Search

Objective: Recent observations in systemic juvenile idiopathic arthritis (JIA) suggest an increasing incidence of high‐mortality interstitial lung disease often characterized by a variant of pulmonary alveolar proteinosis (PAP). Co‐occurrence of macrophage activation syndrome (MAS) and PAP in systemic JIA suggests a shared pathology, but patients with lung disease associated with systemic JIA (designated SJIA‐LD) also commonly experience features of drug reaction such as atypical rashes and eosinophilia. This study was undertaken to investigate immunopathology and identify biomarkers in systemic JIA, MAS, and SJIA‐LD. Methods: We used SOMAscan to measure ~1,300 analytes in sera from healthy controls and patients with systemic JIA, MAS, SJIA‐LD, or other related diseases. We verified selected findings by enzyme‐linked immunosorbent assay and lung immunostaining. Because the proteome of a sample may reflect multiple states (systemic JIA, MAS, or SJIA‐LD), we used regression modeling to identify subsets of altered proteins associated with each state. We tested key findings in a validation cohort. Results: Proteome alterations in active systemic JIA and MAS overlapped substantially, including known systemic JIA biomarkers such as serum amyloid A and S100A9, and novel elevations in the levels of heat‐shock proteins and glycolytic enzymes. Interleukin‐18 levels were elevated in all systemic JIA groups, particularly MAS and SJIA‐LD. We also identified an MAS‐independent SJIA‐LD signature notable for elevated levels of intercellular adhesion molecule 5 (ICAM‐5), matrix metalloproteinase 7 (MMP‐7), and allergic/eosinophilic chemokines, which have been previously associated with lung damage. Immunohistochemistry localized ICAM‐5 and MMP‐7 in the lungs of patients with SJIA‐LD. The ability of ICAM‐5 to distinguish SJIA‐LD from systemic JIA/MAS was independently validated. Conclusion: Serum proteins support a systemic JIA–to‐MAS continuum; help distinguish systemic JIA, systemic JIA/MAS, and SJIA‐LD; and suggest etiologic hypotheses. Select biomarkers, such as ICAM‐5, could aid in early detection and management of SJIA‐LD. [ABSTRACT FROM AUTHOR]

The sympathetic and parasympathetic nervous systems regulate the activities of internal organs1, but the molecular and functional diversity of their constituent neurons and circuits remains largely unknown. Here we use retrograde neuronal tracing, single-cell RNA sequencing, optogenetics and physiological experiments to dissect the cardiac parasympathetic control circuit in mice. We show that cardiac-innervating neurons in the brainstem nucleus ambiguus (Amb) are comprised of two molecularly, anatomically and functionally distinct subtypes. The first, which we call ambiguus cardiovascular (ACV) neurons (approximately 35 neurons per Amb), define the classical cardiac parasympathetic circuit. They selectively innervate a subset of cardiac parasympathetic ganglion neurons and mediate the baroreceptor reflex, slowing heart rate and atrioventricular node conduction in response to increased blood pressure. The other, ambiguus cardiopulmonary (ACP) neurons (approximately 15 neurons per Amb) innervate cardiac ganglion neurons intermingled with and functionally indistinguishable from those innervated by ACV neurons. ACP neurons also innervate most or all lung parasympathetic ganglion neurons—clonal labelling shows that individual ACP neurons innervate both organs. ACP neurons mediate the dive reflex, the simultaneous bradycardia and bronchoconstriction that follows water immersion. Thus, parasympathetic control of the heart is organized into two parallel circuits, one that selectively controls cardiac function (ACV circuit) and another that coordinates cardiac and pulmonary function (ACP circuit). This new understanding of cardiac control has implications for treating cardiac and pulmonary diseases and for elucidating the control and coordination circuits of other organs.In mouse, two distinct types of neurons from the brainstem nucleus ambiguus, one that innervates the heart and another that innervates both the heart and lung, collectively control cardiac function and coordinate cardiac and pulmonary function. [ABSTRACT FROM AUTHOR]

Cell-free RNA from liquid biopsies can be analyzed to determine disease tissue of origin. We extend this concept to identify cell types of origin using the Tabula Sapiens transcriptomic cell atlas as well as individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset. We define cell type signature scores, which allow the inference of cell types that contribute to cell-free RNA for a variety of diseases. Cell types affected by various diseases are inferred from cell-free RNA. [ABSTRACT FROM AUTHOR]

