Data Exploration, Quality Control and Testing in Single-Cell qPCR-Based Gene Expression Experiments

Cell populations are never truly homogeneous; individual cells exist in biochemical states that define functional differences between them. New technology based on microfluidic arrays combined with multiplexed quantitative polymerase chain reactions (qPCR) now enables high-throughput single-cell gene expression measurement, allowing assessment of cellular heterogeneity. However very little analytic tools have been developed specifically for the statistical and analytical challenges of single-cell qPCR data. We present a statistical framework for the exploration, quality control, and analysis of single-cell gene expression data from microfluidic arrays. We assess accuracy and within-sample heterogeneity of single-cell expression and develop quality control criteria to filter unreliable cell measurements. We propose a statistical model accounting for the fact that genes at the single-cell level can be on (and for which a continuous expression measure is recorded) or dichotomously off (and the recorded expression is zero). Based on this model, we derive a combined likelihood-ratio test for differential expression that incorporates both the discrete and continuous components. Using an experiment that examines treatment-specific changes in expression, we show that this combined test is more powerful than either the continuous or dichotomous component in isolation, or a t-test on the zero-inflated data. While developed for measurements from a specific platform (Fluidigm), these tools are generalizable to other multi-parametric measures over large numbers of events.Comment: 9 pages, 5 figure

A metagenomic assessment of the bacteria associated with Lucilia sericata and Lucilia cuprina (Diptera: Calliphoridae)

Lucilia Robineau-Desvoidy (Diptera: Calliphoridae) is a blow fly genus of forensic, medical, veterinary, and agricultural importance. This genus is also famous because of its beneficial uses in maggot debridement therapy (MDT). Although the genus is of considerable economic importance, our knowledge about microbes associated with these flies and how these bacteria are horizontally and trans-generationally transmitted is limited. In this study, we characterized bacteria associated with different life stages of Lucilia sericata (Meigen) and Lucilia cuprina(Wiedemann) and in the salivary gland of L. sericata by using 16S rDNA 454 pyrosequencing. Bacteria associated with the salivary gland of L. sericata were also characterized using light and transmission electron microscopy (TEM). Results from this study suggest that the majority of bacteria associated with these flies belong to phyla Proteobacteria, Firmicutes, andBacteroidetes, and most bacteria are maintained intragenerationally, with a considerable degree of turnover from generation to generation. In both species, second-generation eggs exhibited the highest bacterial phylum diversity (20 % genetic distance) than other life stages. The Lucilia sister species shared the majority of their classified genera. Of the shared bacterial genera, Providencia, Ignatzschineria, Lactobacillus, Lactococcus, Vagococcus, Morganella, and Myroides were present at relatively high abundances. Lactobacillus, Proteus,Diaphorobacter, and Morganella were the dominant bacterial genera associated with a survey of the salivary gland of L. sericata. TEM analysis showed a sparse distribution of both Gram-positive and Gram-negative bacteria in the salivary gland of L. sericata. There was more evidence for horizontal transmission of bacteria than there was for trans-generational inheritance. Several pathogenic genera were either amplified or reduced by the larval feeding on decomposing liver as a resource. Overall, this study provides information on bacterial communities associated with different life stages of Lucilia and their horizontal and trans-generational transmission, which may help in the development of better vector-borne disease management and MDT methods

Charge Fluctuations in the Edge States of N-S hybrid Nano-Structures

In this work we show how to calculate the equilibrium and non-equilibrium charge fluctuations in a gated normal mesoscopic conductor which is attached to one normal lead and one superconducting lead. We then consider an example where the structure is placed in a high magnetic field, such that the transport is dominated by edge states. We calculate the equilibrium and non-equilibrium charge fluctuations in the gate, for a single edge state, comparing our results to those for the same system, but with two normal leads. We then consider the specific example of a quantum point contact and calculate the charge fluctuations in the gate for more than one edge state.Comment: 4 pages with 1 figure. In published version the high magnetic field dynamics of the holes is treated incorrectly. An erratum is in preparatio

Broadly conserved roles of TMEM131 family proteins in intracellular collagen assembly and secretory cargo trafficking.

Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, we identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. We find that amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. We provide evidence that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans

In silico and in vitro drug screening identifies new therapeutic approaches for Ewing sarcoma.

The long-term overall survival of Ewing sarcoma (EWS) patients remains poor; less than 30% of patients with metastatic or recurrent disease survive despite aggressive combinations of chemotherapy, radiation and surgery. To identify new therapeutic options, we employed a multi-pronged approach using in silico predictions of drug activity via an integrated bioinformatics approach in parallel with an in vitro screen of FDA-approved drugs. Twenty-seven drugs and forty-six drugs were identified, respectively, to have anti-proliferative effects for EWS, including several classes of drugs in both screening approaches. Among these drugs, 30 were extensively validated as mono-therapeutic agents and 9 in 14 various combinations in vitro. Two drugs, auranofin, a thioredoxin reductase inhibitor, and ganetespib, an HSP90 inhibitor, were predicted to have anti-cancer activities in silico and were confirmed active across a panel of genetically diverse EWS cells. When given in combination, the survival rate in vivo was superior compared to auranofin or ganetespib alone. Importantly, extensive formulations, dose tolerance, and pharmacokinetics studies demonstrated that auranofin requires alternative delivery routes to achieve therapeutically effective levels of the gold compound. These combined screening approaches provide a rapid means to identify new treatment options for patients with a rare and often-fatal disease

