Sensitivity measurement and compensation in spectral imaging.

BACKGROUND: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented. METHODS: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer. Four single color fluorescent control samples of cells were run to quantify spectral overlap. An additional sample, labeled with all colors was run and compensated in six spectral channels. RESULTS: Analysis of empirical data for sensitivity and dynamic range matched theoretical predictions. The ImageStream system demonstrated fluorescence sensitivity comparable to a PMT-based flow cytometer. A methodology for addressing spectral overlap, individual pixel anomalies, and multiple imaging modalities was demonstrated for spectral compensation of K562 cells. Imagery is shown pre- and post-compensation. CONCLUSIONS: Unlike intensity measurements made with conventional flow cytometers, object size impacts both dynamic range and fluorescence sensitivity in systems that utilize pixilated detection. Simultaneous imaging of alternate modalities can be employed to increase fluorescent sensitivity. Effective compensation of complex multimode imagery spanning six spectral bands is accomplished in a semi-automated manner.     

Variation in reaction norms: Statistical considerations and biological interpretation

Analysis of reaction norms, the functions by which the phenotype produced by a given genotype depends on the environment, is critical to studying many aspects of phenotypic evolution. Different techniques are available for quantifying different aspects of reaction norm variation. We examine what biological inferences can be drawn from some of the more readily applicable analyses for studying reaction norms. We adopt a strongly biologically motivated view, but draw on statistical theory to highlight strengths and drawbacks of different techniques. In particular, consideration of some formal statistical theory leads to revision of some recently, and forcefully, advocated opinions on reaction norm analysis. We clarify what simple analysis of the slope between mean phenotype in two environments can tell us about reaction norms, explore the conditions under which polynomial regression can provide robust inferences about reaction norm shape, and explore how different existing approaches may be used to draw inferences about variation in reaction norm shape. We show how mixed model‐based approaches can provide more robust inferences than more commonly used multistep statistical approaches, and derive new metrics of the relative importance of variation in reaction norm intercepts, slopes, and curvatures.     

Improved Luciferase Tagging System for Listeria monocytogenes Allows Real-Time Monitoring In Vivo and In Vitro

An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux-tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.     

Bacterial polysaccharides suppress induced innate immunity by calcium chelation

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.     

Overstory species composition of naturally regenerated clearcuts in an ecological classification framework

Multifactor ecological classification systems are being developed for many regions. An element of these systems not yet well understood is how disturbances, clearcutting in this instance, may alter the vegetative component of the classification units at the stand and landscape levels. We sampled 1,096 plots in 21–35 year old naturally regenerated clearcuts on the Hoosier National Forest (HNF) in south-central Indiana, USA. We examined overstory species composition of clearcut plots in comparison to reference plots (80+ years old), both within and among six Ecological Landtype Phases (ELTPs) of two ecological sections using non-metric multidimensional scaling and non-metric multi-response permutation procedures. Clearcutting drastically changed species composition in comparison to reference plots within ELTPs ranging from mixed oak-dominated ridges and slopes to bottomland, cove hardwood communities; Quercus species on ridges and slopes were replaced by Liriodendron tulipifera L. and, to a lesser degree, Prunus serotina Ehrh. and Acer rubrum L., in ELTPs of both sections. Contrasts of overstory species composition of reference plots exhibited differences among ELTPs, but clearcut plots showed mixed results and indicated very similar species composition across all ELTPs. Autogenic factors are likely the main drivers of overstory composition of clearcut sites. Species composition of ELTPs will continue to develop in response to autogenic and allogenic factors over time, and differences among ELTPs may emerge in later stages of stand development as the effects of allogenic factors accumulate. It is expected that L. tulipifera, a long-lived species, will be a dominant species in terms of basal area and density of all ELTPs in mature stands. Classification systems not designed to deal with changes related to disturbance and a failure to predict successional pathways after disturbance may limit their usefulness as a management tool in terms of overstory vegetation. For ecological classification systems to be fully effective, we must better understand the role of disturbance in ecosystem function at many different scales and integrate that knowledge into our decision-making and planning regimes to establish realistic and attainable objectives at multiple scales.     

Endoglucanase activities and growth of marine‐derived fungi isolated from the sponge Haliclona simulans

Aims: The conversion of cheap cellulosic biomass to more easily fermentable sugars requires the use of costly cellulases. We have isolated a series of marine sponge‐derived fungi and screened these for cellulolytic activity to determine the potential of this unique environmental niche as a source of novel cellulase activities. Methods and Results: Fungi were isolated from the marine sponge Haliclona simulans. Phylogenetic analysis of these and other fungi previously isolated from H. simulans showed fungi from three phyla with very few duplicate species. Cellulase activities were determined using plate‐based assays using different media and sea water concentrations while extracellular cellulase activities were determined using 3,5‐dinitrosalicylic acid (DNSA)‐based assays. Total and specific cellulase activities were determined using a range of incubation temperatures and compared to those for the cellulase overproducing mutant Hypocrea jecorina QM9414. Several of the strains assayed produced total or relative endoglucanase activities that were higher than H. jecorina, particularly at lower reaction temperatures. Conclusions: Marine sponges harbour diverse fungal species and these fungi are a good source of endoglucanase activities. Analysis of the extracellular endoglucanase activities revealed that some of the marine‐derived fungi produced high endoglucanase activities that were especially active at lower temperatures. Significance and Impact of the Study: Marine‐derived fungi associated with coastal marine sponges are a novel source of highly active endoglucanases with significant activity at low temperatures and could be a source of novel cellulase activities.