Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins

Keck Interferometer Nuller Data Reduction and On-Sky Performance

We describe the Keck Interferometer nuller theory of operation, data reduction, and on-sky performance, particularly as it applies to the nuller exozodiacal dust key science program that was carried out between 2008 February and 2009 January. We review the nuller implementation, including the detailed phasor processing involved in implementing the null-peak mode used for science data and the sequencing used for science observing. We then describe the Level 1 reduction to convert the instrument telemetry streams to raw null leakages, and the Level 2 reduction to provide calibrated null leakages. The Level 1 reduction uses conservative, primarily linear processing, implemented consistently for science and calibrator stars. The Level 2 processing is more flexible, and uses diameters for the calibrator stars measured contemporaneously with the interferometer’s K-band cophasing system in order to provide the requisite accuracy. Using the key science data set of 462 total scans, we assess the instrument performance for sensitivity and systematic error. At 2.0 Jy we achieve a photometrically-limited null leakage uncertainty of 0.25% rms per 10 minutes of integration time in our broadband channel. From analysis of the Level 2 reductions, we estimate a systematic noise floor for bright stars of ~0.2% rms null leakage uncertainty per observing cluster in the broadband channel. A similar analysis is performed for the narrowband channels. We also provide additional information needed for science reduction, including details on the instrument beam pattern and the basic astrophysical response of the system, and references to the data reduction and modeling tools

Milliarcsecond N-Band Observations of the Nova RS Ophiuchi: First Science with the Keck Interferometer Nuller

We report observations of the nova RS Ophiuchi (RS Oph) using the Keck Interferometer Nuller (KIN), approximately 3.8 days following the most recent outburst that occurred on 2006 February 12. These observations represent the first scientific results from the KIN, which operates in N-band from 8 to 12.5 microns in a nulling mode. By fitting the unique KIN data, we have obtained an angular size of the mid-infrared continuum of 6.2, 4.0, or 5.4 mas for a disk profile, gaussian profile (FWHM), and shell profile respectively. The data show evidence of enhanced neutral atomic hydrogen emission and atomic metals including silicon located in the inner spatial regime near the white dwarf (WD) relative to the outer regime. There are also nebular emission lines and evidence of hot silicate dust in the outer spatial region, centered at ! 17 AU from the WD, that are not found in the inner regime. Our evidence suggests that these features have been excited by the nova flash in the outer spatial regime before the blast wave reached these regions. These identifications support a model in which the dust appears to be present between outbursts and is not created during the outburst event. We further discuss the present results in terms of a unifying model of the system that includes an increase in density in the plane of the orbit of the two stars created by a spiral shock wave caused by the motion of the stars through the cool wind of the red giant star. These data show the power and potential of the nulling technique which has been developed for the detection of Earth-like planets around nearby stars for the Terrestrial Planet Finder Mission and Darwin missions.Comment: 41 pages, 10 figure

Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer

BACKGROUND: Circulating tumour cells (CTCs) offer a non-invasive approach to obtain and characterise metastatic tumour cells, but their usefulness has been limited by low CTC yields from conventional isolation methods. METHODS: To improve CTC yields and facilitate their molecular characterisation we compared the Food and Drug Administration-approved CellSearch Epithelial Kit (CEK) to a simplified CTC capture method, CellSearch Profile Kit (CPK), on paired blood samples from patients with metastatic breast (n=75) and lung (n=71) cancer. Molecular markers including Human Epidermal growth factor Receptor 2 (HER2) were evaluated on CTCs by fluorescence in situ hybridisation (FISH) and compared to patients' primary and metastatic cancer. RESULTS: The median cell count from patients with breast cancer using the CPK was 117 vs 4 for CEK (P<0.0001). Lung cancer samples were similar; CPK: 145 cells vs CEK:4 cells (P<0.0001). Recovered CTCs were relatively pure (60-70%) and were evaluable by FISH and immunofluorescence. A total of 10 of 30 (33%) breast cancer patients with HER2-negative primary and metastatic tissue had HER2-amplified CTCs. CONCLUSION: The CPK method provides a high yield of relatively pure CTCs, facilitating their molecular characterisation. Circulating tumour cells obtained using CPK technology demonstrate that significant discordance exists between HER2 amplification of a patient's CTCs and that of the primary and metastatic tumour