In the mammalian lung, an apparently homogenous mesh of capillary vessels surrounds each alveolus, forming the vast respiratory surface across which oxygen transfers to the blood1. Here we use single-cell analysis to elucidate the cell types, development, renewal and evolution of the alveolar capillary endothelium. We show that alveolar capillaries are mosaics; similar to the epithelium that lines the alveolus, the alveolar endothelium is made up of two intermingled cell types, with complex 'Swiss-cheese'-like morphologies and distinct functions. The first cell type, which we term the 'aerocyte', is specialized for gas exchange and the trafficking of leukocytes, and is unique to the lung. The other cell type, termed gCap ('general' capillary), is specialized to regulate vasomotor tone, and functions as a stem/progenitor cell in capillary homeostasis and repair. The two cell types develop from bipotent progenitors, mature gradually and are affected differently in disease and during ageing. This cell-type specialization is conserved between mouse and human lungs but is not found in alligator or turtle lungs, suggesting it arose during the evolution of the mammalian lung. The discovery of cell type specialization in alveolar capillaries transforms our understanding of the structure, function, regulation and maintenance of the air–blood barrier and gas exchange in health, disease and evolution. Single-cell analysis of blood vessels in the alveolus, the site of chronic disease and virus-induced lung injury, reveals two intermingled endothelial cell types with specialized gas exchange and stem cell functions. [ABSTRACT FROM AUTHOR]

Adult stem cells are rare, undifferentiated cells found in all tissues of the body. Although normally kept in a quiescent, nondividing state, these cells can proliferate and differentiate to replace naturally dying cells within their tissue and to repair its wounds in response to injury. Due to their proliferative nature and ability to regenerate tissue, adult stem cells have the potential to treat a variety of degenerative diseases as well as aging. In addition, since stem cells are often thought to be the source of malignant tumors, understanding the mechanisms that keep their proliferative abilities in check can pave the way for new cancer therapies. While adult stem cells have had limited practical and clinical applications to date, several clinical trials of stem cell–based therapies are underway. This report details recent research presented at the New York Academy of Sciences on March 14, 2019 on understanding the factors that regulate stem cell activity and differentiation, with the hope of translating these findings into the clinic. [ABSTRACT FROM AUTHOR]

Summary In the developing brain, neurons expressing VEGF-A and blood vessels grow in close apposition, but many of the molecular pathways regulating neuronal VEGF-A and neurovascular system development remain to be deciphered. Here, we show that miR-9 links neurogenesis and angiogenesis through the formation of neurons expressing VEGF-A. We found that miR-9 directly targets the transcription factors TLX and ONECUTs to regulate VEGF-A expression. miR-9 inhibition leads to increased TLX and ONECUT expression, resulting in VEGF-A overexpression. This untimely increase of neuronal VEGF-A signal leads to the thickening of blood vessels at the expense of the normal formation of the neurovascular network in the brain and retina. Thus, this conserved transcriptional cascade is critical for proper brain development in vertebrates. Because of this dual role on neural stem cell proliferation and angiogenesis, miR-9 and its downstream targets are promising factors for cellular regenerative therapy following stroke and for brain tumor treatment. [ABSTRACT FROM AUTHOR]

Shriveled fruit in vineyards has several origins including sunburn, dehydration, bunchstem necrosis and the recently described sugar accumulation disorder. These disorders are often confused with one another, but they can easily be distinguished by the location or composition of shriveled fruit and the condition of the rachis (the stem structure of a cluster). Sunburn is typically exhibited only on berries that are exposed to direct sunlight, and bunchstem necrosis is typified by necrotic rachis tissue. Berries with sugar accumulation disorder exhibit low sugar concentration, whereas berries with late-season dehydration typically have above-normal sugar concentration. Berries with sugar accumulation disorder and bunchstem necrosis exhibit the sugar content when sugar accumulation ceases or stem necrosis occurs, respectively. In tests, berries with sugar accumulation disorder exhibited lower berry weight, pH and anthocyanins, as well as differences in many nitrogenous compounds compared to normally developing fruit. In one location, sugar accumulation disorder was expressed at the whole-vine level, but none of the commonly known pathogenic organisms were found. [ABSTRACT FROM AUTHOR]

