Recombination signatures distinguish embryonic stem cells derived by parthenogenesis and somatic cell nuclear transfer

Parthenogenesis and somatic cell nuclear transfer (SCNT) are two methods for deriving embryonic stem (ES) cells that are genetically matched to the oocyte donor or somatic cell donor, respectively. Using genome-wide single nucleotide polymorphism (SNP) analysis, we demonstrate distinct signatures of genetic recombination that distinguish parthenogenetic ES cells from those generated by SCNT. We applied SNP analysis to the human ES cell line SCNT-hES-1, previously claimed to have been derived by SCNT, and present evidence that it represents a human parthenogenetic ES cell line. Genome-wide SNP analysis represents a means to validate the genetic provenance of an ES cell line.     

Improving the diagnostic accuracy of N-terminal B-type natriuretic peptide in human systolic heart failure by plasma profiling using mass spectrometry

We have combined the measurement of N-terminal pro-B type natriuretic peptide (NTproBNP) with plasma peptide profiling to evaluate the effect on sensitivity and specificity of systolic heart failure (SHF) diagnosis. Plasma NTproBNP levels were measured from 100 SHF patients and 100 age/gender matched controls and plasma protein profiles obtained using MALDI-MS. Sixty-seven m/z peaks were significantly different between SHF and normals, and following logistic regression analysis with NTproBNP values, 6 peaks retained independent predictive value. Receiver operating characteristic (ROC) curves for SHF diagnosis had areas of 0.91 for NTproBNP, improving to 0.99 with the model. In a separate validation test set (32 SHF, 20 normals), the model remained highly accurate (ROC area 0.995). An artificial neural network with these 6 peak intensities and NTproBNP produced ROC areas of 0.99 in both training and test sets. The sensitivity and specificity of SHF diagnosis using NTproBNP in training (85, 85%) and test (93, 75%) sets was improved in the model for both training (96, 96%) and test (100, 95%) sets. The accuracy of SHF diagnosis using NTproBNP is improved by the use of a plasma profile of 6 peptide peaks, reducing the uncertainty in the diagnostic gray zone of using NTproBNP alone.     

Flow analysis and sorting of microchromosomes (<3 Mb)

BACKGROUND: The analysis and isolation of high numbers of chromosomes smaller than 3 Mb in size (microchromosomes) with good purity is dependent primarily on the detection sensitivity of the flow cytometer and the precision of the sort unit. The aim of this study was to investigate the capability of using a conventional flow cytometer for the detection and sorting at high purity microchromosomes with an estimated size of 2.7 Mb. METHODS: Chromosomes were isolated from a human cell line containing a pair of X-derived microchromosomes, using a modified polyamine isolation buffer. The chromosome preparation was labeled with Hoechst and Chromomycin and analyzed and purified using a MoFlo sorter (DAKO) configured for high-speed sorting. The purity of the flow-sorted microchromosomes was assessed by reverse chromosome painting. RESULTS: Improved resolution of the peak of microchromosomes in a bivariate plot of Hoechst versus Chromomycin fluorescence was obtainable after discriminating clumps and debris based on gating data within a FSC versus pulse width plot. CONCLUSIONS: Chromosomes of smaller size, less than 3 Mb, can be detected with high resolution and flow-sorted with high purity using a conventional flow sorter.     

Quantification of isoniazid and acetylisoniazid in rat plasma and alveolar macrophages by liquid chromatography-tandem mass spectrometry with on-line extraction

To evaluate if pulmonary delivery of microparticles loaded with a prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), can target alveolar macrophages (AM) and reduce metabolism of INH, an HPLC-MS/MS assay with automated online extraction for quantification of INH and its metabolite acetylisoniazid (AcINH) in plasma and AMs was developed and validated. Reproducibility in rat plasma and homogenate of a rat AM cell line, NR8383, for INH and AcINH showed excellent precision and accuracy with calibration curves exhibiting linearity within a range of 1-250ng/ml of INH and 0.05-50ng/ml of AcINH (r(2)>0.99). The validated methods were successfully applied to pharmacokinetic study of INHMS-loaded microparticles in rats, demonstrating efficient targeting of AMs and reduction of INH metabolism.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Imaging proteins in vivo using fluorescence lifetime microscopy

Fluorescence lifetime imaging (FLIM) represents a key optical technique for imaging proteins and protein interaction in vivo. We review the principles and recent advances in the application of the technique, instrumentation and molecular probe development.     

BMI and waist circumference in predicting cardiovascular risk factor clustering in Chinese adolescents

Objective: To derive the optimal BMI and waist circumference (WC) cut‐off values to predict clustering of cardiovascular risk factors in Hong Kong Chinese adolescents. Research Methods and Procedures: A total of 2102 Hong Kong Chinese 12 to 19 years of age were recruited. Participants were considered to have clustering of risk factors if at least three of the following risk factors were present: 1) high‐density lipoprotein cholesterol (HDL‐C) ≤1.03 mM, 2) low‐density lipoprotein cholesterol (LDL‐C) ≥2.6 mM, 3) triglyceride (TG) ≥1.24 mM, 4) fasting plasma glucose (FPG) ≥6.1 mM, and 5) age‐, sex‐, and height‐adjusted systolic or diastolic blood pressure (BP) ≥ 90th percentile. Receiver operating characteristics (ROC) curves were generated to identify the optimal age‐adjusted BMI and WC cut‐off values to predict clustering of risk factors in boys and girls separately. These age‐adjusted BMI and WC cut‐offs were transformed to percentile values. Cole's lambda‐mu‐sigma (LMS) method was used to obtain smoothed age‐specific BMI and WC at these percentile values. Results: The areas under ROC curves for BMI in girls and boys were 0.85 [95% confidence interval (CI), 0.77 to 0.92] and 0.76 (95% CI, 0.66 to 0.85), respectively. The respective areas under ROC curves for WC in girls and boys were 0.82 (95% CI, 0.74 to 0.91) and 0.78 (95% CI, 0.68 to 0.87). The optimal BMI thresholds were at the 78th percentile for girls and the 72nd percentile for boys. The respective values for WC were at the 77th percentile for girls and the 76th percentile for boys. The sensitivities and specificities of these cut‐off values ranged from 72% to 80%. Discussion: Age‐ and sex‐specific BMI and WC cut‐off values can be used to identify adolescents with clustering of cardiovascular risk factors.