Leishmania chagasi naturally resistant to nitric oxide isolated from humans and dogs with visceral leishmaniasis in Brazil

Nitric oxide (NO) plays an important role as a leishmanicidal agent in murine macrophages. NO resistant Escherichia coli and Mycobacterium tuberculosis have been associated with poor outcomes of their resulting diseases. NO resistant Leishmania braziliensis has also been identified and exacerbates the clinical course of human leishmaniasis. We report, for the first time, natural resistance of Leishmania chagasi promastigotes to NO. These parasites were isolated from humans and dogs with visceral leishmaniasis. We also demonstrate that this resistance profile was associated with a greater survival capacity and a greater parasite burden in murine macrophages, independent of activation and after activation by IFN-γ and LPS.     

Simulation of nitroxide electron paramagnetic resonance spectra from brownian trajectories and molecular dynamics simulations

A simulated continuous wave electron paramagnetic resonance spectrum of a nitroxide spin label can be obtained from the Fourier transform of a free induction decay. It has been previously shown that the free induction decay can be calculated by solving the time-dependent stochastic Liouville equation for a set of Brownian trajectories defining the rotational dynamics of the label. In this work, a quaternion-based Monte Carlo algorithm has been developed to generate Brownian trajectories describing the global rotational diffusion of a spin-labeled protein. Also, molecular dynamics simulations of two spin-labeled mutants of T4 lysozyme, T4L F153R1, and T4L K65R1 have been used to generate trajectories describing the internal dynamics of the protein and the local dynamics of the spin-label side chain. Trajectories from the molecular dynamics simulations combined with trajectories describing the global rotational diffusion of the protein are used to account for all of the dynamics of a spin-labeled protein. Spectra calculated from these combined trajectories correspond well to the experimental spectra for the buried site T4L F153R1 and the helix surface site T4L K65R1. This work provides a framework to further explore the modeling of the dynamics of the spin-label side chain in the wide variety of labeling environments encountered in site-directed spin labeling studies.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Damage and recovery from UV-B exposure in conidia of the entomopathogens Verticillium lecanii and Aphanocladium album

We evaluated the effects of exposure to doses supplied at an environmentally realistic intensity of UV-B radiation (800 mW m(-2) weighted irradiance) on the culturability and germination of selected strains of the entomopathogenic Hyphomycetes Verticillium lecanii and Aphanocladium album. Increased UV-B exposure decreased relative percent culturability for all strains. Four hours of exposure to UV-B were sufficient to reduce the culturability close to zero. The LT(50) (50% lethal time) ranged from 120 ± 5 min for the V. lecanii strain ARSEF 6430 to 86 ± 14 min for the A. album strain ARSEF 6433. A strong delay in the germination of surviving conidia was observed. To determine the occurrence of photoreactivation in these two genera, we evaluated the effect of exposure to visible light after exposure to UV-B radiation. There was no significant difference in relative culturability between conidia exposed to visible light after UV-B exposure compared to those incubated in the dark after UV-B exposure. This indicates that photoreactivation, if it occurs, must have limited importance in the repair of the damage induced by UV-B radiation in these two genera.     

Molecular PET imaging of HSV1-tk reporter gene expression using [18F]FEAU

Non-invasive imaging of transgene expression requires the appropriate combination of a reporter gene and a reporter probe. [18F]FEAU positron emission tomography (PET) is used for the assessment of herpes simplex virus type-1 thymidine kinase gene expression. Hybrid AAV phage (termed AAVP) can be adapted to transduce mammalian cells by targeting to a specific receptor. We evaluated a targeted AAVP vector using [18F]FEAU PET. This protocol describes [18F]FEAU production and dosing, micro-PET imaging and image analysis. 2-Deoxy-2-trifluoromethanesulfonyl-1,3,5-tri-O-benzoyl-alpha-D-ribofuranose is radio-fluorinated, converted into its 1-bromo derivative and coupled with protected 5-ethyl uracil. The coupled product is hydrolyzed and purified using HPLC. Tumor-bearing animals targeted with either retroviral or AAVP vectors are anesthetized and injected with [18F]FEAU (0.1 mCi per mouse); this is followed 2 h after injection by imaging on a micro-PET. Production of [18F]FEAU requires approximately 3.5 h from the end of bombardment. PET imaging studies require 2-3 h (depending on the number of animals) after synthesis of [18F]FEAU.     

<Emphasis Type="Italic">ScreenMill</Emphasis>: A freely available software suite for growth measurement,analysis and visualization of high-throughput screen data

Background  

Many high-throughput genomic experiments, such as Synthetic Genetic Array and yeast two-hybrid, use colony growth on solid media as a screen metric. These experiments routinely generate over 100,000 data points, making data analysis a time consuming and painstaking process. Here we describe ScreenMill, a new software suite that automates image analysis and simplifies data review and analysis for high-throughput biological experiments.     

Widespread positive but weak assortative mating by diet within stickleback populations

Assortative mating – correlation between male and female traits – is common within populations and has the potential to promote genetic diversity and in some cases speciation. Despite its importance, few studies have sought to explain variation in the extent of assortativeness across populations. Here, we measure assortative mating based on an ecologically important trait, diet as inferred from stable isotopes, in 16 unmanipulated lake populations of three‐spine stickleback. As predicted, we find a tendency toward positive assortment on the littoral–pelagic axis, although the magnitude is consistently weak. These populations vary relatively little in the strength of assortativeness, and what variation occurs is not explained by hypothesized drivers including habitat cosegregation, the potential for disruptive selection, costs to choosiness, and the strength of the relationship between diet and body size. Our results support recent findings that most assortative mating is positive, while suggesting that new approaches may be required to identify the environmental variables that drive the evolution of nonrandom mating within populations.     

Transposon mutagenesis identifies cooperating genetic drivers during keratinocyte transformation and cutaneous squamous cell carcinoma progression

The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates.