Gradient microfluidics enables rapid bacterial growth inhibition testing

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask)

High spatial resolution imaging of methane and other trace gases with the airborne Hyperspectral Thermal Emission Spectrometer (HyTES)

Currently large uncertainties exist associated with the attribution and quantification of fugitive emissions of criteria pollutants and greenhouse gases such as methane across large regions and key economic sectors. In this study, data from the airborne Hyperspectral Thermal Emission Spectrometer (HyTES) have been used to develop robust and reliable techniques for the detection and wide-area mapping of emission plumes of methane and other atmospheric trace gas species over challenging and diverse environmental conditions with high spatial resolution that permits direct attribution to sources. HyTES is a pushbroom imaging spectrometer with high spectral resolution (256 bands from 7.5 to 12 µm), wide swath (1–2 km), and high spatial resolution (∼ 2 m at 1 km altitude) that incorporates new thermal infrared (TIR) remote sensing technologies. In this study we introduce a hybrid clutter matched filter (CMF) and plume dilation algorithm applied to HyTES observations to efficiently detect and characterize the spatial structures of individual plumes of CH4, H2S, NH3, NO2, and SO2 emitters. The sensitivity and field of regard of HyTES allows rapid and frequent airborne surveys of large areas including facilities not readily accessible from the surface. The HyTES CMF algorithm produces plume intensity images of methane and other gases from strong emission sources. The combination of high spatial resolution and multi-species imaging capability provides source attribution in complex environments. The CMF-based detection of strong emission sources over large areas is a fast and powerful tool needed to focus on more computationally intensive retrieval algorithms to quantify emissions with error estimates, and is useful for expediting mitigation efforts and addressing critical science questions

Evidence for core exosome independent function of the nuclear exoribonuclease Rrp6p

The RNA exosome processes and degrades RNAs in archaeal and eukaryotic cells. Exosomes from yeast and humans contain two active exoribonuclease components, Rrp6p and Dis3p/Rrp44p. Rrp6p is concentrated in the nucleus and the dependence of its function on the nine-subunit core exosome and Dis3p remains unclear. We found that cells lacking Rrp6p accumulate poly(A)+ rRNA degradation intermediates distinct from those found in cells depleted of Dis3p, or the core exosome component Rrp43p. Depletion of Dis3p in the absence of Rrp6p causes a synergistic increase in the levels of degradation substrates common to the core exosome and Rrp6p, but has no effect on Rrp6p-specific substrates. Rrp6p lacking a portion of its C-terminal domain no longer co-purifies with the core exosome, but continues to carry out RNA 3′-end processing of 5.8S rRNA and snoRNAs, as well as the degradation of certain truncated Rrp6-specific rRNA intermediates. However, disruption of Rrp6p–core exosome interaction results in the inability of the cell to efficiently degrade certain poly(A)+ rRNA processing products that require the combined activities of Dis3p and Rrp6p. These findings indicate that Rrp6p may carry out some of its critical functions without physical association with the core exosome

Genome-wide search for breast cancer linkage in large Icelandic non-BRCA1/2 families

Abstract Introduction: A significant proportion of high-risk breast cancer families are not explained by mutations in known genes. Recent genome-wide searches (GWS) have not revealed any single major locus reminiscent of BRCA1 and BRCA2, indicating that still unidentified genes may explain relatively few families each or interact in a way obscure to linkage analyses. This has drawn attention to possible benefits of studying populations where genetic heterogeneity might be reduced. We thus performed a GWS for linkage on nine Icelandic multiple-case non-BRCA1/2 families of desirable size for mapping highly penetrant loci. To follow up suggestive loci, an additional 13 families from other Nordic countries were genotyped for selected markers. Methods: GWS was performed using 811 microsatellite markers providing about five centiMorgan (cM) resolution. Multipoint logarithm of odds (LOD) scores were calculated using parametric and nonparametric methods. For selected markers and cases, tumour tissue was compared to normal tissue to look for allelic loss indicative of a tumour suppressor gene. Results: The three highest signals were located at chromosomes 6q, 2p and 14q. One family contributed suggestive LOD scores (LOD 2.63 to 3.03, dominant model) at all these regions, without consistent evidence of a tumour suppressor gene. Haplotypes in nine affected family members mapped the loci to 2p23.2 to p21, 6q14.2 to q23.2 and 14q21.3 to q24.3. No evidence of a highly penetrant locus was found among the remaining families. The heterogeneity LOD (HLOD) at the 6q, 2p and 14q loci in all families was 3.27, 1.66 and 1.24, respectively. The subset of 13 Nordic families showed supportive HLODs at chromosome 6q (ranging from 0.34 to 1.37 by country subset). The 2p and 14q loci overlap with regions indicated by large families in previous GWS studies of breast cancer. Conclusions: Chromosomes 2p, 6q and 14q are candidate sites for genes contributing together to high breast cancer risk. A polygenic model is supported, suggesting the joint effect of genes in contributing to breast cancer risk to be rather common in non-BRCA1/2 families. For genetic counselling it would seem important to resolve the mode of genetic interaction