The Role of Socioemotional Wealth in Entrepreneurial Persistence Decisions for Family Businesses

Many factors may influence entrepreneurial persistence in various contexts. For example, scholars find that family business entrepreneurs are more persistent than other entrepreneurs. However, the reasons why they are more persistent are not as well known. Utilizing a conjoint experiment with 64 entrepreneurs and 376 decisions, this paper examines the influence of socioemotional wealth (SEW) on persistence decisions in a family business context. The results of the Hierarchical Linear Modelling show that the expected financial returns, expected non-financial benefits, expected switching costs, and probability of expected outcomes influence entrepreneurial persistence decisions. Further, family business entrepreneurs with higher levels of SEW focus more on non-financial benefits when facing alternative opportunities. This study also provided empirical evidence for different dimensions of SEW. The results show that the emotional attachment of family members and the renewal of family bonds to the firm are effective indicators, which provide a direct measurement of SEW. The findings of this study increase scholarly understanding of both entrepreneurial persistence literature founded in threshold theory and SEW literature

Magnetic resonance imaging detects significant sex differences in human myocardial strain

<p>Abstract</p> <p>Background</p> <p>The pathophysiology responsible for the significant outcome disparities between men and women with cardiac disease is largely unknown. Further investigation into basic cardiac physiological differences between the sexes is needed. This study utilized magnetic resonance imaging (MRI)-based multiparametric strain analysis to search for sex-based differences in regional myocardial contractile function.</p> <p>Methods</p> <p>End-systolic strain (circumferential, longitudinal, and radial) was interpolated from MRI-based radiofrequency tissue tagging grid point displacements in each of 60 normal adult volunteers (32 females).</p> <p>Results</p> <p>The average global left ventricular (LV) strain among normal female volunteers (n = 32) was significantly larger in absolute value (functionally better) than in normal male volunteers (n = 28) in both the circumferential direction (Male/Female = -0.19 ± 0.02 vs. -0.21 ± 0.02; p = 0.025) and longitudinal direction (Male/Female = -0.14 ± 0.03 vs. -0.16 ± 0.02; p = 0.007).</p> <p>Conclusions</p> <p>The finding of significantly larger circumferential and longitudinal LV strain among normal female volunteers suggests that baseline contractile differences between the sexes may contribute to the well-recognized divergence in cardiovascular disease outcomes. Further work is needed in order to determine the pathologic changes that occur in LV strain between women and men with the onset of cardiovascular disease.</p

Comparative Distribution of Relaxin-3 Inputs and Calcium-Binding Protein-Positive Neurons in Rat Amygdala

The neural circuits involved in mediating complex behaviors are being rapidly elucidated using various newly developed and powerful anatomical and molecular techniques, providing insights into the neural basis for anxiety disorders, depression, addiction, and dysfunctional social behaviors. Many of these behaviors and associated physiological processes involve the activation of the amygdala in conjunction with cortical and hippocampal circuits. Ascending subcortical projections provide modulatory inputs to the extended amygdala and its related nodes (or ‘hubs’) within these key circuits. One such input arises from the nucleus incertus (NI) in the tegmentum, which sends amino acid- and peptide-containing projections throughout the forebrain. Notably, a distinct population of GABAergic NI neurons expresses the highly-conserved neuropeptide, relaxin-3, and relaxin-3 signaling has been implicated in the modulation of reward/motivation and anxiety- and depressive-like behaviors in rodents via actions within the extended amygdala. Thus, a detailed description of the relaxin-3 innervation of the extended amygdala would provide an anatomical framework for an improved understanding of NI and relaxin-3 modulation of these and other specific amygdala-related functions. Therefore, in this study, we examined the distribution of NI projections and relaxin-3-positive elements (axons/fibers/terminals) within the amygdala, relative to the distribution of neurons expressing the calcium-binding proteins, parvalbumin, calretinin and/or calbindin. Anterograde tracer injections into the NI revealed a topographic distribution of NI efferents within the amygdala that was near identical to the distribution of relaxin-3-immunoreactive fibers. Highest densities of anterogradely-labeled elements and relaxin-3-immunoreactive fibers were observed in the medial nucleus of the amygdala, medial divisions of the bed nucleus of the stria terminalis (BST) and in the endopiriform nucleus. In contrast, sparse anterogradely-labeled and relaxin-3-immunoreactive fibers were observed in other amygdala nuclei, including the lateral, central and basal nuclei and the nucleus accumbens lacked any innervation. Using synaptophysin as a synaptic marker, we identified relaxin-3 positive synaptic terminals in the medial amygdala, BST and endopiriform nucleus of amygdala. Our findings demonstrate the existence of topographic NI and relaxin-3-containing projections to specific nuclei of the extended amygdala, consistent with a likely role for this putative integrative arousal system in the regulation of amygdala-dependent social and emotional behaviors