Ehrenzweig and the Statute of Frauds: An Inquiry Into the Rule of Validation

Graphics processing units (GPUs) are used today in a wide range of applications, mainly because they can dramatically accelerate parallel computing, are affordable and energy efficient. In the field of medical imaging, GPUs are in some cases crucial for enabling practical use of computationally demanding algorithms. This review presents the past and present work on GPU accelerated medical image processing, and is meant to serve as an overview and introduction to existing GPU implementations. The review covers GPU acceleration of basic image processing operations (filtering, interpolation, histogram estimation and distance transforms), the most commonly used algorithms in medical imaging (image registration, image segmentation and image denoising) and algorithms that are specific to individual modalities (CT, PET, SPECT, MRI, fMRI, DTI, ultrasound, optical imaging and microscopy). The review ends by highlighting some future possibilities and challenges

Uterine Nodal expression supports maternal immunotolerance and establishment of the FOXP3+ regulatory T cell population during the preimplantation period

Pregnancy success is dependent on the establishment of maternal tolerance during the preimplantation period. The immunosuppressive function of regulatory T cells is critical to limit inflammation arising from implantation of the semi-allogeneic blastocyst. Insufficient maternal immune adaptations to pregnancy have been frequently associated with cases of female infertility and recurrent implantation failure. The role of Nodal, a secreted morphogen of the TGFβ superfamily, was recently implicated during murine pregnancy as its conditional deletion (NodalΔ/Δ) in the female reproductive tract resulted in severe subfertility. Here, it was determined that despite normal preimplantation processes and healthy, viable embryos, NodalΔ/Δ females had a 50% implantation failure rate compared to NodalloxP/loxP controls. Prior to implantation, the expression of inflammatory cytokines MCP-1, G-CSF, IFN-γ and IL-10 was dysregulated in the NodalΔ/Δ uterus. Further analysis of the preimplantation leukocyte populations in NodalΔ/Δ uteri showed an overabundance of infiltrating, pro-inflammatory CD11bhigh Ly6C+ macrophages coupled with the absence of CD4+ FOXP3+ regulatory T cells. Therefore, it is proposed that uterine Nodal expression during the preimplantation period has a novel role in the establishment of maternal immunotolerance, and its dysregulation should be considered as a potential contributor to cases of female infertility and recurrent implantation failure

Uterine WNTS modulates fibronectin binding activity required for blastocyst attachment through the WNT/CA2+ signaling pathway in mice

Abstract Adhesion of the implanting blastocyst involves the interaction between integrin proteins expressed by trophoblast cells and components present in the basement membrane of the endometrial luminal epithelium. Although several factors regulating integrins and their adhesion to fibronectin are already known, we showed that Wnt signaling is involved in the regulation of blastocyst adhesion through the trafficking of integrins expressed by trophoblast cells. Localization of Itgα5β1 by immunofluorescence and FN-binding assays were conducted on peri-implantation blastocysts treated with either Wnt5a or Wnt7a proteins. Both Wnt5a and Wnt7a induced a translocation of Itgα5β1 at the surface of the blastocyst and an increase in FN-binding activity. We further demonstrated that uterine fluid is capable of inducing integrin translocation and this activity can be specifically inhibited by the Wnt inhibitor sFRP2. To identify the Wnt signaling pathway involved in this activity, blastocysts were incubated with inhibitors of either p38MAPK, PI3K pathway or CamKII prior to the addition of Wnts. Whereas inhibition of p38MAPK and PI3K had not effect, inhibition of CamKII reduced FN-binding activity induced by Wnts. Finally, we demonstrated that inhibition of Wnts by sFRP2 reduced the binding efficiency of the blastocyst to uterine epithelial cells. Our findings provide new insight into the mechanism that regulates integrin trafficking and FN-binding activity and identifies Wnts as a key player in blastocyst attachment to the uterine epithelium

A sensitive and bright single-cell resolution live imaging reporter of Wnt/ß-catenin signaling in the mouse

<p>Abstract</p> <p>Background</p> <p>Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placing <it>cis</it>-regulatory elements of interest to direct the expression of a desired reporter gene. In the case of canonical Wnt signaling, also referred to as Wnt/β-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/β-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies.</p> <p>Results</p> <p>We have placed six copies of a TCF/Lef responsive element and an <it>hsp68 </it>minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/β-catenin signaling activity. Characterization of developmental and adult stages of the resulting <it>TCF/Lef:H2B-GFP </it>strain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/β-catenin signaling. In support of the increased sensitivity of the <it>TCF/Lef:H2B-GFP </it>reporter, additional sites of Wnt/β-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis.</p> <p>Conclusion</p> <p>By combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. The <it>TCF/Lef:H2B-GFP </it>reporter represents a unique tool for live imaging the <it>in vivo </it>processes triggered by Wnt/β-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.</p

Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples

Fast Multispectral Optoacoustic Tomography (MSOT) for Dynamic Imaging of Pharmacokinetics and Biodistribution in Multiple Organs

