Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging

Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels. Images of the same molecule in the two channels are then combined to determine both the location and the identity of the molecule. The single-objective configuration of our approach allows for flexible sample geometry and the use of a live-cell incubation chamber required for live-cell SMT. Despite a lower photon yield, we achieve excellent spatial (20–40 nm) and spectral (10–15 nm) resolutions comparable to those obtained with dual-objective, spectrally resolved Stochastic Optical Reconstruction Microscopy. Furthermore, motions of the fluorescent molecules did not cause loss of spectral resolution owing to the dual-channel spectral calibration. We demonstrate SMT in three (and potentially more) colors using spectrally proximal fluorophores and single-laser excitation, and show that trajectories of each species can be reliably extracted with minimal cross talk.     

Infectious disease in fish: global risk of viral hemorrhagic septicemia virus

As the global human population continues to increase and become more industrialized, the need for safe, secure, and sustainable protein production is critical. One sector of particular importance is seafood production, where capture fishery and aquaculture industries provide 15–20% of the global protein supply. However, fish production can be severely affected by diseases. Notably, viral hemorrhagic septicemia, caused by the viral hemorrhagic septicemia virus (VHSv; Rhabdoviridae), may be one of the most devastating viral diseases of fishes worldwide. We explored the ecology and epidemiology of VHSv using an ecological niche modeling approach to identify vulnerable disease-free regions. Results showed an impressive ecological plasticity of VHSv. The virus was found in >?140 fish species in marine and freshwater ecosystems, with high diversity of lineages in Eurasia. Sub-genotypes from marine and fresh waters were ecologically similar, suggesting broad ecological niches, rather than rapid evolutive adaptation to novel environments. Ecological niche models predicted that VHSv may have favorable physical (e.g., temperature, runoff), chemical (e.g., salinity, pH, phosphate), and biotic (i.e., chlorophyll) conditions for establishing into areas with important fish industries that, so far, are believed to be disease-free (i.e., freshwater and marine ecosystems of Africa, Latin America, Australia, and inland China). The model and our review suggest fish species from the Perciformes, Salmoniformes, and Gadiformes orders are likely to be infected with VHSv in novel regions as the virus expands its range to areas predicted to be at risk. In conclusion, VHSv remains an emerging disease threat to global food security and aquatic biodiversity.     

Insight in schizophrenia spectrum disorders: relationship with behavior,mood and perceived quality of life,underlying causes and emerging treatments

Poor insight in schizophrenia is prevalent across cultures and phases of illness. In this review, we examine the recent research on the relationship of insight with behavior, mood and perceived quality of life, on its complex roots, and on the effects of existing and emerging treatments. This research indicates that poor insight predicts poorer treatment adherence and therapeutic alliance, higher symptom severity and more impaired community function, while good insight predicts a higher frequency of depression and demoralization, especially when coupled with stigma and social disadvantage. This research also suggests that poor insight may arise in response to biological, experiential, neuropsychological, social‐cognitive, metacognitive and socio‐political factors. Studies of the effects of existing and developing treatments indicate that they may influence insight. In the context of earlier research and historical models, these findings support an integrative model of poor insight. This model suggests that insight requires the integration of information about changes in internal states, external circumstances, others’ perspectives and life trajectory as well as the multifaceted consequences and causes of each of those changes. One implication is that treatments should, beyond providing education, seek to assist persons with schizophrenia to integrate the broad range of complex and potentially deeply painful experiences which are associated with mental illness into their own personally meaningful, coherent and adaptive picture.     

Grain size and depth constraints on microbial variability in coastal plain subsurface sediments

We have examined the effects of sediment grain size and depth on the abundance and activity of aerobic bacteria at two coastal plain sites in Virginia. Samples were collected at centimeter intervals as well as meter intervals because fine‐scale sampling can be essential to assess microbial variability. At the Oyster site, grain size varied from 0.12 to 0.25 mm below 1.5 m depth and did not correlate with either bacterial abundance or activity. Perhaps due to the fairly uniform grain size at this site, variations in bacterial numbers were less than fivefold between replicate samples of 0,1 to 100 g and generally less than 15‐fold among closely spaced intervals (～5 cm). At the Abbott Pit site, grain size was about threefold greater (0.50 ± 0.17mm) in an interval of 4.35 to 5.0m below land surface than grain size in the surrounding sediments. In the same interval, bacterial abundance increased by 11‐fold and activity increased by 217‐fold relative to the surrounding sediments. Overall, grain size correlated significantly with bacterial abundance and activity below the soil zone at the Abbott Pit site. This suggests that changes in grain size, even at the centimeter scale, could have a predominant effect on microbial variability in sandy aquifers of the coastal plain. Besides grain size, depth correlated significantly with total organic carbon and bacterial abundance and activity at both sites, suggesting that depth is also an important factor controlling microbial variability in the subsurface environments.     

Use of a novel Förster resonance energy transfer method to identify locations of site-bound metal ions in the U2-U6 snRNA complex

U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its folding and catalytic activity. We used a novel Förster resonance energy transfer (FRET) method between site-bound luminescent lanthanide ions and a covalently attached fluorescent dye, combined with supporting stoichiometric and mutational studies, to determine locations of site-bound Tb(III) within the human U2–U6 complex. At pH 7.2, we detected three metal-ion-binding sites in: (1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; (2) the four-way junction, which contains the conserved AGC triad; and (3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which may impact on splicing activity.     

Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens

Coat protein of the cowpea chlorotic mottle virus (CCMV), a plant bromovirus, has been expressed in a soluble form in a prokaryote, Pseudomonas fluorescens, and assembled into virus-like particles (VLPs) in vivo that were structurally similar to the native CCMV particles derived from plants. The CCMV VLPs were purified by PEG precipitation followed by separation on a sucrose density gradient and analyzed by size exclusion chromatography, UV spectrometry, and transmission electron microscopy. DNA microarray experiments revealed that the VLPs encapsulated very large numbers of different host RNAs in a non-specific manner. The development of a P. fluorescens expression system now enables production of CCMV VLPs by bacterial fermentation for use in pharmaceutical or nanotechnology applications.     

Exposure to degraded coral habitat depresses oxygen uptake rate during exercise of a juvenile reef fish

Coral Reefs - Coral reef ecosystems are currently under unprecedented stress due to anthropogenic induced climate change. Such stress causes coral habitats to degrade, which has been found to...     

Evidence for an early evolutionary emergence of γ-type carbonic anhydrases as components of mitochondrial respiratory complex I

Background  

The complexity of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase) has increased considerably relative to the homologous complex in bacteria. Comparative analyses of CI composition in animals, fungi and land plants/green algae suggest that novel components of mitochondrial CI include a set of 18 proteins common to all eukaryotes and a variable number of lineage-specific subunits. In plants and green algae, several purportedly plant-specific proteins homologous to γ-type carbonic anhydrases (γCA) have been identified as components of CI. However, relatively little is known about CI composition in the unicellular protists, the characterizations of which are essential to our understanding of CI evolution.