Phenotype sequencing: identifying the genes that cause a phenotype directly from pooled sequencing of independent mutants

Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50-100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a "Phenotype Sequencing" approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110-$340.     

Subject-specific models of the hindfoot reveal a relationship between morphology and passive mechanical properties

The morphology of the bones, articular surfaces and ligaments and the passive mechanical characteristics of the ankle complex were reported to vary greatly among individuals. The goal of this study was to test the hypothesis that the variations observed in the passive mechanical properties of the healthy ankle complex are strongly influenced by morphological variations. To evaluate this hypothesis six numerical models of the ankle joint complex were developed from morphological data obtained from MRI of six cadaveric lower limbs, and from average reported data on the mechanical properties of ligaments and articular cartilage. The passive mechanical behavior of each model, under a variety of loading conditions, was found to closely match the experimental data obtained from each corresponding specimen. Since all models used identical material properties and were subjected to identical loads and boundary conditions, it was concluded that the observed variations in passive mechanical characteristics were due to variations in morphology, thus confirming the hypothesis. In addition, the average and large variations in passive mechanical behavior observed between the models were similar to those observed experimentally between cadaveric specimens. The results suggest that individualized subject-specific treatment procedures for ankle complex disorders are potentially superior to a one-size-fits-all approach.     

Occlusive Impedance Plethysmography: A Noninvasive Method of Diagnosis of Proximal Deep Vein Thrombosis

The purpose of this study was to assess and confirm the accuracy of impedance plethysmography (IPG) by the occlusive cuff method, in detecting proximal (popliteal, femoral and iliac) deep vein thrombosis in patients with symptomatic limbs. In 27 patients 30 consecutive limbs were studied with concurrent venography and IPG. The IPG result was normal in 9 of 9 limbs which were normal on venography, and abnormal in 15 of 16 limbs which showed venographic evidence of proximal deep vein thrombosis (DVT). An abnormal IPG strongly suggests proximal DVT (predictive value 0.88). A normal IPG virtually rules out proximal DVT (predictive value 1.0).     

Viral Events Necessary for the Induction of Interferon in Chick Embryo Cells

Temperature-sensitive mutants of Sindbis virus were employed to investigate the nature of the viral event(s) which induces chick-embryo cells to produce interferon. Chick embryo cells induced by the parental heat-resistant strain of Sindbis virus produced essentially equal amounts of interferon at 29 and 42 C. An RNA⁻ and three RNA⁺ strains [temperature-sensitive mutants unable (RNA⁻) and able (RNA⁺) to make ribonucleic acid] produced interferon at 29 C but not at 42 C. It is concluded that viral RNA per se and the replication of viral RNA do not induce interferon production by chick embryo cells.     

Beta-blocker timolol alleviates hyperglycemia-induced cardiac damage via inhibition of endoplasmic reticulum stress

Current data support that pharmacological modulators of endoplasmic reticulum stress (ERS) have therapeutic potential for diabetic individuals. Therefore, we aimed to examine whether timolol, having free radical-scavenger action, besides being a β-blocker, exerts a cardioprotective effect via inhibition of ERS response in diabetic rats in a comparison with an antioxidant N-acetylcysteine (NAC). Histopathological data showed that either timolol- or NAC-treatment of diabetic rats prevented the changes in mitochondria and nucleus of the cardiac tissue while they enhanced the cellular redox-state in heart as well. The levels of ER-targeted cytoprotective chaperones GRP78 and calnexin, unfolded protein response signaling protein CHO/Gadd153 besides the levels of calpain, BCL-2, phospho-Akt, PUMA, and PML in the hearts from diabetic rats, treated with either timolol or NAC, are found to be similar among these groups, although all these parameters were markedly preserved in the untreated diabetics compared to those of the controls. Taken into consideration how important a balanced-ratio between anti-apoptotic and pro-apoptotic proteins for the maintenance mitochondria/ER function, our results suggest that ERS in diabetic rat heart is mediated by increased oxidative damage, which in turn triggers cardiac dysfunction. Moreover, we also demonstrated that timolol treatment of diabetic rats, similar to NAC treatment, induced a well-controlled redox-state and apoptosis in cardiac myocardium. We, thus for the first time, report that cardioprotective effect of timolol seems to be associated with normalization of ER function due to its antioxidant action in cardiomyocytes even under hyperglycemia.     

A practical method to determine the amount of tissue to analyze using laser scanning cytometry.

BACKGROUND: Laser scanning cytometry (LSC) is a new technology similar to flow cytometry but generates data from analysis of successive microscopic fields. Unlike its use in other applications, LSC-generated data are not random when used for tissue sections, but are dependent on the microanatomy of the tissue and the distribution and expression of the protein under investigation. For valid LSC analysis, the data generated requires the evaluation of a sufficient tissue area to ensure an accurate representation of expression within the tissue of interest. METHODS: In this report, we describe a simple and common sense method for determining the area of tissue required for sound LSC analysis by tracking the variation in the measure of target expression with increasing number of fields until it approaches zero. RESULTS: This approach was used to evaluate the expression of immunohistochemical markers with differing tissue distributions in liver (PMP70, CYP1A2, and Ki67 positive macrophages) and a colorectal adenocarcinoma (activated caspase-3 positive cells), which exhibited diffuse, regional (centrilobular), random, and irregular distribution patterns respectively. CONCLUSIONS: Analyses of these markers demonstrated that the amount of tissue area required to reach a steady measure of a parameter increased with increasing variability of the tissue distribution.