Mass-selected polyatomic cations and anions, produced by electrosonic spray ionization (ESSI), were deposited onto polycrystalline Au or fluorinated self-assembled monolayer (FSAM) surfaces by soft landing (SL), using a rectilinear ion trap (RIT) mass spectrometer. Protonated and deprotonated molecules, as well as intact cations and anions generated from such molecules as peptides, inorganic catalysts, and fluorescent dyes, were soft-landed onto the surfaces. Analysis of the modified surfaces was performed in situ by Cs(+) secondary ion mass spectrometry (SIMS) using the same RIT mass analyzer to characterize the sputtered ions as that used to mass select the primary ions for SL. Soft-landing times as short as 30 s provided surfaces that yielded good quality SIMS spectra. Chemical reactions of the surfaces modified by SL were generated in an attached reaction chamber into which the surface was transferred under vacuum. For example, a surface on which protonated triethanolamine had been soft landed was silylated using vapor-phase chlorotrimethylsilane before being returned still under vacuum to the preparation chamber where SIMS analysis revealed the silyloxy functionalization. SL and vapor-phase reactions are complementary methods of surface modification and in situ surface analysis by SIMS is a simple way to characterize the products produced by either technique.

SUMMARY: INFERNAL builds consensus RNA secondary structure profiles called covariance models (CMs), and uses them to search nucleic acid sequence databases for homologous RNAs, or to create new sequence- and structure-based multiple sequence alignments. AVAILABILITY: Source code, documentation and benchmark downloadable from http://infernal.janelia.org. INFERNAL is freely licensed under the GNU GPLv3 and should be portable to any POSIX-compliant operating system, including Linux and Mac OS/X.

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

Ever since the integration of Mendelian genetics into evolutionary biology in the early 20th century, evolutionary geneticists have for the most part treated genes and mutations as generic entities. However, recent observations indicate that all genes are not equal in the eyes of evolution. Evolutionarily relevant mutations tend to accumulate in hotspot genes and at specific positions within genes. Genetic evolution is constrained by gene function, the structure of genetic networks, and population biology. The genetic basis of evolution may be predictable to some extent, and further understanding of this predictability requires incorporation of the specific functions and characteristics of genes into evolutionary theory.

Crustaceans possess remarkably diverse appendages, both between segments of a single individual as well as between species. Previous studies in a wide range of crustaceans have demonstrated a correlation between the anterior expression boundary of the homeotic (Hox) gene Ultrabithorax (Ubx) and the location and number of specialized thoracic feeding appendages, called maxillipeds. Given that Hox genes regulate regional identity in organisms as diverse as mice and flies, these observations in crustaceans led to the hypothesis that Ubx expression regulates the number of maxillipeds and that evolutionary changes in Ubx expression have generated various aspects of crustacean appendage diversity. Specifically, evolutionary changes in the expression boundary of Ubx have resulted in crustacean species with either 0, 1, 2, or 3 pairs of thoracic maxillipeds. Here we test this hypothesis by altering the expression of Ubx in Parhyale hawaiensis, a crustacean that normally possesses a single pair of maxillipeds. By reducing Ubx expression, we can generate Parhyale with additional maxillipeds in a pattern reminiscent of that seen in other crustacean species, and these morphological alterations are maintained as the animals molt and mature. These results provide critical evidence supporting the proposition that changes in Ubx expression have played a role in generating crustacean appendage diversity and lend general insights into the mechanisms of morphological evolution.

The dramatic transformation from a larva to an adult must be accompanied by a coordinated activity of genes and hormones that enable an orchestrated transformation from larval to pupal/adult tissues. The maintenance of larval appendages and their subsequent transformation to appendages in holometabolous insects remains elusive at the developmental genetic level. Here the role of a key appendage patterning gene Distal-less (Dll) was examined in mid- to late-larval stages of the flour beetle, Tribolium castaneum. During late larval development, Dll was expressed in appendages in a similar manner as previously reported for the tobacco hornworm, Manduca sexta. Removal of this late Dll expression resulted in disruption of adult appendage patterning. Intriguingly, earlier removal resulted in dramatic loss of structural integrity and identity of larval appendages. A large amount of variability in appendage morphology was observed following Dll dsRNA injection, unlike larvae injected with dachshund dsRNA. These Dll dsRNA-injected larvae underwent numerous supernumerary molts, which could be terminated with injection of either JH methyltransferase or Methoprene-tolerant dsRNA. Apparently, the partial dedifferentiation of the appendages in these larvae acts to maintain high JH and, hence, prevents metamorphosis.

The central actions of leptin are essential for homeostatic control of adipose tissue mass, glucose metabolism, and many autonomic and neuroendocrine systems. In the brain, leptin acts on numerous different cell types via the long-form leptin receptor (LepRb) to elicit its effects. The precise identification of leptin’s cellular targets is fundamental to understanding the mechanism of its pleiotropic central actions. We have systematically characterized LepRb distribution in the mouse brain using in situ hybridization in wildtype mice as well as by EYFP immunoreactivity in a novel LepRb-IRES-Cre EYFP reporter mouse line showing high levels of LepRb mRNA/EYFP coexpression. We found substantial LepRb mRNA and EYFP expression in hypothalamic and extrahypothalamic sites described before, including the dorsomedial nucleus of the hypothalamus, ventral premammillary nucleus, ventral tegmental area, parabrachial nucleus, and the dorsal vagal complex. Expression in insular cortex, lateral septal nucleus, medial preoptic area, rostral linear nucleus, and in the Edinger-Westphal nucleus was also observed and had been previously unreported. The LepRb-IRES-Cre reporter line was used to chemically characterize a population of leptin receptor-expressing neurons in the midbrain. Tyrosine hydroxylase and Cre reporter were found to be coexpressed in the ventral tegmental area and in other midbrain dopaminergic neurons. Lastly, the LepRb-IRES-Cre reporter line was used to map the extent of peripheral leptin sensing by central nervous system (CNS) LepRb neurons. Thus, we provide data supporting the use of the LepRb-IRES-Cre line for the assessment of the anatomic and functional characteristics of neurons expressing leptin receptor.

Although the vinegar fly, Drosophila melanogaster, has been a biological model organism for over a century, its emergence as a model system for the study of neurophysiology is comparatively recent. The primary reason for this is that the vinegar fly and its neurons are tiny; up until 5 years ago, it was prohibitively difficult to record intracellularly from individual neurons in the intact Drosophila brain (Wilson et al., 2004). Today, fly electrophysiologists can genetically label neurons with GFP and reliably record from many (but not all) neurons in the fruit fly brain. Using genetic tools to drive expression of fluorescent calcium indicators, light-sensitive ion channels, or cell activity suppressors, we are beginning to understand how the external environment is represented with electrical potentials in Drosophila neurons (for review, see Olsen and Wilson, 2008).

Two-dimensional dispersions of colloidal particles with a range of surface chemistries and electrostatic potentials are characterized under a series of solution ionic strengths. A combination of optical imaging techniques are employed to monitor both the colloid structure and the electrostatic surface potential of individual particles in situ. We find that like-charge multiparticle interactions can be tuned from exclusively repulsive to long-range attractive by changing the particle surface composition. This behavior is strongly asymmetric with respect to the sign of the surface potential. Collective long-range attractive interactions are only observed among negatively charged particles.

Electron crystallography is arguably the only electron cryomicroscopy (cryoEM) technique able to deliver an atomic-resolution structure of membrane proteins embedded in the lipid bilayer. In the electron crystallographic structures of the light driven ion pump, bacteriorhodopsin, and the water channel, aquaporin-0, sufficiently high resolution was obtained and both lipid and protein were visualized, modeled, and described in detail. An extensive network of lipid-protein interactions mimicking native membranes is established and maintained in two-dimensional (2D) crystalline vesicles used for structural analysis by electron crystallography. Lipids are tightly integrated into the protein’s architecture where they can affect the function, structure, quaternary assembly, and the stability of the membrane protein.