The characterization of pharmacokinetic and biodistribution profiles is an essential step in the development process of new candidate drugs or imaging agents. Simultaneously, the assessment of organ function related to the uptake and clearance of drugs is of great importance. To this end, we demonstrate an imaging platform capable of high-rate characterization of the dynamics of fluorescent agents in multiple organs using multispectral optoacoustic tomography (MSOT). A spatial resolution of approximately 150 µm through mouse cross-sections allowed us to image blood vessels, the kidneys, the liver and the gall bladder. In particular, MSOT was employed to characterize the removal of indocyanine green from the systemic circulation and its time-resolved uptake in the liver and gallbladder. Furthermore, it was possible to track the uptake of a carboxylate dye in separate regions of the kidneys. The results demonstrate the acquisition of agent concentration metrics at rates of 10 samples per second at a single wavelength and 17 s per multispectral sample with 10 signal averages at each of 5 wavelengths. Overall, such imaging performance introduces previously undocumented capabilities of fast, high resolution in vivo imaging of the fate of optical agents for drug discovery and basic biological research

Elevated AKR1C3 expression promotes prostate cancer cell survival and prostate cell-mediated endothelial cell tube formation: implications for prostate cancer progressioan

<p>Abstract</p> <p>Background</p> <p>Aldo-keto reductase (AKR) 1C family member 3 (AKR1C3), one of four identified human AKR1C enzymes, catalyzes steroid, prostaglandin, and xenobiotic metabolism. In the prostate, AKR1C3 is up-regulated in localized and advanced prostate adenocarcinoma, and is associated with prostate cancer (PCa) aggressiveness. Here we propose a novel pathological function of AKR1C3 in tumor angiogenesis and its potential role in promoting PCa progression.</p> <p>Methods</p> <p>To recapitulate elevated AKR1C3 expression in cancerous prostate, the human PCa PC-3 cell line was stably transfected with an AKR1C3 expression construct to establish PC3-AKR1C3 transfectants. Microarray and bioinformatics analysis were performed to identify AKR1C3-mediated pathways of activation and their potential biological consequences in PC-3 cells. Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and an <it>in vitro </it>Matrigel angiogenesis assays were applied to validate the pro-angiogenic activity of PC3-AKR1C3 transfectants identified by bioinformatics analysis.</p> <p>Results</p> <p>Microarray and bioinformatics analysis suggested that overexpression of AKR1C3 in PC-3 cells modulates estrogen and androgen metabolism, activates insulin-like growth factor (IGF)-1 and Akt signaling pathways, as well as promotes tumor angiogenesis and aggressiveness. Levels of IGF-1 receptor (IGF-1R) and Akt activation as well as vascular endothelial growth factor (VEGF) expression and secretion were significantly elevated in PC3-AKR1C3 transfectants in comparison to PC3-mock transfectants. PC3-AKR1C3 transfectants also promoted endothelial cell (EC) tube formation on Matrigel as compared to the AKR1C3-negative parental PC-3 cells and PC3-mock transfectants. Pre-treatment of PC3-AKR1C3 transfectants with a selective IGF-1R kinase inhibitor (AG1024) or a non-selective phosphoinositide 3-kinases (PI3K) inhibitor (LY294002) abolished ability of the cells to promote EC tube formation.</p> <p>Conclusions</p> <p>Bioinformatics analysis followed by functional genomics demonstrated that AKR1C3 overexpression promotes angiogenesis and aggressiveness of PC-3 cells. These results also suggest that AKR1C3-mediated tumor angiogenesis is regulated by estrogen and androgen metabolism with subsequent IGF-1R and Akt activation followed by VEGF expression in PCa cells.</p

Characterization of a protein binding site involved in the regulation of transcription elongation within the murine c-myc gene

The c-myc gene was previously shown to be regulated by a conditional block to transcription elongation and sequences from its promoter were implicated in this regulation. The objective of this project was to define the promoter elements involved in the control of transcription elongation. Using heterologous promoter constructs, the ME1a1 protein binding site located in the c-myc promoter was shown to be required for the block to transcription elongation. From mutation analysis, a correlation was established between the binding of nuclear factors to ME1a1 sites in vitro and the ability of these sites to confer block to transcription elongation in vivo, strongly implicating trans-acting factors in this process. Fractionation studies demonstrated that three nuclear factors interact with the ME1a1 site, thus generating three protein-DNA complexes termed "a", "b", and "c". These factors were characterized and a cDNA encoding the protein responsible for complex "c" was isolated. This protein was shown to represent the human homologue of the Drosophila Cut homeodomain protein (hu-Cut) and to repress expression from the c-myc promoter in transient transfection assays

Correspondence between factor binding sites and replication activation in the polyomavirus enhancer

Polyomavirus DNA replication requires three distinct elements, the origin core, and two functionally redundant auxillary elements alpha ($alpha$) and beta ($beta$). To correlate protein binding sites within the $alpha$ and $beta$ elements and the ability to activate polyomavirus DNA replication, I performed both DNA replication assays and gel retardation assays using constructs containing unidirectional deletions of the $alpha$ and $beta$ elements. To map the sequences required of polyomavirus DNA replication in FM3A cells, the deletion mutants of $alpha$ and $beta$ were cotransfected along with a plasmid expressing polyomavirus large T antigen, in FM3A cells. As previously identified in MOP-8 cells, in FM3A cells the alpha element can be subdivided in a core subelement and an auxillary subelement. The beta element in FM3A cells can be subdivided in three core subelements termed core subelement one, two, and three. Band retardation assays using nuclear extracts from FM3A cells illustrate that cellular factors can interact to all subelements that were functional in DNA replication