Macrophages are specialized phagocytic cells, present in all tissues, which engulf and digest pathogens, infected and dying cells, and debris, and can recruit and regulate other immune cells and the inflammatory response and aid in tissue repair. Macrophage subpopulations play distinct roles in these processes and in disease, and are typically recognized by differences in marker expression, immune function, or tissue of residency. Although macrophage subpopulations in the brain have been found to have distinct developmental origins, the extent to which development contributes to macrophage diversity between tissues and within tissues is not well understood. Here, we investigate the development and maintenance of mouse lung macrophages by marker expression patterns, genetic lineage tracing and parabiosis. We show that macrophages populate the lung in three developmental waves, each giving rise to a distinct lineage. These lineages express different markers, reside in different locations, renew in different ways, and show little or no interconversion. Thus, development contributes significantly to lung macrophage diversity and targets each lineage to a different anatomical domain. [ABSTRACT FROM AUTHOR]

Along with sugar accumulation and colour development, softening is an important physiological change during the onset of ripening in fruits. In this work, we investigated the relationships among major events during softening in grape (Vitis vinifera L.) by quantifying elasticity in individual berries. In addition, we delayed softening and inhibited sugar accumulation using a mechanical growth-preventing treatment in order to identify processes that are sugar and/or growth dependent. Ripening processes commenced on various days after anthesis, but always at similarly low elasticity and turgor. Much of the softening occurred in the absence of other changes in berry physiology investigated here. Several genes encoding key cell wall-modifying enzymes were not up-regulated until softening was largely completed, suggesting softening may result primarily from decreases in turgor. Similarly, there was no decrease in solute potential, increase in sugar concentration, or colour development until elasticity and turgor were near minimum values, and these processes were inhibited when berry growth was prevented. Increases in abscisic acid occurred early during softening and in the absence of significant expression of the V. vinifera 9-cis-epoxycarotenoid dioxygenases. However, these increases were coincident with decreases in the abscisic acid catabolite diphasic acid, indicating that initial increases in abscisic acid may result from decreases in catabolism and/or exogenous import. These data suggest that softening, decreases in turgor, and increases in abscisic acid represent some of the earliest events during the onset of ripening. Later, physical growth, further increases in abscisic acid, and the accumulation of sugar are integral for colour development. [ABSTRACT FROM AUTHOR]

Animals have evolved homeostatic responses to changes in oxygen availability that act on different timescales. Although the hypoxia-inducible factor (HIF) transcriptional pathway that controls long-term responses to low oxygen (hypoxia) has been established, the pathway that mediates acute responses to hypoxia in mammals is not well understood. Here we show that the olfactory receptor gene Olfr78 is highly and selectively expressed in oxygen-sensitive glomus cells of the carotid body, a chemosensory organ at the carotid artery bifurcation that monitors blood oxygen and stimulates breathing within seconds when oxygen declines. Olfr78 mutants fail to increase ventilation in hypoxia but respond normally to hypercapnia. Glomus cells are present in normal numbers and appear structurally intact, but hypoxia-induced carotid body activity is diminished. Lactate, a metabolite that rapidly accumulates in hypoxia and induces hyperventilation, activates Olfr78 in heterologous expression experiments, induces calcium transients in glomus cells, and stimulates carotid sinus nerve activity through Olfr78. We propose that, in addition to its role in olfaction, Olfr78 acts as a hypoxia sensor in the breathing circuit by sensing lactate produced when oxygen levels decline. [ABSTRACT FROM AUTHOR]

Tempered chocolate was hardened on surfaces of differing glosses, including polypropylene transfer sheets, Plexiglas®, marble, textured polyethylene, 180 grit sandpaper, and 400 grit sandpaper. Lower gloss surfaces, such as the sandpapers and polyethylene induced lower gloss in the chocolates hardened on them compared with the high gloss polypropylene and Plexiglas®. There was a strong correlation between the gloss of the surface and the resulting gloss of the chocolate hardened on that surface. The topography of the surface was maintained on the hardened chocolate. Glossier chocolates were perceived to be of significantly higher value than less glossy samples, however the surface gloss had no significant effect on panelist’s liking, perceived sweetness, bitterness, and chocolate flavor intensity. These data suggest that molds can be created or manipulated to impart specific gloss and texture to chocolates to better express the artistic vision of the chocolatier. [ABSTRACT FROM PUBLISHER]