The dusty AGB star RS CrB: first mid-infrared interferometric observations with the Keck Telescopes

We report interferometric observations of the semi-regular variable star RS CrB, a red giant with strong silicate emission features. The data were among the first long baseline mid-infrared stellar fringes obtained between the Keck telescopes, using parts of the new nulling beam combiner. The light was dispersed by a low-resolution spectrometer, allowing simultaneous measurement of the source visibility and intensity spectra from 8 to 12 microns. The interferometric observations allow a non-ambiguous determination of the dust shell spatial scale and relative flux contribution. Using a simple spherically-symmetric model, in which a geometrically thin shell surrounds the stellar photosphere, we find that ~30% to ~70% of the overall mid-infrared flux - depending on the wavelength - originates from 7-8 stellar radii. The derived shell opacity profile shows a broad peak around 11 microns (tau ~ 0.06), characteristic of Mg-rich silicate dust particles.Comment: Accepted for publication in ApJ Letter

The near-infrared size-luminosity relations for Herbig Ae/Be disks

We report the results of a sensitive K-band survey of Herbig Ae/Be disk sizes using the 85-m baseline Keck Interferometer. Targets were chosen to span the maximum range of stellar properties to probe the disk size dependence on luminosity and effective temperature. For most targets, the measured near-infrared sizes (ranging from 0.2 to 4 AU) support a simple disk model possessing a central optically-thin (dust-free) cavity, ringed by hot dust emitting at the expected sublimation temperatures (T_sub~1000-1500K). Furthermore, we find a tight correlation of disk size with source luminosity R propto L^(1/2) for Ae and late Be systems (valid over more than 2 decades in luminosity), confirming earlier suggestions based on lower-quality data. Interestingly, the inferred dust-free inner cavities of the highest luminosity sources (Herbig B0-B3 stars) are under-sized compared to predictions of the optically-thin cavity model, likely due to optically-thick gas within the inner AU.Comment: Accepted by Astrophysical Journal; 24 pages, 4 figures, 4 table

Id4 and FABP7 are preferentially expressed in cells with astrocytic features in oligodendrogliomas and oligoastrocytomas

BACKGROUND: Oligodendroglioma (ODG) and oligoastrocytoma (OAC) are diffusely infiltrating primary brain tumors whose pathogenesis remains unclear. We previously identified a group of genes whose expression was inversely correlated with survival in a cohort of patients with glioblastoma (GBM), and some of these genes are also reportedly expressed in ODG and OAC. We examined the expression patterns and localization of these survival-associated genes in ODG and OAC in order to analyze their possible roles in the oncogenesis of these two tumor types. METHODS: We used UniGene libraries derived from GBM and ODG specimens to examine the expression levels of the transcripts for each of the 50 GBM survival-associated genes. We used immunohistochemistry and cDNA microarrays to examine expression of selected survival-associated genes and Id4, a gene believed to control the timing of oligodendrocyte development. The expression of FABP7 and Id4 and the survival of patients with ODG and OAC were also analyzed. RESULTS: Transcripts of most survival-associated genes as well as Id4 were present in both GBM and ODG tumors, whereas protein expression of Id4 and one of the survival-associated genes, brain-type fatty acid-binding protein (FABP7), was present in cells with astrocytic features, including reactive and neoplastic astrocytes, but not in neoplastic oligodendrocytes. Id4 was co-expressed with FABP7 in microgemistocytes in ODG and in neoplastic astrocytes in OAC. Id4 and FABP7 expression, however, did not correlate with the clinical outcome of patients with ODG or OAC tumors. CONCLUSION: Expression of Id4 and some of our previously identified GBM survival-associated genes is present in developing or mature oligodendrocytes. However, protein expression of Id4 and FABP7 in GBM, ODG, and OAC suggests that this group of functionally important genes might demonstrate two patterns of expression in these glioma subtypes: one group is universally expressed in glioma cells, and the other group of genes is expressed primarily in neoplastic astrocytes but not in neoplastic oligodendrocytes. Differential protein expression of these two groups of genes in ODG and OAC may be related to the cellular origins and the histological features of the neoplastic cells

