The Good,the Bad,and the Ugly: in search of gold standards for assessing functional genetic screen quality

Variable screen quality, off‐target effects, and unclear false discovery rates often hamper large‐scale functional genomic screens in mammalian cells. Hart et al (2014) introduce gold standard reference sets of essential and non‐essential genes, aiming at standardizing the analysis of genome‐wide screens. This work provides a framework to compare both the quality and analysis methods of functional genetic screens.     

SlideToolkit: An Assistive Toolset for the Histological Quantification of Whole Slide Images

The demand for accurate and reproducible phenotyping of a disease trait increases with the rising number of biobanks and genome wide association studies. Detailed analysis of histology is a powerful way of phenotyping human tissues. Nonetheless, purely visual assessment of histological slides is time-consuming and liable to sampling variation and optical illusions and thereby observer variation, and external validation may be cumbersome. Therefore, within our own biobank, computerized quantification of digitized histological slides is often preferred as a more precise and reproducible, and sometimes more sensitive approach. Relatively few free toolkits are, however, available for fully digitized microscopic slides, usually known as whole slides images. In order to comply with this need, we developed the slideToolkit as a fast method to handle large quantities of low contrast whole slides images using advanced cell detecting algorithms. The slideToolkit has been developed for modern personal computers and high-performance clusters (HPCs) and is available as an open-source project on github.com. We here illustrate the power of slideToolkit by a repeated measurement of 303 digital slides containing CD3 stained (DAB) abdominal aortic aneurysm tissue from a tissue biobank. Our workflow consists of four consecutive steps. In the first step (acquisition), whole slide images are collected and converted to TIFF files. In the second step (preparation), files are organized. The third step (tiles), creates multiple manageable tiles to count. In the fourth step (analysis), tissue is analyzed and results are stored in a data set. Using this method, two consecutive measurements of 303 slides showed an intraclass correlation of 0.99. In conclusion, slideToolkit provides a free, powerful and versatile collection of tools for automated feature analysis of whole slide images to create reproducible and meaningful phenotypic data sets.     

Irrational time allocation in decision-making

Time is an extremely valuable resource but little is known about the efficiency of time allocation in decision-making. Empirical evidence suggests that in many ecologically relevant situations, decision difficulty and the relative reward from making a correct choice, compared to an incorrect one, are inversely linked, implying that it is optimal to use relatively less time for difficult choice problems. This applies, in particular, to value-based choices, in which the relative reward from choosing the higher valued item shrinks as the values of the other options get closer to the best option and are thus more difficult to discriminate. Here, we experimentally show that people behave sub-optimally in such contexts. They do not respond to incentives that favour the allocation of time to choice problems in which the relative reward for choosing the best option is high; instead they spend too much time on problems in which the reward difference between the options is low. We demonstrate this by showing that it is possible to improve subjects'' time allocation with a simple intervention that cuts them off when their decisions take too long. Thus, we provide a novel form of evidence that organisms systematically spend their valuable time in an inefficient way, and simultaneously offer a potential solution to the problem.     

Assessment of Myocardial Fibrosis in Mice Using a T2*-Weighted 3D Radial Magnetic Resonance Imaging Sequence

Background

Myocardial fibrosis is a common hallmark of many diseases of the heart. Late gadolinium enhanced MRI is a powerful tool to image replacement fibrosis after myocardial infarction (MI). Interstitial fibrosis can be assessed indirectly from an extracellular volume fraction measurement using contrast-enhanced T1 mapping. Detection of short T2* species resulting from fibrotic tissue may provide an attractive non-contrast-enhanced alternative to directly visualize the presence of both replacement and interstitial fibrosis.

Objective

To goal of this paper was to explore the use of a T2*-weighted radial sequence for the visualization of fibrosis in mouse heart.

Methods

C57BL/6 mice were studied with MI (n = 20, replacement fibrosis), transverse aortic constriction (TAC) (n = 18, diffuse fibrosis), and as control (n = 10). 3D center-out radial T2*-weighted images with varying TE were acquired in vivo and ex vivo (TE = 21 μs-4 ms). Ex vivo T2*-weighted signal decay with TE was analyzed using a 3-component model. Subtraction of short- and long-TE images was used to highlight fibrotic tissue with short T2*. The presence of fibrosis was validated using histology and correlated to MRI findings.