The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors. [ABSTRACT FROM AUTHOR]

Alveoli are gas-exchange sacs lined by squamous alveolar type (AT) 1 cells and cuboidal, surfactant-secreting AT2 cells. Classical studies suggested that AT1 arise from AT2 cells, but recent studies propose other sources. Here we use molecular markers, lineage tracing and clonal analysis to map alveolar progenitors throughout the mouse lifespan. We show that, during development, AT1 and AT2 cells arise directly from a bipotent progenitor, whereas after birth new AT1 cells derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. This stem-cell function is broadly activated by AT1 injury, and AT2 self-renewal is selectively induced by EGFR (epidermal growth factor receptor) ligands in vitro and oncogenic Kras(G12D) in vivo, efficiently generating multifocal, clonal adenomas. Thus, there is a switch after birth, when AT2 cells function as stem cells that contribute to alveolar renewal, repair and cancer. We propose that local signals regulate AT2 stem-cell activity: a signal transduced by EGFR-KRAS controls self-renewal and is hijacked during oncogenesis, whereas another signal controls reprogramming to AT1 fate. [ABSTRACT FROM AUTHOR]

The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1. [ABSTRACT FROM AUTHOR]

Turkey breasts were brined under two salt concentrations for 24 hours (4°C). Moisture uptake during brining and loss after roasting (177°C) was measured. Seventy-eight culinary students evaluated juiciness, saltiness, sweetness, turkey flavor, and overall liking. Moisture uptake was greater in the normal salt brine (7.2% of initial weight) compared to the low salt brine (6.0%) and water (2.3%). Turkey brined in high salt lost less water during cooking compared to control and water soaked samples. Brined turkey breast, regardless of the salt content of the brine, had higher scores for liking and the perception of saltiness, sweetness, juiciness, and turkey flavor compared to control samples. Turkey soaked in higher salt brine was more liked, had more turkey flavor, and was perceived to be saltier than turkey in a lower salt brine. The use of experiments such as this as teaching tools in culinary and food science classes is also discussed. [ABSTRACT FROM PUBLISHER]

Spots of blood are routinely collected from newborn babies onto filter paper called Guthrie cards and used to screen for metabolic and genetic disorders. The archived dried blood spots are an important and precious resource for genomic research. Whole genome amplification of dried blood spot DNA has been used to provide DNA for genome-wide SNP genotyping. Here we describe a 96 well format procedure to extract DNA from a portion of a dried blood spot that provides sufficient unamplified genomic DNA for genome-wide single nucleotide polymorphism (SNP) genotyping. We show that SNP genotyping of the unamplified DNA is more robust than genotyping amplified dried blood spot DNA, is comparable in cost, and can be done with thousands of samples. This procedure can be used for genome-wide association studies and other large-scale genomic analyses that require robust, high-accuracy genotyping of dried blood spot DNA. [ABSTRACT FROM AUTHOR]

Epithelia are classified as either simple, a single cell layer thick, or stratified (multilayered). Stratified epithelia arise from simple epithelia during development, and transcription factor p63 functions as a key positive regulator of epidermal stratification. Here we show that deletion of integrin beta 1 (Itgb1) in the developing mouse airway epithelium abrogates airway branching and converts this monolayer epithelium into a multilayer epithelium with more than 10 extra layers. Mutant lung epithelial cells change mitotic spindle orientation to seed outer layers, and cells in different layers become molecularly and functionally distinct, hallmarks of normal stratification. However, mutant lung epithelial cells do not activate p63 and do not switch to the stratified keratin profile of epidermal cells. These data, together with previous data implicating Itgb1 in regulation of epidermal stratification, suggest that the simple-versus-stratified developmental decision may involve not only stratification inducers like p63 but suppressors like Itgb1 that prevent simple epithelia from inappropriately activating key steps in the stratification program. [ABSTRACT FROM AUTHOR]

