Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin

There is a lack of available methods to noninvasively quantify lymphatic function in small experimental animals, a necessity for studies on lymphatic system pathophysiology. We present a new method to quantify lymph flow in mice and rats, based on optically monitoring the depot clearance of near-infrared fluorescently labeled albumin and subsequent calculation of removal rate constants (k). BSA was conjugated with Alexa680 NHS ester and remained stable in protein-rich solutions without free dye dissociation. To assess lymph flow, mice or rats were imaged every 30 or 60 min during a 3- to 6-h period following an intradermal injection of 0.5 or 1 μl Alexa680-albumin. Mice were awake between measurements, whereas rats were anesthetized throughout the experiment. The k, a parameter defined as equivalent to lymph flow, was calculated from the slopes of the resultant log-linear washout curves and averaged -0.40 ± 0.03 and -0.30 ± 0.02%/min for control C57BL/6 and C3H mice, respectively. Local administration of the vasoconstrictor endothelin-1 in mice led to a significant reduction in k, whereas overhydration in rats increased k, reflecting the coupling between capillary filtration and lymph flow. Furthermore, k was 50% of wild type in lymphedema Chy mice where dermal lymphatics are absent. We conclude that lymph flow can be determined as its rate constant k by optical imaging of depot clearance of submicroliter amounts of Alexa680-albumin. The method offers a minimally invasive, reproducible, and simple alternative to assess lymphatic function in mice and rats.     

Evaluating bronchodilator effects in chronic obstructive pulmonary disease using diffusion-weighted hyperpolarized helium-3 magnetic resonance imaging

The objective of this study was to evaluate the regional effects of bronchodilator administration in chronic obstructive pulmonary disease (COPD) using hyperpolarized helium-3 ((3)He) MRI apparent diffusion coefficient (ADC). Ten COPD ex-smokers provided written, informed consent and underwent diffusion-weighted, hyperpolarized (3)He MRI, spirometry, and plethysmography before and 25 ± 2 min after bronchodilator administration. Pre- and postsalbutamol whole-lung (WL) ADC maps were generated and registered together to identify the lung regions containing the (3)He signal at both time points, and mean ADC within those regions of interest (ROI) was determined for a measurement of previously ventilated ROI ADC (ADC(P)). Lung ROI with (3)He signal at both time points was used as a binary mask on postsalbutamol WL ADC maps to obtain an ADC measurement for newly ventilated ROI (ADC(N)). Postsalbutamol, no significant differences were detected in WL ADC (P = 0.516). There were no significant differences between ADC(N) and ADC(P) postsalbutamol (P = 1.00), suggesting that the ADC(N) lung regions were not more emphysematous than the lung ROI participating in ventilation before bronchodilator administration. Postsalbutamol, a statistically significant decrease in ADC(P) (P = 0.01) was detected, and there were significant differences between ADC(P) in the most anterior and most posterior image slices (P = 0.02), suggesting a reduction in regional gas trapping following bronchodilator administration. Regional evaluation of tissue microstructure using hyperpolarized (3)He MRI ADC provides insights into lung alterations that accompany improvements in regional (3)He gas distribution after bronchodilator administration.     

New phylogenetic information suggests both an increase and at least one loss of cooperative breeding during the evolutionary history of Aphelocoma jays

Efforts to identify ecological and life history factors associated with cooperative breeding have been largely unsuccessful, and interest is growing in the role of phylogenetic history in determining the distribution of this social system among lineages. In birds, cooperative breeding is distributed non-randomly among lineages, suggesting that phylogenetic inertia may play an important role in determining its distribution. The bird genus Aphelocoma has been particularly well studied because, although it is a relatively small genus, it shows broad among-lineage variation in level of cooperation. Previous analyses described an unusual unidirectional pattern of evolutionary loss of cooperation in Aphelocoma. Here, historical reconstructions based on new phylogenetic data suggest that evolutionary changes in cooperation have been bidirectional, with at least one gain and at least one loss over relatively recent timescales. This result emphasizes that, although history plays an important role in determining the incidence of cooperative breeding, cooperative behavior can switch relatively quickly in evolutionary time and may be influenced by the ecological context within which particular populations are distributed.     