Host Reproductive Phenology Drives Seasonal Patterns of Host Use in Mosquitoes

Seasonal shifts in host use by mosquitoes from birds to mammals drive the timing and intensity of annual epidemics of mosquito-borne viruses, such as West Nile virus, in North America. The biological mechanism underlying these shifts has been a matter of debate, with hypotheses falling into two camps: (1) the shift is driven by changes in host abundance, or (2) the shift is driven by seasonal changes in the foraging behavior of mosquitoes. Here we explored the idea that seasonal changes in host use by mosquitoes are driven by temporal patterns of host reproduction. We investigated the relationship between seasonal patterns of host use by mosquitoes and host reproductive phenology by examining a seven-year dataset of blood meal identifications from a site in Tuskegee National Forest, Alabama USA and data on reproduction from the most commonly utilized endothermic (white-tailed deer, great blue heron, yellow-crowned night heron) and ectothermic (frogs) hosts. Our analysis revealed that feeding on each host peaked during periods of reproductive activity. Specifically, mosquitoes utilized herons in the spring and early summer, during periods of peak nest occupancy, whereas deer were fed upon most during the late summer and fall, the period corresponding to the peak in births for deer. For frogs, however, feeding on early- and late-season breeders paralleled peaks in male vocalization. We demonstrate for the first time that seasonal patterns of host use by mosquitoes track the reproductive phenology of the hosts. Peaks in relative mosquito feeding on each host during reproductive phases are likely the result of increased tolerance and decreased vigilance to attacking mosquitoes by nestlings and brooding adults (avian hosts), quiescent young (avian and mammalian hosts), and mate-seeking males (frogs)

PLEKHA7 Is an Adherens Junction Protein with a Tissue Distribution and Subcellular Localization Distinct from ZO-1 and E-Cadherin

The pleckstrin-homology-domain-containing protein PLEKHA7 was recently identified as a protein linking the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Here we characterize the expression, tissue distribution and subcellular localization of PLEKHA7 by immunoblotting, immunofluorescence microscopy, immunoelectron microscopy, and northern blotting in mammalian tissues. Anti-PLEKHA7 antibodies label the junctional regions of cultured kidney epithelial cells by immunofluorescence microscopy, and major polypeptides of Mr ∼135 kDa and ∼145 kDa by immunoblotting of lysates of cells and tissues. Two PLEKHA7 transcripts (∼5.5 kb and ∼6.5 kb) are detected in epithelial tissues. PLEKHA7 is detected at epithelial junctions in sections of kidney, liver, pancreas, intestine, retina, and cornea, and its tissue distribution and subcellular localization are distinct from ZO-1. For example, PLEKHA7 is not detected within kidney glomeruli. Similarly to E-cadherin, p120 ctn, β-catenin and α-catenin, PLEKHA7 is concentrated in the apical junctional belt, but unlike these adherens junction markers, and similarly to afadin, PLEKHA7 is not localized along the lateral region of polarized epithelial cells. Immunoelectron microscopy definitively establishes that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of 28 nm from the plasma membrane. In summary, we show that PLEKHA7 is a cytoplasmic component of the epithelial adherens junction belt, with a subcellular localization and tissue distribution that is distinct from that of ZO-1 and most AJ proteins, and we provide the first description of its distribution and localization in several tissues