Hedgehog pathway dysregulation contributes to the pathogenesis of human gastrointestinal stromal tumors via GLI-mediated activation of KIT expression.

Gastrointestinal stromal tumors (GIST) arise within the interstitial cell of Cajal (ICC) lineage due to activating KIT/PDGFRA mutations. Both ICC and GIST possess primary cilia (PC), which coordinate PDGFRA and Hedgehog signaling, regulators of gastrointestinal mesenchymal development. Therefore, we hypothesized that Hedgehog signaling may be altered in human GIST and controls KIT expression. Quantitative RT-PCR, microarrays, and next generation sequencing were used to describe Hedgehog/PC-related genes in purified human ICC and GIST. Genetic and pharmacologic approaches were employed to investigate the effects of GLI manipulation on KIT expression and GIST cell viability. We report that Hedgehog pathway and PC components are expressed in ICC and GIST and subject to dysregulation during GIST oncogenesis, irrespective of KIT/PDGFRA mutation status. Using genomic profiling, 10.2% of 186 GIST studied had potentially deleterious genomic alterations in 5 Hedgehog-related genes analyzed, including in the PTCH1 tumor suppressor (1.6%). Expression of the predominantly repressive GLI isoform, GLI3, was inversely correlated with KIT mRNA levels in GIST cells and non-KIT/non-PDGFRA mutant GIST. Overexpression of the 83-kDa repressive form of GLI3 or small interfering RNA-mediated knockdown of the activating isoforms GLI1/2 reduced KIT mRNA. Treatment with GLI1/2 inhibitors, including arsenic trioxide, significantly increased GLI3 binding to the KIT promoter, decreased KIT expression, and reduced viability in imatinib-sensitive and imatinib-resistant GIST cells. These data offer new evidence that genes necessary for Hedgehog signaling and PC function in ICC are dysregulated in GIST. Hedgehog signaling activates KIT expression irrespective of mutation status, offering a novel approach to treat imatinib-resistant GIST

The signed loop approach to the Ising model: foundations and critical point

The signed loop method is a beautiful way to rigorously study the two-dimensional Ising model with no external field. In this paper, we explore the foundations of the method, including details that have so far been neglected or overlooked in the literature. We demonstrate how the method can be applied to the Ising model on the square lattice to derive explicit formal expressions for the free energy density and two-point functions in terms of sums over loops, valid all the way up to the self-dual point. As a corollary, it follows that the self-dual point is critical both for the behaviour of the free energy density, and for the decay of the two-point functions.Comment: 38 pages, 7 figures, with an improved Introduction. The final publication is available at link.springer.co

Ground states of two-dimensional ±\pmJ Edwards-Anderson spin glasses

We present an exact algorithm for finding all the ground states of the two-dimensional Edwards-Anderson $\pm J$ spin glass and characterize its performance. We investigate how the ground states change with increasing system size and and with increasing antiferromagnetic bond ratio $x$. We find that that some system properties have very large and strongly non-Gaussian variations between realizations.Comment: 15 pages, 21 figures, 2 tables, uses revtex4 macro

Demonstration of Binding of Neuronal Calcium Sensor-1 to the Ca(v)2.1 P/Q-Type Calcium Channel

[Image: see text] In neurons, entry of extracellular calcium (Ca(2+)) into synaptic terminals through Ca(v)2.1 (P/Q-type) Ca(2+) channels is the driving force for exocytosis of neurotransmitter-containing synaptic vesicles. This class of Ca(2+) channel is, therefore, pivotal during normal neurotransmission in higher organisms. In response to channel opening and Ca(2+) influx, specific Ca(2+)-binding proteins associate with cytoplasmic regulatory domains of the P/Q channel to modulate subsequent channel opening. Channel modulation in this way influences synaptic plasticity with consequences for higher-level processes such as learning and memory acquisition. The ubiquitous Ca(2+)-sensing protein calmodulin (CaM) regulates the activity of all types of mammalian voltage-gated Ca(2+) channels, including the P/Q class, by direct binding to specific regulatory motifs. More recently, experimental evidence has highlighted a role for additional Ca(2+)-binding proteins, particularly of the CaBP and NCS families in the regulation of P/Q channels. NCS-1 is a protein found from yeast to humans and that regulates a diverse number of cellular functions. Physiological and genetic evidence indicates that NCS-1 regulates P/Q channel activity, including calcium-dependent facilitation, although a direct physical association between the proteins has yet to be demonstrated. In this study, we aimed to determine if there is a direct interaction between NCS-1 and the C-terminal cytoplasmic tail of the Ca(v)2.1 α-subunit. Using distinct but complementary approaches, including in vitro binding of bacterially expressed recombinant proteins, fluorescence spectrophotometry, isothermal titration calorimetry, nuclear magnetic resonance, and expression of fluorescently tagged proteins in mammalian cells, we show direct binding and demonstrate that CaM can compete for it. We speculate about how NCS-1/Ca(v)2.1 association might add to the complexity of calcium channel regulation mediated by other known calcium-sensing proteins and how this might help to fine-tune neurotransmission in the mammalian central nervous system

Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon‐induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS‐CoV‐2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS‐CoV‐2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS‐CoV‐2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS‐CoV‐2 and highlight the multiple distinct mechanisms by which SARS‐CoV‐2 subverts tetherin function

Reproducibility of in-vivo OCT measured three-dimensional human lamina cribrosa microarchitecture

Purpose: To determine the reproducibility of automated segmentation of the three-dimensional (3D) lamina cribrosa (LC) microarchitecture scanned in-vivo using optical coherence tomography (OCT). Methods: Thirty-nine eyes (8 healthy, 19 glaucoma suspects and 12 glaucoma) from 49 subjects were scanned twice using swept-source (SS-) OCT in a 3.5x3.5x3.64 mm (400x400x896 pixels) volume centered on the optic nerve head, with the focus readjusted after each scan. The LC was automatically segmented and analyzed for microarchitectural parameters, including pore diameter, pore diameter standard deviation (SD), pore aspect ratio, pore area, beam thickness, beam thickness SD, and beam thickness to pore diameter ratio. Reproducibility of the parameters was assessed by computing the imprecision of the parameters between the scans. Results: The automated segmentation demonstrated excellent reproducibility. All LC microarchitecture parameters had an imprecision of less or equal to 4.2%. There was little variability in imprecision with respect to diagnostic category, although the method tends to show higher imprecision amongst healthy subjects. Conclusion: The proposed automated segmentation of the LC demonstrated high reproducibility for 3D LC parameters. This segmentation analysis tool will be useful for in-vivo studies of the LC. © 2014 Wang et al

The medical student

The Medical Student was published from 1888-1921 by the students of Boston University School of Medicine

Disulfide-activated protein kinase G I alpha regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response

British Heart Foundation, European Research Council (ERC Advanced award), Medical Research Council and the Department of Health via the NIHR cBRC award to Guy’s & St Thomas’ NHS Foundation Trust. Part supported by the NIHR University College London Hospitals Biomedical Research Centre

Genomic and expression analysis of multiple Sry loci from a single Rattus norvegicus Y chromosome

BACKGROUND: Sry is a gene known to be essential for testis determination but is also transcribed in adult male tissues. The laboratory rat, Rattus norvegicus, has multiple Y chromosome copies of Sry while most mammals have only a single copy. DNA sequence comparisons with other rodents with multiple Sry copies are inconsistent in divergence patterns and functionality of the multiple copies. To address hypotheses of divergence, gene conversion and functional constraints, we sequenced Sry loci from a single R. norvegicus Y chromosome from the Spontaneously Hypertensive Rat strain (SHR) and analyzed DNA sequences for homology among copies. Next, to determine whether all copies of Sry are expressed, we developed a modification of the fluorescent marked capillary electrophoresis method to generate three different sized amplification products to identify Sry copies. We applied this fragment analysis method to both genomic DNA and cDNA prepared from mRNA from testis and adrenal gland of adult male rats. RESULTS: Y chromosome fragments were amplified and sequenced using primers that included the entire Sry coding region and flanking sequences. The analysis of these sequences identified six Sry loci on the Y chromosome. These are paralogous copies consistent with a single phylogeny and the divergence between any two copies is less than 2%. All copies have a conserved reading frame and amino acid sequence consistent with function. Fragment analysis of genomic DNA showed close approximations of experimental with predicted values, validating the use of this method to identify proportions of each copy. Using the fragment analysis procedure with cDNA samples showed the Sry copies expressed were significantly different from the genomic distribution (testis p < 0.001, adrenal gland p < 0.001), and the testis and adrenal copy distribution in the transcripts were also significantly different from each other (p < 0.001). Total Sry transcript expression, analyzed by real-time PCR, showed significantly higher levels of Sry in testis than adrenal gland (p, 0.001). CONCLUSION: The SHR Y chromosome contains at least 6 full length copies of the Sry gene. These copies have a conserved coding region and conserved amino acid sequence. The pattern of divergence is not consistent with gene conversion as the mechanism for this conservation. Expression studies show multiple copies expressed in the adult male testis and adrenal glands, with tissue specific differences in expression patterns. Both the DNA sequence analysis and RNA transcript expression analysis are consistent with more than one copy having function and selection preventing divergence although we have no functional evidence

HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment