The biofilm on untreated and treated LLDPE was determined after 48 and 168 h. The comparison of the results indicated that the ability of Listeria monocytogenes to form biofilms was completely suppressed by poly(TBAMS) (Δ_168 h 3.2 log₁₀ cfu cm^?2) and colonization of Staphylococcus aureus and Escherichia coli was significantly delayed, but no effect on Pseudomonas fluorescens was observed. The results of dual-species biofilms showed complex interactions between the microorganisms, but comparable effects on the individual bacteria by poly(TBAMS) were identified. Antimicrobial treatment with poly(TBAMS) shows great potential to prevent biofilms on polymeric surfaces. However, a further development of the material is necessary to reduce the colonization of strong biofilm formers.     

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata)

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240- bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.      

A Positive Stable Frailty Model for Clustered Failure Time Data with Covariate‐Dependent Frailty

Summary In this article, we propose a positive stable shared frailty Cox model for clustered failure time data where the frailty distribution varies with cluster‐level covariates. The proposed model accounts for covariate‐dependent intracluster correlation and permits both conditional and marginal inferences. We obtain marginal inference directly from a marginal model, then use a stratified Cox‐type pseudo‐partial likelihood approach to estimate the regression coefficient for the frailty parameter. The proposed estimators are consistent and asymptotically normal and a consistent estimator of the covariance matrix is provided. Simulation studies show that the proposed estimation procedure is appropriate for practical use with a realistic number of clusters. Finally, we present an application of the proposed method to kidney transplantation data from the Scientific Registry of Transplant Recipients.     

Proportional Hazards Regression for the Analysis of Clustered Survival Data from Case–Cohort Studies

Summary Case–cohort sampling is a commonly used and efficient method for studying large cohorts. Most existing methods of analysis for case–cohort data have concerned the analysis of univariate failure time data. However, clustered failure time data are commonly encountered in public health studies. For example, patients treated at the same center are unlikely to be independent. In this article, we consider methods based on estimating equations for case–cohort designs for clustered failure time data. We assume a marginal hazards model, with a common baseline hazard and common regression coefficient across clusters. The proposed estimators of the regression parameter and cumulative baseline hazard are shown to be consistent and asymptotically normal, and consistent estimators of the asymptotic covariance matrices are derived. The regression parameter estimator is easily computed using any standard Cox regression software that allows for offset terms. The proposed estimators are investigated in simulation studies, and demonstrated empirically to have increased efficiency relative to some existing methods. The proposed methods are applied to a study of mortality among Canadian dialysis patients.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.