As a base for sauces, soups, and cooking liquids for meats, grains, and vegetables, stocks can be integral to the overall quality of restaurant menu items, however, science-based studies on the effects of cooking methods on the physiochemical and sensory properties of stock are lacking. The effects of starting (22 °C, 85 °C, and 99 °C) and cooking temperatures (85 °C and 99 °C) of chicken stock on clarity, color, viscosity, protein content, amino acid content, mineral content, and overall liking were measured. Protein content and viscosity were significantly higher for stocks cooked at 99 °C, but no effect on amino acid content, color, or clarity was observed. Calcium concentration in stocks cooked at 99 °C was significantly ( P < 0.0001) lower (9.3 and 10.1 mg/mL, for stocks started at temperatures of 22 and 99 °C, respectively) than stock cooked at 85 °C (16.6 and 17.5 mg/mL for stocks started at temperatures of 22 and 85 °C, respectively). Stocks prepared at 99 °C scored higher on overall liking compared to commercial samples and those cooked at 85 °C ( P= 0.0101). These data can be used by culinary scientists and professionals to develop more efficient techniques in the kitchen, and by product developers to optimize the overall quality and acceptance of stock. Practical Application: This work documents the effects of preparation method on the physical and chemical properties, and consumer acceptance of chicken stock. This information can be used by product developers, culinary scientists, and professional chefs to optimize stock-based products. Culinary educators can use this information to provide students with objective evidence-based rationale for the techniques underlying a celebrated culinary tradition. This is also an example of how research can facilitate collaboration between culinary and food science professionals. [ABSTRACT FROM AUTHOR]

Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third chromosome genes (~40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general "house-keepin" genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ~70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system. [ABSTRACT FROM AUTHOR]

Coronary artery disease is the leading cause of death worldwide. Determining the coronary artery developmental program could aid understanding of the disease and lead to new treatments, but many aspects of the process, including their developmental origin, remain obscure. Here we show, using histological and clonal analysis in mice and cardiac organ culture, that coronary vessels arise from angiogenic sprouts of the sinus venosus—the vein that returns blood to the embryonic heart. Sprouting venous endothelial cells dedifferentiate as they migrate over and invade the myocardium. Invading cells differentiate into arteries and capillaries; cells on the surface redifferentiate into veins. These results show that some differentiated venous cells retain developmental plasticity, and indicate that position-specific cardiac signals trigger their dedifferentiation and conversion into coronary arteries, capillaries and veins. Understanding this new reprogramming process and identifying the endogenous signals should suggest more natural ways of engineering coronary bypass grafts and revascularizing the heart. [ABSTRACT FROM AUTHOR]

Insects have an open circulatory system in which the heart pumps blood (hemolymph) into the body cavity, where it directly bathes the internal organs and epidermis. The blood contains free and tissue-bound immune cells that function in the inflammatory response. Here, we use live imaging of transgenic Drosophila larvae with fluorescently labeled blood cells (hemocytes) to investigate the circulatory dynamics of larval blood cells and their response to tissue injury. We find that, under normal conditions, the free cells rapidly circulate, whereas the tissue-bound cells are sessile. After epidermal wounding, tissue-bound cells around the wound site remain sessile and unresponsive, whereas circulating cells are rapidly recruited to the site of damage by adhesive capture. After capture, these cells distribute across the wound, appear phagocytically active, and are subsequently released back into circulation by the healing epidermis. The results demonstrate that circulating cells function as a surveillance system that monitors larval tissues for damage, and that adhesive capture, an important mechanism of recruitment of circulating cells to inflammatory sites in vertebrates, is shared by insects and vertebrates despite the vastly different architectures of their circulatory systems. [ABSTRACT FROM AUTHOR]

Mammalian lungs are branched networks containing thousands to millions of airways arrayed in intricate patterns that are crucial for respiration. How such trees are generated during development, and how the developmental patterning information is encoded, have long fascinated biologists and mathematicians. However, models have been limited by a lack of information on the normal sequence and pattern of branching events. Here we present the complete three-dimensional branching pattern and lineage of the mouse bronchial tree, reconstructed from an analysis of hundreds of developmental intermediates. The branching process is remarkably stereotyped and elegant: the tree is generated by three geometrically simple local modes of branching used in three different orders throughout the lung. We propose that each mode of branching is controlled by a genetically encoded subroutine, a series of local patterning and morphogenesis operations, which are themselves controlled by a more global master routine. We show that this hierarchical and modular programme is genetically tractable, and it is ideally suited to encoding and evolving the complex networks of the lung and other branched organs. [ABSTRACT FROM AUTHOR]

