Numerical Simulation of AFP Nip Point Temperature Prediction for Complex Geometries

Material placement at the ideal nip point temperature over complex surfaces with uniformity across the width of the compaction rollers results in optimized part properties for Automated Fiber Placement (AFP) processes. However, current AFP systems utilize heat control models and methodologies, based on multiple process parameters such as feed-rate and orientation, that are mostly open-loop. Here, infrared (IR) heater input is calibrated as a function of process parameters during machine qualification. This work presents a numerical simulation to predict arrayed-infrared (AIR) emitter radiation onto a substrate that includes view factor implementation, IR radiative heat flow calculation, energy rate balance, and a transient heat transfer model. The purpose of this numerical model is to predict nip point temperature on complex surfaces, serving as a baseline for a new arrayed-infrared (AIR) thermoset heater to improve AFP process control. It is anticipated that this simulation will accurately control the temperature for high-speed AFP layup of complex geometries. An anticipated result of an AIR heater system is that material calibration and testing will be reduced as temperature is instantaneously monitored and controlled. Therefore, temperature across the roller width will be uniform during placement of complex parts, independent of their geometry

Pathological extracellular matrix suppresses microRNA-29 at the level of microRNA processing in Idiopathic Pulmonary Fibrosis

University of Minnesota Ph.D. dissertation. June 2015. Major: Microbiology, Immunology and Cancer Biology. Advisor: Kaylee Schwertfeger. 1 computer file (PDF); vi, 65 pages.Introduction: Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disorder that kills 40,000 Americans each year. It is characterized by pathological accumulation of activated myofibroblasts and extracellular matrix (ECM) which compromises gas exchange leading to death by asphyxiation. The IPF lung is an order of magnitude stiffer than normal. When primary human lung fibroblasts are cultured on IPF decellularized lung tissue (IPF-ECM), translational repression of ECM genes is disabled due to down-regulation of microRNA-29 (miR-29); however, the molecular mechanism is undefined. Stiffness drives fibroblast-to-myofibroblast differentiation; and stiffness has been shown to mediate microRNA expression. We therefore hypothesized that IPF-ECM decreases miR-29 due to increased matrix stiffness. Methodology: We cultured primary human lung fibroblasts on polyacrylamide hydrogels corresponding to physiological or IPF stiffness, or on Control- or IPF-ECM; and quantified precursor or mature miR-29 (Q-PCR). On hydrogels and decellularized ECM, we defined microRNA processing activity using a luciferase based construct. Results: IPF-ECM caused decreased mature miR-29 and increased precursor miR-29. In contrast, stiffness increased mature miR-29, and decreased precursor miR-29. MicroRNA processing activity was increased by both IPF-ECM and stiffness. Conclusion: On hydrogels simulating pathological stiffness, a stiffness sensor provides a physiological negative feedback signal to increase miR-29, which in turn represses connective tissue synthesis. IPF-ECM mediates decreased miR-29, overriding what should be a negative feedback response to stiffness. Both IPF-ECM and pathological stiffness increases microRNA processing activity, which suggests primary microRNA is converted to precursor microRNA and that IPF-ECM deregulates microRNA processing at the level of post-microprocessor complex. Thus, our data is the first to identify corruption of microRNA processing by the IPF fibrotic matrix itself as one mechanism for sustained connective tissue production in IPF

A third-person perspective on co-speech action gestures in Parkinson's disease

A combination of impaired motor and cognitive function in Parkinson’s disease (PD) can impact on language and communication, with patients exhibiting a particular difficulty processing action verbs. Co-speech gestures embody a link between action and language and contribute significantly to communication in healthy people. Here, we investigated how co-speech gestures depicting actions are affected in PD, in particular with respect to the visual perspective—or the viewpoint – they depict. Gestures are closely related to mental imagery and motor simulations, but people with PD may be impaired in the way they simulate actions from a first-person perspective and may compensate for this by relying more on third-person visual features. We analysed the action-depicting gestures produced by mild-moderate PD patients and age-matched controls on an action description task and examined the relationship between gesture-viewpoint, action-naming, and performance on an action observation task (weight judgement). Healthy controls produced the majority of their action-gestures from a first person perspective, whereas PD patients produced a greater proportion of gestures produced from a third person perspective. We propose that this reflects a compensatory reliance on third-person visual features in the simulation of actions in PD. Performance was also impaired in action-naming and weight judgement, although this was unrelated to gesture viewpoint. Our findings provide a more comprehensive understanding of how action-language impairments in PD impact on action communication, on the cognitive underpinnings of this impairment, as well as elucidating the role of action simulation in gesture production

