Whole Slide Image Analysis Quantification using Aperio Digital Imaging in a Mouse Lung Metastasis Model

poster abstractDigital whole slide imaging is the technique of digitizing a microscope slide at the highest resolution to produce a “digital virtual microscope slide”. This digital image can be viewed in three or four fields, from low to high power, which can be commonly used to evaluate the tissue. Many of these systems have whole slide software image analysis capability. The goal of this study was to determine if the Aperio positive pixel algorithm (image analysis) could effectively quantitate metastatic mouse lung tumors in a lung section using a H&E stain. Lung sections from a mouse lung metastasis model of 8 mice per group were evaluated: control, 50mg/kg, and 75mg/kg carboplatin. H&E and Ki67 immunostain slides were scanned using the Aperio whole slide scanning system (Scanscope CS). A single field of view from each slide representing a whole lung lobe with multiple lung metastases was selected for image analysis. The standard positive pixel algorithm was altered to read the H&E slides. Various histology slides were used to validate the altered algorithm. The immunostain (Ki67) was generated using the standard positive pixel algorithm analysis. The Aperio automated positive pixel count for a Ki67 immunostain was consistent with the H&E image analysis. The values decreased with a dose dependent treatment (control vs. 50mg/kg and 75mg/kg carboplatin) and were (H&E) 37%, 28%, and 22%, and (Ki67) 9%, 5%, and 3%. The analysis had decreasing values for both the H&E and Ki67 analysis on a dose dependent drug treatment. The metastases decreased in both treatment groups compared to controls with both the H&E and Ki67 analyses. The Aperio Image Analysis positive pixel algorithm allows large areas of the lung tissue section to be examined and not just a single 25x or 40x field like many common image analyses systems

Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates

Nitrogen-containing-bisphosphonates (N-BPs) are widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance

Casimir effect due to a single boundary as a manifestation of the Weyl problem

The Casimir self-energy of a boundary is ultraviolet-divergent. In many cases the divergences can be eliminated by methods such as zeta-function regularization or through physical arguments (ultraviolet transparency of the boundary would provide a cutoff). Using the example of a massless scalar field theory with a single Dirichlet boundary we explore the relationship between such approaches, with the goal of better understanding the origin of the divergences. We are guided by the insight due to Dowker and Kennedy (1978) and Deutsch and Candelas (1979), that the divergences represent measurable effects that can be interpreted with the aid of the theory of the asymptotic distribution of eigenvalues of the Laplacian discussed by Weyl. In many cases the Casimir self-energy is the sum of cutoff-dependent (Weyl) terms having geometrical origin, and an "intrinsic" term that is independent of the cutoff. The Weyl terms make a measurable contribution to the physical situation even when regularization methods succeed in isolating the intrinsic part. Regularization methods fail when the Weyl terms and intrinsic parts of the Casimir effect cannot be clearly separated. Specifically, we demonstrate that the Casimir self-energy of a smooth boundary in two dimensions is a sum of two Weyl terms (exhibiting quadratic and logarithmic cutoff dependence), a geometrical term that is independent of cutoff, and a non-geometrical intrinsic term. As by-products we resolve the puzzle of the divergent Casimir force on a ring and correct the sign of the coefficient of linear tension of the Dirichlet line predicted in earlier treatments.Comment: 13 pages, 1 figure, minor changes to the text, extra references added, version to be published in J. Phys.

On Density of State of Quantized Willmore Surface-A Way to Quantized Extrinsic String in R^3

Recently I quantized an elastica with Bernoulli-Euler functional in two-dimensional space using the modified KdV hierarchy. In this article, I will quantize a Willmore surface, or equivalently a surface with the Polyakov extrinsic curvature action, using the modified Novikov-Veselov (MNV) equation. In other words, I show that the density of state of the partition function for the quantized Willmore surface is expressed by volume of a subspace of the moduli of the MNV equation.Comment: AMS-Tex Us

Assessment of an All-Particle Hybrid Method for Hypersonic Rarefied Flow

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106450/1/AIAA2013-1203.pd

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

H2A.Z Acidic Patch Couples Chromatin Dynamics to Regulation of Gene Expression Programs during ESC Differentiation

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z[superscript AP3]) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z[superscript AP3] interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z[superscript AP3] was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z[superscript AP3] ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z[superscript AP3] ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z[superscript AP3] displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z[superscript AP3] mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment.Massachusetts Life Sciences Center (David H. Koch Institute for Integrative Cancer Research at MIT Core Grant P30-CA14051)National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (Grant CBET-0939511)MIT Faculty Start-up FundMassachusetts Institute of Technology. Computational and Systems Biology Initiative (Merck & Co. Postdoctoral Fellowship

Development of risk maps to minimize uranium exposures in the Navajo Churchrock mining district

© 2009 deLemos et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Stem cells in ectodermal development

Tissue-specific stem cells sustain organs for a lifetime through self-renewal and generating differentiated progeny. Although tissue stem cells are established during organogenesis, the precise origin of most adult stem cells in the developing embryo is unclear. Mammalian skin is one of the best-studied epithelial systems containing stem cells to date, however the origin of most of the stem cell populations found in the adult epidermis is unknown. Here, we try to recapitulate the emergence and genesis of an ectodermal stem cell during development until the formation of an adult skin. We ask whether skin stem cells share key transcriptional regulators with their embryonic counterparts and discuss whether embryonic-like stem cells may persist through to adulthood in vivo

Polycomb-Like 3 Promotes Polycomb Repressive Complex 2 Binding to CpG Islands and Embryonic Stem Cell Self-Renewal

Polycomb repressive complex 2 (PRC2) trimethylates lysine 27 of histone H3 (H3K27me3) to regulate gene expression during diverse biological transitions in development, embryonic stem cell (ESC) differentiation, and cancer. Here, we show that Polycomb-like 3 (Pcl3) is a component of PRC2 that promotes ESC self-renewal. Using mass spectrometry, we identified Pcl3 as a Suz12 binding partner and confirmed Pcl3 interactions with core PRC2 components by co-immunoprecipitation. Knockdown of Pcl3 in ESCs increases spontaneous differentiation, yet does not affect early differentiation decisions as assessed in teratomas and embryoid bodies, indicating that Pcl3 has a specific role in regulating ESC self-renewal. Consistent with Pcl3 promoting PRC2 function, decreasing Pcl3 levels reduces H3K27me3 levels while overexpressing Pcl3 increases H3K27me3 levels. Furthermore, chromatin immunoprecipitation and sequencing (ChIP-seq) reveal that Pcl3 co-localizes with PRC2 core component, Suz12, and depletion of Pcl3 decreases Suz12 binding at over 60% of PRC2 targets. Mutation of conserved residues within the Pcl3 Tudor domain, a domain implicated in recognizing methylated histones, compromises H3K27me3 formation, suggesting that the Tudor domain of Pcl3 is essential for function. We also show that Pcl3 and its paralog, Pcl2, exist in different PRC2 complexes but bind many of the same PRC2 targets, particularly CpG islands regulated by Pcl3. Thus, Pcl3 is a component of PRC2 critical for ESC self-renewal, histone methylation, and recruitment of PRC2 to a subset of its genomic sites