Fluorescein diacetate (FDA) was used as a vital stain to assay membrane integrity (cell viability) in mesocarp tissue of the developing grape (Vitis vinifera L.) berry in order to test the hypothesis that there is a substantial loss of compartmentation in these cells during ripening. This technique was also used to determine whether loss of viability was associated with symptoms of a ripening disorder known as berry shrivel. FDA fluorescence of berry cells was rapid, bright, and stable for over 1 h at room temperature. Confocal microscopy detected FDA staining through two to three intact surface cell layers (300–400 μm) of bisected berries, and showed that the fluorescence was confined to the cytoplasm, indicating the maintenance of integrity in both cytoplasmic as well as vacuolar membranes, and the presence of active cytoplasmic esterases. FDA clearly discriminated between living cells and freeze-killed cells, and exhibited little, if any, non-specific staining. Propidium iodide and DAPI, both widely used to assess cell viability, were unable to discriminate between living and freeze-killed cells, and did not specifically stain the nuclei of dead cells. For normally developing berries under field conditions there was no evidence of viability loss until about 40 d after veraison, and the majority (80%) of mesocarp cells remained viable past commercial harvest (26 °Brix). These results are inconsistent with current models of grape berry development which hypothesize that veraison is associated with a general loss of compartmentation in mesocarp cells. The observed viability loss was primarily in the locule area around the seeds, suggesting that a localized loss of viability and compartmentation may occur as part of normal fruit development. The cell viability of berry shrivel-affected berries was similar to that of normally developing berries until the onset of visible symptoms (i.e. shrivelling), at which time viability declined in visibly shrivelled berries. Berries with extensive shrivelling exhibited very low cell viability (15%). [ABSTRACT FROM PUBLISHER]

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of Drosophila ‘photoshop’ mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3′ UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells. [ABSTRACT FROM AUTHOR]

Many organs are composed of tubular networks that arise by branching morphogenesis in which cells bud from an epithelium and organize into a tube. Fibroblast growth factors (FGFs) and other signalling molecules have been shown to guide branch budding and outgrowth, but it is not known how epithelial cells coordinate their movements and morphogenesis. Here we use genetic mosaic analysis in Drosophila melanogaster to show that there are two functionally distinct classes of cells in budding tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive a secondary signal to follow the lead cells and form a tube. These roles are not pre-specified; rather, there is competition between cells such that those with the highest FGF receptor activity take the lead positions, whereas those with less FGF receptor activity assume subsidiary positions and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibition that prevents extra cells from assuming the lead. There may also be cooperation between budding cells, because in a mosaic epithelium, cells that cannot respond to the chemoattractant, or respond only poorly, allow other cells in the epithelium to move ahead of them. [ABSTRACT FROM AUTHOR]

Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3'-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins. [ABSTRACT FROM AUTHOR]

Many organs are composed of branched networks of epithelial tubes that transport vital fluids or gases. The proper size and shape of tubes are crucial for their transport function, but the molecular processes that govern tube size and shape are not well understood. Here we show that three genes required for tracheal tube morphogenesis in Drosophila melanogaster encode proteins involved in the synthesis and accumulation of chitin, a polymer of N-acetyl-β-D-glucosamine that serves as a scaffold in the rigid extracellular matrix of insect cuticle. In all three mutants, developing tracheal tubes bud and extend normally, but the epithelial walls of the tubes do not expand uniformly, and the resultant tubes are grossly misshapen, with constricted and distended regions all along their lengths. The genes are expressed in tracheal cells during the expansion process, and chitin accumulates in the lumen of tubes, forming an expanding cylinder that we propose coordinates the behavior of the surrounding tracheal cells and stabilizes the expanding epithelium. These findings show that chitin regulates epithelial tube morphogenesis, in addition to its classical role protecting mature epithelia. [ABSTRACT FROM AUTHOR]