IL-13 deficiency exacerbates lung damage and impairs epithelial-derived type 2 molecules during nematode infection

Acknowledgements This work was supported by the Wellcome Trust (203128/Z/16/Z, 110126/Z/ 15/Z, and 106898/A/15/Z) and the Medical Research Council UK (MR/ K01207X/2). TE Sutherland was supported by Medical Research Founda- tion UK joint funding with Asthma UK (MRFAUK-2015-302). We thank Andrew McKenzie (Cambridge) for providing the Il13 tm3.1Anjm mice. We further thank the Flow Cytometry, Bioimaging, Genomic Technologies, BioMS, and Bio- logical Services core facilities at the University of Manchester.Peer reviewedPublisher PD

Release of miR-29 Target Laminin C2 Improves Skin Repair

miRNAs are small noncoding RNAs that regulate mRNA targets in a cell-specific manner. miR-29 is expressed in murine and human skin, where it may regulate functions in skin repair. Cutaneous wound healing model in miR-29a/b1 gene knockout mice was used to identify miR-29 targets in the wound matrix, where angiogenesis and maturation of provisional granulation tissue was enhanced in response to genetic deletion of miR-29. Consistently, antisense-mediated inhibition of miR-29 promoted angiogenesis in vitro by autocrine and paracrine mechanisms. These processes are likely mediated by miR-29 target mRNAs released upon removal of miR-29 to improve cell–matrix adhesion. One of these, laminin (Lam)-c2 (also known as laminin γ2), was strongly up-regulated during skin repair in the wound matrix of knockout mice. Unexpectedly, Lamc2 was deposited in the basal membrane of endothelial cells in blood vessels forming in the granulation tissue of knockout mice. New blood vessels showed punctate interactions between Lamc2 and integrin α6 (Itga6) along the length of the proto-vessels, suggesting that greater levels of Lamc2 may contribute to the adhesion of endothelial cells, thus assisting angiogenesis within the wound. These findings may be of translational relevance, as LAMC2 was deposited at the leading edge in human wounds, where it formed a basal membrane for endothelial cells and assisted neovascularization. These results suggest a link between LAMC2, improved angiogenesis, and re-epithelialization

Identification of a Cell-of-Origin for Fibroblasts Comprising the Fibrotic Reticulum in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the middle aged and elderly with a prevalence of one million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli, creating a reticular network that leads to death by asphyxiation. Lung fibroblasts from patients with IPF have phenotypic hallmarks, distinguishing them from their normal counterparts: pathologically activated Akt signaling axis, increased collagen and α-smooth muscle actin expression, distinct gene expression profile, and ability to form fibrotic lesions in model organisms. Despite the centrality of these fibroblasts in disease pathogenesis, their origin remains uncertain. Here, we report the identification of cells in the lungs of patients with IPF with the properties of mesenchymal progenitors. In contrast to progenitors isolated from nonfibrotic lungs, IPF mesenchymal progenitor cells produce daughter cells manifesting the full spectrum of IPF hallmarks, including the ability to form fibrotic lesions in zebrafish embryos and mouse lungs, and a transcriptional profile reflecting these properties. Morphological analysis of IPF lung tissue revealed that mesenchymal progenitor cells and cells with the characteristics of their progeny comprised the fibrotic reticulum. These data establish that the lungs of patients with IPF contain pathological mesenchymal progenitor cells that are cells of origin for fibrosis-mediating fibroblasts. These fibrogenic mesenchymal progenitors and their progeny represent an unexplored target for novel therapies to interdict fibrosis

Developmental programming of cardiovascular dysfunction by prenatal hypoxia and oxidative stress.