Results

Detailed ex vivo T2*-weighted signal analysis revealed a fast (T2*_fast), slow (T2*_slow) and lipid (T2*_lipid) pool. T2*_fast remained essentially constant. Infarct T2*_slow decreased significantly, while a moderate decrease was observed in remote tissue in post-MI hearts and in TAC hearts. T2*_slow correlated with the presence of diffuse fibrosis in TAC hearts (r = 0.82, P = 0.01). Ex vivo and in vivo subtraction images depicted a positive contrast in the infarct co-localizing with the scar. Infarct volumes from histology and subtraction images linearly correlated (r = 0.94, P<0.001). Region-of-interest analysis in the in vivo post-MI and TAC hearts revealed significant T2* shortening due to fibrosis, in agreement with the ex vivo results. However, in vivo contrast on subtraction images was rather poor, hampering a straightforward visual assessment of the spatial distribution of the fibrotic tissue.     

Effects of corticosteroid treatment on airway inflammation, mechanics, and hyperpolarized ³He magnetic resonance imaging in an allergic mouse model

The purpose of this study was to assess the effects of corticosteroid therapy on a murine model of allergic asthma using hyperpolarized (3)He magnetic resonance imaging (MRI) and respiratory mechanics measurements before, during, and after methacholine (MCh) challenge. Three groups of mice were prepared, consisting of ovalbumin sensitized/ovalbumin challenged (Ova/Ova, n = 5), Ova/Ova challenged but treated with the corticosteroid dexamethasone (Ova/Ova+Dex, n = 3), and ovalbumin-sensitized/saline-challenged (Ova/PBS, n = 4) control animals. All mice underwent baseline 3D (3)He MRI, then received a MCh challenge while 10 2D (3)He MR images were acquired for 2 min, followed by post-MCh 3D (3)He MRI. Identically treated groups underwent respiratory mechanics evaluation (n = 4/group) and inflammatory cell counts (n = 4/group). Ova/Ova animals exhibited predominantly large whole lobar defects at baseline, with significantly higher ventilation defect percentage (VDP = 19 ± 4%) than Ova/PBS (+2 ± 1%, P = 0.01) animals. Such baseline defects were suppressed by dexamethasone (0%, P = 0.009). In the Ova/Ova group, MCh challenge increased VDP on both 2D (+30 ± 8%) and 3D MRI scans (+14 ± 2%). MCh-induced VDP changes were diminished in Ova/Ova+Dex animals on both 2D (+21 ± 9%, P = 0.63) and 3D scans (+7 ± 2%, P = 0.11) and also in Ova/PBS animals on 2D (+6 ± 3%, P = 0.07) and 3D (+4 ± 1%, P = 0.01) scans. Because MCh challenge caused near complete cessation of ventilation in four of five Ova/Ova animals, even as large airways remained patent, this implies that small airway (<188 μm) obstruction predominates in this model. This corresponds with respiratory mechanics observations that MCh challenge significantly increases elastance and tissue damping but only modestly affects Newtonian airway resistance.     

Human Monoclonal Antibodies against West Nile Virus Induced by Natural Infection Neutralize at a Postattachment Step