To establish a genetic system to study postembryonic wound healing, we characterized epidermal wound healing in Drosophila larvae. Following puncture wounding, larvae begin to bleed but within an hour a plug forms in the wound gap. Over the next couple of hours the outer part of the plug melanizes to form a scab, and epidermal cells surrounding the plug orient toward it and then fuse to form a syncytium. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. During this time, the Jun N-terminal kinase (JNK) pathway is activated in a gradient emanating out from the wound, and the epidermal cells spread along or through the wound plug to reestablish a continuous epithelium and its basal lamina and apical cuticle lining. Inactivation of the JNK pathway inhibits epidermal spreading and reepithelialization but does not affect scab formation or other wound healing responses. Conversely, mutations that block scab formation, and a scabless wounding procedure, provide evidence that the scab stabilizes the wound site but is not required to initiate other wound responses. However, in the absence of a scab, the JNK pathway is hyperinduced, reepithelialization initiates but is not always completed, and a chronic wound ensues. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that the responses are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. Cell biological and molecular parallels to vertebrate wound healing lead us to speculate that wound healing is an ancient response that has diversified during evolution. [ABSTRACT FROM AUTHOR]

To establish a genetic system to study postembryonic wound healing, we characterized epidermal wound healing in Drosophila larvae. Following puncture wounding, larvae begin to bleed but within an hour a plug forms in the wound gap. Over the next couple of hours the outer part of the plug melanizes to form a scab, and epidermal cells surrounding the plug orient toward it and then fuse to form a syncytium. Subsequently, more-peripheral cells orient toward and fuse with the central syncytium. During this time, the Jun N-terminal kinase (JNK) pathway is activated in a gradient emanating out from the wound, and the epidermal cells spread along or through the wound plug to reestablish a continuous epithelium and its basal lamina and apical cuticle lining. Inactivation of the JNK pathway inhibits epidermal spreading and reepithelialization but does not affect scab formation or other wound healing responses. Conversely, mutations that block scab formation, and a scabless wounding procedure, provide evidence that the scab stabilizes the wound site but is not required to initiate other wound responses. However, in the absence of a scab, the JNK pathway is hyperinduced, reepithelialization initiates but is not always completed, and a chronic wound ensues. The results demonstrate that the cellular responses of wound healing are under separate genetic control, and that the responses are coordinated by multiple signals emanating from the wound site, including a negative feedback signal between scab formation and the JNK pathway. Cell biological and molecular parallels to vertebrate wound healing lead us to speculate that wound healing is an ancient response that has diversified during evolution. A powerful new system for studying wound healing in the fruitfly is helping to unearth the genetic and cellular requirements of the healing process. [ABSTRACT FROM AUTHOR]

Many organs including the mammalian lung and vascular system consist of branched tubular networks that transport essential gases or fluids, but the genetic programs that control the development of these complex three-dimensional structures are not well understood. The Drosophila melanogaster tracheal (respiratory) system is a network of interconnected epithelial tubes that transports oxygen and other gases in the body and provides a paradigm of branching morphogenesis. It develops by sequential sprouting of primary, secondary, and terminal branches from an epithelial sac of ∼80 cells in each body segment of the embryo. Mapping of the cell movements and shape changes during the sprouting process has revealed that distinct mechanisms of epithelial migration and tube formation are used at each stage of branching. Genetic dissection of the process has identified a general program in which a fibroblast growth factor (FGF) and fibroblast growth factor receptor (FGFR) are used repeatedly to control branch budding and outgrowth. At each stage of branching, the mechanisms controlling FGF expression and the downstream signal transduction pathway change, altering the pattern and structure of the branches that form. During terminal branching, FGF expression is regulated by hypoxia, ensuring that tracheal structure matches cellular oxygen need. A branch diversification program operates in parallel to the general budding program: Regional signals locally modify the general program, conferring specific structural features and other properties on individual branches, such as their substrate outgrowth preferences, differences in tube size and shape, and the ability to fuse to other branches to interconnect the network. [ABSTRACT FROM AUTHOR]