Fetal hypoxia is a common complication of pregnancy. It has been shown to programme cardiac and endothelial dysfunction in the offspring in adult life. However, the mechanisms via which this occurs remain elusive, precluding the identification of potential therapy. Using an integrative approach at the isolated organ, cellular and molecular levels, we tested the hypothesis that oxidative stress in the fetal heart and vasculature underlies the molecular basis via which prenatal hypoxia programmes cardiovascular dysfunction in later life. In a longitudinal study, the effects of maternal treatment of hypoxic (13% O(2)) pregnancy with an antioxidant on the cardiovascular system of the offspring at the end of gestation and at adulthood were studied. On day 6 of pregnancy, rats (n = 20 per group) were exposed to normoxia or hypoxia ± vitamin C. At gestational day 20, tissues were collected from 1 male fetus per litter per group (n = 10). The remaining 10 litters per group were allowed to deliver. At 4 months, tissues from 1 male adult offspring per litter per group were either perfusion fixed, frozen, or dissected for isolated organ preparations. In the fetus, hypoxic pregnancy promoted aortic thickening with enhanced nitrotyrosine staining and an increase in cardiac HSP70 expression. By adulthood, offspring of hypoxic pregnancy had markedly impaired NO-dependent relaxation in femoral resistance arteries, and increased myocardial contractility with sympathetic dominance. Maternal vitamin C prevented these effects in fetal and adult offspring of hypoxic pregnancy. The data offer insight to mechanism and thereby possible targets for intervention against developmental origins of cardiac and peripheral vascular dysfunction in offspring of risky pregnancy

Laser capture microdissection coupled mass spectrometry (LCM-MS) for spatially resolved analysis of formalin-fixed and stained human lung tissues

From Springer Nature via Jisc Publications RouterHistory: received 2019-11-06, accepted 2020-06-11, registration 2020-06-11, pub-electronic 2020-06-17, online 2020-06-17, pub-print 2020-12Publication status: PublishedFunder: Wellcome Trust; doi: http://dx.doi.org/10.13039/100004440; Grant(s): 203128/Z/16/ZFunder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/L024551/1Abstract: Background: Haematoxylin and eosin (H&E)—which respectively stain nuclei blue and other cellular and stromal material pink—are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing and H&E staining of tissues provides challenges with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique to analyse H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. Methods: Herein, we utilize heat extraction, physical disruption, and in column digestion for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm3) and human lung blood vessels (0.094 mm3) from FFPE-fixed H&E-stained sections from Idiopathic Pulmonary Fibrosis (IPF) specimens (n = 3 IPF specimens) were then subject to a qualitative and then quantitative proteomics approach using BayesENproteomics. In addition, we tested the sensitivity of this method by processing and analysing a range of micro-dissected human lung blood vessel tissue volumes. Results: This approach yields 1252 uniquely expressed proteins (at a protein identification threshold of 3 unique peptides) with 892 differentially expressed proteins between these regions. In accord with prior knowledge, our methodology approach confirms that human lung blood vessels are enriched with smoothelin, CNN1, ITGA7, MYH11, TAGLN, and PTGIS; whereas morphologically normal human lung alveoli are enriched with cytokeratin-7, -8, -18, -19, 14, and -17. In addition, we identify a total of 137 extracellular matrix (ECM) proteins and immunohistologically validate that laminin subunit beta-1 localizes to morphologically normal human lung alveoli and tenascin localizes to human lung blood vessels. Lastly, we show that this micro-proteomics technique can be applied to tissue volumes as low as 0.0125 mm3. Conclusion: Herein we show that our multistep sample preparation methodology of LCM-MS can identify distinct, characteristic proteomic compositions of anatomical features within complex fixed and stained tissues

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.Comment: 41 page

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication

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