The Effect of Viewing Eccentricity on Enumeration

Visual acuity and contrast sensitivity progressively diminish with increasing viewing eccentricity. Here we evaluated how visual enumeration is affected by visual eccentricity, and whether subitizing capacity, the accurate enumeration of a small number (∼3) of items, decreases with more eccentric viewing. Participants enumerated gratings whose (1) stimulus size was constant across eccentricity, and (2) whose stimulus size scaled by a cortical magnification factor across eccentricity. While we found that enumeration accuracy and precision decreased with increasing eccentricity, cortical magnification scaling of size neutralized the deleterious effects of increasing eccentricity. We found that size scaling did not affect subitizing capacities, which were nearly constant across all eccentricities. We also found that size scaling modulated the variation coefficients, a normalized metric of enumeration precision, defined as the standard deviation divided by the mean response. Our results show that the inaccuracy and imprecision associated with increasing viewing eccentricity is due to limitations in spatial resolution. Moreover, our results also support the notion that the precise number system is restricted to small numerosities (represented by the subitizing limit), while the approximate number system extends across both small and large numerosities (indexed by variation coefficients) at large eccentricities

HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-κB activation through the ubiquitin-dependent activation of IKKγ. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-κB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling

The Essential Nucleolar Yeast Protein Nop8p Controls the Exosome Function during 60S Ribosomal Subunit Maturation

The yeast nucleolar protein Nop8p has previously been shown to interact with Nip7p and to be required for 60S ribosomal subunit formation. Although depletion of Nop8p in yeast cells leads to premature degradation of rRNAs, the biochemical mechanism responsible for this phenotype is still not known. In this work, we show that the Nop8p amino-terminal region mediates interaction with the 5.8S rRNA, while its carboxyl-terminal portion interacts with Nip7p and can partially complement the growth defect of the conditional mutant strain Δnop8/GAL::NOP8. Interestingly, Nop8p mediates association of Nip7p to pre-ribosomal particles. Nop8p also interacts with the exosome subunit Rrp6p and inhibits the complex activity in vitro, suggesting that the decrease in 60S ribosomal subunit levels detected upon depletion of Nop8p may result from degradation of pre-rRNAs by the exosome. These results strongly indicate that Nop8p may control the exosome function during pre-rRNA processing

Embedding mRNA Stability in Correlation Analysis of Time-Series Gene Expression Data

Current methods for the identification of putatively co-regulated genes directly from gene expression time profiles are based on the similarity of the time profile. Such association metrics, despite their central role in gene network inference and machine learning, have largely ignored the impact of dynamics or variation in mRNA stability. Here we introduce a simple, but powerful, new similarity metric called lead-lag R2 that successfully accounts for the properties of gene dynamics, including varying mRNA degradation and delays. Using yeast cell-cycle time-series gene expression data, we demonstrate that the predictive power of lead-lag R2 for the identification of co-regulated genes is significantly higher than that of standard similarity measures, thus allowing the selection of a large number of entirely new putatively co-regulated genes. Furthermore, the lead-lag metric can also be used to uncover the relationship between gene expression time-series and the dynamics of formation of multiple protein complexes. Remarkably, we found a high lead-lag R2 value among genes coding for a transient complex