Nuclear lamins are intermediate filaments that compose the nuclear lamina ? the filamentous meshwork underlying the inner nuclear membrane ? and are required for nuclear assembly, organization and maintenance. Here we present evidence that a nuclear lamin is also required for cytoplasmic organization in two highly polarized cell types. Zygotic loss-of-function mutations in the Drosophila gene encoding the principal lamin (Dm0) disrupt the directed outgrowth of cytoplasmic extensions from terminal cells of the tracheal system. Germline mutant clones disrupt dorsal?ventral polarity of the oocyte. In mutant oocytes, transcripts of the dorsal determinant Gurken, a transforming growth factor-α homologue, fail to localize properly around the anterodorsal surface of the oocyte nucleus; their ventral spread results in dorsalized eggs that resemble those of the classical dorsalizing mutations squid and fs(1)K10. The requirement of a nuclear lamin for cytoplasmic as well as nuclear organization has important implications for both the cellular functions of lamins and the pathogenesis of human diseases caused by lamin mutations. [ABSTRACT FROM AUTHOR]

Tests the model that intercellular signalling is involved in intrasegmental patterning in Drosophila melonagaster development. Switching off of engrailed (en) expression; Locally active signal preventing loss of expression in purified wingless (wg)-expressing cells; Signalling activity of heterologous cells engineered to produce wg, indicating that wg protein is involved in the signal.

The article discusses a study on back-up role of differentiated stem cells of the lung and stomach in context of two research published in 2013 issue of the journal "Cell." The study included genetic techniques for finding expression of marker of intestinal stem cells Troy, differentiation of airway secretory cells Clara cells, and bulk-labelling strategy. The study concluded that regenerative medicine will eliminate the need for introduction of cellular reprogramming factors.

Epithelial tubes that compose many organs are typically long lasting, except under specific developmental and physiological conditions when network remodeling occurs. Although there has been progress elucidating mechanisms of tube formation, little is known of the mechanisms that maintain tubes and destabilize them during network remodeling. Here, we describe Drosophila tendrils mutations that compromise maintenance of tracheal terminal branches, fine gauge tubes formed by tracheal terminal cells that ramify on and adhere tightly to tissues in order to supply them with oxygen. Homozygous tendrils terminal cell clones have fewer terminal branches than normal but individual branches contain multiple convoluted lumens. The phenotype arises late in development: terminal branches bud and form lumens normally early in development, but during larval life lumens become convoluted and mature branches degenerate. Their lumens, however, are retained in the remaining branches, resulting in the distinctive multi-lumen phenotype. Mapping and molecular studies demonstrate that tendrils is allelic to rhea, which encodes Drosophila talin, a large cytoskeletal protein that links integrins to the cytoskeleton. Terminal cells mutant for myospheroid, the major Drosophila β-integrin, or doubly mutant for multiple edematous wings and inflated α-integrins, also show the tendrils phenotype, and localization of myospheroid β-integrin protein is disrupted in tendrils mutant terminal cells. The results provide evidence that integrin-talin adhesion complexes are necessary to maintain tracheal terminal branches and luminal organization. Similar complexes may stabilize other tubular networks and may be targeted for inactivation during network remodeling events. [ABSTRACT FROM AUTHOR]

Drosophila Corkscrew protein and its vertebrate ortholog SHP-2 (now known as Ptpn11) positively modulate receptor tyrosine kinase (RTK) signaling during development, but how these tyrosine phosphatases promote tyrosine kinase signaling is not well understood. Sprouty proteins are tyrosine-phosphorylated RTK feedback inhibitors, but their regulation and mechanism of action are also poorly understood. Here, we show that Corkscrew/SHP-2 proteins control Sprouty phosphorylation and function. Genetic experiments demonstrate that Corkscrew/SHP-2 and Sprouty proteins have opposite effects on RTK-mediated developmental events in Drosophila and an RTK signaling process in cultured mammalian cells, and the genes display dose-sensitive genetic interactions. In cultured cells, inactivation of SHP-2 increases phosphorylation on the critical tyrosine of Sprouty 1. SHP-2 associates in a complex with Sprouty 1 in cultured cells and in vitro, and a purified SHP-2 protein dephosphorylates the critical tyrosine of Sprouty 1. Substrate-trapping forms of Corkscrew bind Sprouty in cultured Drosophila cells and the developing eye. These results identify Sprouty proteins as in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases and show how Corkscrew/SHP-2 proteins can promote RTK signaling by inactivating a feedback inhibitor. We propose that this double-negative feedback circuit shapes the output profile of RTK signaling events. [ABSTRACT FROM AUTHOR]