West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.West Nile encephalitis virus (WNV) is a positive-polarity, single-stranded RNA virus of the genus Flavivirus within the family Flaviviridae. Other members of this genus that cause significant human disease include dengue virus (DENV), St. Louis encephalitis virus, Japanese encephalitis virus (JEV), yellow fever virus, and tick-borne encephalitis virus (TBEV). Flaviviruses are translated as a single polypeptide, which is then cleaved by host and viral proteases into three structural (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (reviewed in references 42 and 43).WNV cycles in nature between several species of birds and Culex mosquitoes, with humans and other mammals as dead-end hosts (25, 62). Infection causes syndromes ranging from a mild febrile illness to severe encephalitis and death (13, 72). WNV has spread globally and causes outbreaks with thousands of severe human cases annually in the United States. An age of greater than 55 years, a compromised immune status, and a CC5Δ32 genotype have been associated with more-severe disease (15, 20). There is currently no approved vaccine or therapy for WNV infection.The mature WNV virion has a ∼500-Å diameter and consists of a single RNA genome surrounded by the capsid protein, a lipid bilayer, and a shell of the prM/M and E proteins (31, 55). X-ray crystallography studies have elucidated the three-domain structure of the flavivirus E protein (30, 48, 50, 58, 67). Domain I (DI) is a central, eight-stranded β-barrel, which contains the only N-linked glycosylation site in WNV E. Domain II (DII) is a long, finger-like protrusion from DI and contains the highly conserved fusion peptide at its distal end. Domain III (DIII) adopts an immunoglobulin-like fold at the opposite end of DI and is believed to contain a site for receptor attachment (6, 8, 40).Within an infected cell, progeny WNV are assembled initially as immature particles. In immature virions, three pairs of E and prM interact as trimers and form 60 spiked projections with icosahedral symmetry (85, 86). Exposure to mildly acidic conditions in the trans-Golgi secretory pathway promotes virus maturation through a structural rearrangement of the E proteins and cleavage of prM to M by a furin-like protease (41, 83). Mature WNV virions are covered by 90 antiparallel E protein homodimers, which are arranged flat along the surface in a herringbone pattern with quasi-icosahedral symmetry (55).Upon binding to poorly characterized cell surface receptors, internalization of WNV is believed to occur through receptor-mediated, clathrin-dependent endocytosis (1, 79, 80). After trafficking to Rab5- and/or Rab7-positive endosomes (38, 79), the mildly acidic pH within the lumen of the endosome induces structural alterations in the flavivirus E protein (7, 49), which includes changes in its oligomeric state (7, 49, 77). During this process, also known as type II fusion, the hydrophobic peptide on the fusion loop of DII of the E protein inserts into the endosomal membrane, thus physically joining the host and viral membranes, which allows the infectious RNA genome to enter the cytoplasm (32, 33).Humoral immunity is an essential component of the protective host response against flaviviruses including WNV (reviewed in references 64 and 68). Studies by several groups have shown that the neutralization of WNV can occur after antibodies bind to a series of discrete epitopes on all three domains of the E protein (3, 12, 22, 59, 61, 71). To date, the most potently neutralizing monoclonal antibodies (MAbs) localize to an epitope on the lateral ridge of DIII (DIII-lr). One well-characterized strongly neutralizing mouse MAb, E16, blocks infection primarily at a postattachment step (57) and requires the engagement of only a fraction of its epitopes on the surface of the virion (66). Studies of the human antibody response to WNV infection reveal that, in contrast to mice, antibodies that bind the DIII-lr epitope comprise a minor component of the neutralizing humoral response in most individuals (60).In this study, we characterized two strongly neutralizing novel human MAbs (CR4348 and CR4354) that were selected from an antibody phage display library constructed from B cells of subjects that survived WNV infection (78). We demonstrate that both MAbs are WNV specific, bind weakly to recombinant or yeast surface-displayed E proteins, exhibit pH-sensitive binding to viral particles, and protect against lethal infection in mice. Our experiments suggest that these human MAbs map to distinct epitopes and neutralize infection at a postattachment stage, likely by inhibiting the acid-catalyzed viral fusion step.     

Design and baseline characteristics of the ParkFit study,a randomized controlled trial evaluating the effectiveness of a multifaceted behavioral program to increase physical activity in Parkinson patients

Background  

Many patients with Parkinson's disease (PD) lead a sedentary lifestyle. Promotion of physical activities may beneficially affect the clinical presentation of PD, and perhaps even modify the course of PD. However, because of physical and cognitive impairments, patients with PD require specific support to increase their level of physical activity.     

<Emphasis Type="Italic">recA</Emphasis> mediated spontaneous deletions of the <Emphasis Type="Italic">icaADBC</Emphasis> operon of clinical <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> isolates: a new mechanism of phenotypic variations

Phenotypic variation of Staphylococcus epidermidis involving the slime related ica operon results in heterogeneity in surface characteristics of individual bacteria in axenic cultures. Five clinical S. epidermidis isolates demonstrated phenotypic variation, i.e. both black and red colonies on Congo Red agar. Black colonies displayed bi-modal electrophoretic mobility distributions at pH 2, but such phenotypic variation was absent in red colonies of the same strain as well as in control strains without phenotypic variation. All red colonies had lost ica and the ability to form biofilms, in contrast to black colonies of the same strain. Real time PCR targeting icaA indicated a reduction in gene copy number within cultures exhibiting phenotypic variation, which correlated with phenotypic variations in biofilm formation and electrophoretic mobility distribution of cells within a culture. Loss of ica was irreversible and independent of the mobile element IS256. Instead, in high frequency switching strains, spontaneous mutations in lexA were found which resulted in deregulation of recA expression, as shown by real time PCR. RecA is involved in genetic deletions and rearrangements and we postulate a model representing a new mechanism of phenotypic variation in clinical isolates of S. epidermidis. This is the first report of S. epidermidis strains irreversibly switching from biofilm-positive to biofilm-negative phenotype by spontaneous deletion of icaADBC.