Dry collection and culture methods for recovery of methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains from indoor home environments

Staphylococcus aureus in home environments may serve as a reservoir for human colonization, making sampling of indoor surfaces relevant to exposure assessment. Using laboratory experiments and application to homes of asthmatic children in Barbados, we characterize microbiological methods adapted for settings with transportation delays between sampling and initiation of culture.     

Brown seaweed species from Strangford Lough: compositional analyses of seaweed species and biostimulant formulations by rapid instrumental methods

The aims of this study were to characterise the composition of five seaweed species (Ascophyllum nodosum, Fucus serratus, Fucus vesiculosus, Laminaria hyperborea and Sargassum muticum), their extracts and commercial formulations, using thermogravimetry (TGA), energy dispersive X-ray microanalysis (EDX), Fourier-transform infrared spectroscopy (FTIR) and pyrolysis gas chromatography/mass spectrometry (Py-GC/MS). Analyses of the samples by TGA and EDX provided information on the proportions of algal cell wall, inorganic fractions and minerals. The main carbohydrate constituents of the five species and extracts were identified by their pyrolysis products, e.g. 1-(2-furanyl) ethanone, 5-methyl-2-furcarboxaldehyde, 2-hydroxy-3-methyl-2-cyclopenten-1-one, diannhydromannitol, 1,6-anhydromannopyranose and 1,6-anhydromannofuranose, using Py-GC/MS. The differences in relative intensities of the infrared bands of the five species were enhanced, especially after acid extraction compared with alkaline or neutral treatments, resulting in improved understanding of the compositional changes. In addition four commercial formulations and two acidic extracts of A. nodosum were evaluated for composition using the techniques. The dry matter, pH, electrical conductivity, ash, carbon and nitrogen content of the six preparations showed significant differences in composition. Variations in fatty acid, alginic acid, mannitol, laminarin and fucoidan content of the six formulations were reported. The results have shown that TGA, EDX, Py-GC/MS and FTIR are complementary techniques for rapid evaluation of seaweed materials and products.     

Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin

1. Download : Download high-res image (307KB)
2. Download : Download full-size image

Highlights? The fungal secondary metabolite Cladosporin inhibits liver- and blood-stage malaria parasites ? Cladosporin specifically targets lysyl-tRNA synthetase (Krs1) ? Cladosporin is >100-fold more potent against parasite Krs1 relative to the human enzyme ? Two amino acids in the Krs1 ATP-binding pocket confer species-selective inhibition     

Protein kinase A regulatory subunit isoforms regulate growth and differentiation in Mucor circinelloides: essential role of PKAR4

The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway.     

Cross-Species Functional Genomic Analysis Identifies Resistance Genes of the Histone Deacetylase Inhibitor Valproic Acid

The mechanisms of successful epigenetic reprogramming in cancer are not well characterized as they involve coordinated removal of repressive marks and deposition of activating marks by a large number of histone and DNA modification enzymes. Here, we have used a cross-species functional genomic approach to identify conserved genetic interactions to improve therapeutic effect of the histone deacetylase inhibitor (HDACi) valproic acid, which increases survival in more than 20% of patients with advanced acute myeloid leukemia (AML). Using a bidirectional synthetic lethality screen revealing genes that increased or decreased VPA sensitivity in C. elegans, we identified novel conserved sensitizers and synthetic lethal interactors of VPA. One sensitizer identified as a conserved determinant of therapeutic success of HDACi was UTX (KDM6A), which demonstrates a functional relationship between protein acetylation and lysine-specific methylation. The synthetic lethal screen identified resistance programs that compensated for the HDACi-induced global hyper-acetylation, and confirmed MAPKAPK2, HSP90AA1, HSP90AB1 and ACTB as conserved hubs in a resistance program for HDACi that are drugable in human AML cell lines. Hence, these resistance hubs represent promising novel targets for refinement of combinatorial epigenetic anti-cancer therapy.