Next-Generation cDNA Screening for Oncogene and Resistance Phenotypes

There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo

Lorecivivint, a Novel Intraarticular CDC‐like Kinase 2 and Dual‐Specificity Tyrosine Phosphorylation‐Regulated Kinase 1A Inhibitor and Wnt Pathway Modulator for the Treatment of Knee Osteoarthritis: A Phase II Randomized Trial

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162805/2/art41315_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162805/1/art41315.pd

Quaternary structure of a G-protein coupled receptor heterotetramer in complex with Gi and Gs

Background: G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. Results: We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. Conclusions: The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function

The Impact of Manuscript Learning vs. Video Learning on a Surgeon's Confidence in Performing a Difficult Procedure

Introduction: Operative surgical videos are a popular educational resource, not commonly a part of a peer-reviewed article. We wanted to evaluate the impact of either reading a peer-reviewed manuscript or watching an operative video on a surgeon's confidence in performing a complex case.Methods: Pediatric surgeons and fellows were asked to complete an initial questionnaire to assess their confidence (formulated as a score) in the diagnosis and operative repair of anal stenosis and rectal atresia.Results: Of 101 pediatric surgeons and fellows, 52 (51%) were randomized into a “manuscript” group and 49 (49%) into a “video” group. The mean confidence before the intervention was the same in the two groups (6.4 vs. 6.6). Attending surgeons started with more confidence than trainees (7.1 vs. 5.3, p < 0.001). In the manuscript group, the average confidence increased to 7.7 (p = 0.005), and in the video group the average confidence increased to 7.9 (p = 0.001) globally. Trainees in the video group significantly improved their confidence to a score of 6.6 (p = 0.035), as did attending surgeons to 8.5 (p = 0.01). In the manuscript group, only attendings significantly improved their confidence by 1.5–8.3 (p < 0.001), whereas trainees did not with a difference of 1.3 (p = 0.194). When considering experience level, physicians who reported never having performed this surgery improved only by reading the manuscript (3.9–6.2) (p = 0.004), not by watching the video (5.4–6.6) (p = 0.106). Surgeons with experience doing this operation (>5 times) did not improve their confidence by reading the manuscript (p = 0.10), nor by watching the video (p = 0.112).Conclusion: Reviewing either a detailed manuscript or operative video on the surgical management of rectal atresia and anal stenosis demonstrated a significant increase in self-reported confidence. Trainees benefitted the most from operative videos, whereas experienced surgeons did not improve their confidence by reading the manuscript nor watching the video

Darwin -— an experimental astronomy mission to search for extrasolar planets

As a response to ESA call for mission concepts for its Cosmic Vision 2015–2025 plan, we propose a mission called Darwin. Its primary goal is the study of terrestrial extrasolar planets and the search for life on them. In this paper, we describe different characteristics of the instrument

Low-dose TNF augments fracture healing in normal and osteoporotic bone by up-regulating the innate immune response

The mechanism by which trauma initiates healing remains unclear. Precise understanding of these events may define interventions for accelerating healing that could be translated to the clinical arena. We previously reported that addition of low-dose recombinant human TNF (rhTNF) at the fracture site augmented fracture repair in a murine tibial fracture model. Here, we show that local rhTNF treatment is only effective when administered within 24h of injury, when neutrophils are the major inflammatory cell infiltrate. Systemic administration of anti-TNF impaired fracture healing. Addition of rhTNF enhanced neutrophil recruitment and promoted recruitment of monocytes through CCL2 production. Conversely, depletion of neutrophils or inhibition of the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death. Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair. If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization

Operationalizing the centiloid scale for [18F]florbetapir PET studies on PET/MRI

INTRODUCTION: The Centiloid scale aims to harmonize amyloid beta (Aβ) positron emission tomography (PET) measures across different analysis methods. As Centiloids were created using PET/computerized tomography (CT) data and are influenced by scanner differences, we investigated the Centiloid transformation with data from Insight 46 acquired with PET/magnetic resonanceimaging (MRI). METHODS: We transformed standardized uptake value ratios (SUVRs) from 432 florbetapir PET/MRI scans processed using whole cerebellum (WC) and white matter (WM) references, with and without partial volume correction. Gaussian-mixture-modelling-derived cutpoints for Aβ PET positivity were converted. RESULTS: The Centiloid cutpoint was 14.2 for WC SUVRs. The relationship between WM and WC uptake differed between the calibration and testing datasets, producing implausibly low WM-based Centiloids. Linear adjustment produced a WM-based cutpoint of 18.1. DISCUSSION: Transformation of PET/MRI florbetapir data to Centiloids is valid. However, further understanding of the effects of acquisition or biological factors on the transformation using a WM reference is needed