14 research outputs found
Resource: A multiâspecies multiâtimepoint transcriptome database and webpage for the pineal gland and retina
The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.Fil: Chang, Eric. National Instituto of Child Health & Human Development; Estados UnidosFil: Fu, Cong. National Instituto of Child Health & Human Development; Estados UnidosFil: Coon, Steven L.. National Instituto of Child Health & Human Development; Estados UnidosFil: Alon, Shahar. No especifĂca;Fil: Bozinoski, Marjan. No especifĂca;Fil: Breymaier, Matthew. National Instituto of Child Health & Human Development; Estados UnidosFil: Bustos, Diego Martin. Consejo Nacional de Investigaciones CientĂficas y TĂŠcnicas. Centro CientĂfico TecnolĂłgico Conicet - Mendoza. Instituto de HistologĂa y EmbriologĂa de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias MĂŠdicas. Instituto de HistologĂa y EmbriologĂa de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Clokie, Samuel J.. National Instituto of Child Health & Human Development; Estados UnidosFil: Gothilf, Yoav. No especifĂca;Fil: Esnault, Caroline. National Instituto of Child Health & Human Development; Estados UnidosFil: Iuvone, P. Michael. Emory University School of Medicine; Estados UnidosFil: Mason, Christopher E.. No especifĂca;Fil: Ochocinska, Margaret J.. National Instituto of Child Health & Human Development; Estados UnidosFil: Tovin, Adi. No especifĂca;Fil: Wang, Charles. Loma Linda University; Estados UnidosFil: Xu, Pinxian. No especifĂca;Fil: Zhu, Jinhang. No especifĂca;Fil: Dale, Ryan. National Instituto of Child Health & Human Development; Estados UnidosFil: Klein, David C.. National Instituto of Child Health & Human Development; Estados Unido
Using the Tg(nrd:egfp)/albino Zebrafish Line to Characterize In Vivo Expression of neurod
In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in MĂźller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults
NIH Workshop 2018: Towards Minimally Invasive or Noninvasive Approaches to Assess Tissue Oxygenation Pre- and Post-transfusion
Because blood transfusion is one of the most common therapeutic interventions in hospitalized patients, much recent research has focused on improving the storage quality in vitro of donor red blood cells (RBCs) that are then used for transfusion. However, there is a significant need for enhancing our understanding of the efficacy of the transfused RBCs in vivo. To this end, the NIH sponsored a one-and-a-half-day workshop that brought together experts in multiple disciplines relevant to tissue oxygenation (eg, transfusion medicine, critical care medicine, cardiology, neurology, neonatology and pediatrics, bioengineering, biochemistry, and imaging). These individuals presented their latest findings, discussed key challenges, and aimed to identify opportunities for facilitating development of new technologies and/or biomarker panels to assess tissue oxygenation in a minimally-invasive to non-invasive fashion, before and after RBC transfusion
NIH Workshop 2018: Towards Minimally-invasive or Non-invasive Approaches to Assess Tissue Oxygenation Pre- and Post-Transfusion
Because blood transfusion is one of the most common therapeutic interventions in hospitalized patients, much recent research has focused on improving the storage quality in vitro of donor red blood cells (RBCs) that are then used for transfusion. However, there is a significant need for enhancing our understanding of the efficacy of the transfused RBCs in vivo. To this end, the NIH sponsored a one-and-a-half-day workshop that brought together experts in multiple disciplines relevant to tissue oxygenation (e.g., transfusion medicine, critical care medicine, cardiology, neurology, neonatology and pediatrics, bioengineering, biochemistry, and imaging). These individuals presented their latest findings, discussed key challenges, and aimed to construct recommendations for facilitating development of new technologies and/or biomarker panels to assess tissue oxygenation in a minimally-invasive to non-invasive fashion, before and after RBC transfusion.
The workshop was structured into four sessions: (1) Global Perspective; (2) Organ Systems; (3) Neonatology; and (4) Emerging Technologies. The first day provided an overview of current approaches in the clinical setting, both from a global perspective, including the use of metabolomics for studying RBCs and tissue perfusion, and from a more focused perspective, including tissue oxygenation assessments in neonates and in specific adult organ systems. The second day focused on emerging technologies, which could be applied pre- and post-RBC transfusion, to assess tissue oxygenation in minimally-invasive or non-invasive ways. Each day concluded with an open-microphone discussion among the speakers and workshop participants. The workshop presentations and ensuing interdisciplinary discussions highlighted the potential of technologies to combine global âomicsâ signatures with additional measures (e.g., thenar eminence measurements or various imaging methods) to predict which patients could potentially benefit from a RBC transfusion and whether the ensuing RBC transfusion was effective. The discussions highlighted the need for collaborations across the various disciplines represented at the meeting to leverage existing technologies and to develop novel approaches for assessing RBC transfusion efficacy in various clinical settings.
Although the Workshop took place in April, 2018, the concepts described and the ensuing discussions were, perhaps, even more relevant in April, 2020, at the time of writing this manuscript, during the explosive growth of the COVID-19 pandemic in the United States. Thus, issues relating to maintaining and improving tissue oxygenation and perfusion are especially pertinent because of the extensive pulmonary damage resulting from SARS-CoV-2 infection [1], compromises in perfusion caused by thrombotic-embolic phenomena [2], and damage to circulating RBCs, potentially compromising their oxygen-carrying capacity [3]. The severe end organ effects of SARS-CoV-2 infection mandate even more urgency for improving our understanding of tissue perfusion and oxygenation, improve methods for measuring and monitoring them, and develop novel ways of enhancing them
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Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases.
Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.Funding information:
National Heart, Lung, and Blood Institute,
Grant/Award Numbers: HL 122596, HL124021,
HL124074, HL128297, HL141080, HL155346-01,
R35HL150807, R56HL141206
Prithu Sundd was supported by NIH-NHLBI R01 grants (HL128297 and HL141080) and 18TPA34170588 from American Heart
Association. Stephen Y. Chan was supported by NIH grants R01 HL124021 and HL 122596 as well as AHA grant 18EIA33900027.
SuamyaDaswas supported by NIH grants R35HL150807, UH3 TR002878 andAHASFRN35120123. ZhenjiaWangwas supported
by NIH grant (R01EB027078). Pilar MartĂn was supported by MCIN-ISCIII-Fondo de InvestigaciĂłn Sanitaria grant PI22/01759.
KennethW.Witwer was supported in part by NIH grants R01AI144997, R01DA047807, R33MH118164 andUH3CA241694. Tianji
Chen was supported by AHA Career Development Award 18CDA34110301, Gilead Sciences Research Scholars Program in PAH,
NIH-NHLBI grant R56HL141206 and Chicago Biomedical ConsortiumCatalyst Award. EduardoMarbĂĄn was supported byNIH
R01 HL124074 and HL155346-01.S
Retinal sections from adult Tg(<i>nrd:egfp</i>)/<i>albino</i> zebrafish over a time course of light treatment and immunolabeled with EGFP (green) to visualize the <i>nrd:egfp</i> transgene and co-labeled with either PCNA (A, C, D, I, J, Jâ˛, K, L, Lâ˛, M, N) or Glutamine Synthetase (B, G, H).
<p>(A) At 48 hours after light onset, almost all rod photoreceptors have been ablated and proliferating cells can be seen in the in the INL. Nuclei are labeled in blue with TO-PRO-3 (TP3). (B) At this time point, MĂźller glial cells express Glutamine Synthetase (G.S., red, arrow), and do not co-label with EGFP (arrowhead). (C) At 72 hours after light onset, clusters of proliferating progenitors begin to migrate towards the ONL (arrowheads). (D) At 96 hours post light onset, PCNA-positive progenitors (arrowheads) are present in both in INL and ONL, with occasional aberrant migration to the GCL. (EâF) At 72 and 96 hours after light onset, respectively, clusters of progenitors weakly express EGFP (arrowheads). (F) At 96 hours after light onset, EGFP is observed in a newly-formed and disorganized ONL. (GâH) At 72 and 96 hours after light onset, respectively, weakly-EGFP-positive clusters in the INL (arrowheads) down-regulated Glutamine Synthetase. MĂźller glial cells that did not re-enter the cell cycle strongly express G.S. (arrows), but are EGFP-negative. (IâJâ˛) At 72 hours after light onset, weakly-EGFP-positive cells in both the INL and ONL co-label with PCNA. The box in I represents the PCNA and EGFP labeling shown in J and Jâ˛, respectively. (KâLâ˛) At 96 hours after light onset, weakly-EGFP-positive cells in both the INL (arrowheads) and ONL continue to co-label with PCNA. The box in K represents the PCNA and EGFP labeling shown in L and Lâ˛, respectively. (M) At 7 days after light onset, a subset of PCNA-positive progenitors in the ONL co-label with EGFP (N) At 11 days after light onset, only a few PCNA-positive progenitors remain in the ONL. EGFP can be visualized in rod photoreceptors and newly-formed rod inner segments (arrowhead).</p
Retinal sections from embryonic Tg(<i>nrd:egfp</i>)<i>/albino</i> zebrafish immunolabeled with PCNA (red) and EGFP (green).
<p>(A) At 42 hpf, EGFP is detected throughout the retinal neuroepithelium in the central retina. PCNA immunolocalization, showing proliferating cells, is primarily restricted to the CMZ (arrows). In the overlapping region of PCNA and EGFP co-labeling can be visualized (arrowhead). (B) At 72 hpf, EGFP is detected in a subset of ganglion, amacrine and bipolar cells, and is not present in the outer nuclear layer. Proliferating cells are restricted to the CMZ (arrows), and there is no evidence of PCNA and EGFP co-immunolabeling. (C) At 96 hpf, there is persistent expression of EGFP detected in a subset of ganglion, amacrine and bipolar cells. Proliferating cells are restricted to the CMZ (arrows) and do not co-label with the transgene. Scale bars: 25 microns (A) and 50 microns (B, C).</p
Wholemount brightfield and flourescent images showing <i>nrd:egfp</i> transgene expression in the adult caudal tail fin.
<p>(A) Brightfield image of the adult caudal fin. (B) Corresponding fluorescent image to panel A. EGFP expression is visualized in the nerve coursing through each bony hemiray of the caudal fin, however at this level of magnification, it is difficult to visualize. (CâCâ˛) High magnification brightfield and corresponding fluorescent overlay showing multiple bony lepidotrichia. The arrows point to the nerve running within each bony hemiray and arrowheads point to EGFP-positive ganglia associated with the nerve. (D and Dâ˛) A section of a single bony ray immunolabeled with EGFP to show the transgene and co-labeled with TO-PRO-3 to show all nuclei (magenta). The white line in Panel D shows the location of the nerve. Epâ=âEpithelium, Bnâ=âbony ray, CTâ=âconnective tissue.</p
Retinal sections from adult Tg(<i>nrd:egfp</i>)/<i>albino</i> zebrafish at 96 hours after light onset showing transgene expression (green) and EdU labeling (red).
<p>(A). Schematic representation of EdU injections during the light time course with corresponding immunolocalization shown in Panels BâDâ˛. (BâBâ˛) EGFP and EGFP/EdU co-labeling, respectively, showing weakly-EGFP-positive cells in the INL (arrowheads) and ONL co-label with EdU. The boxes in BⲠrepresent the panels shown in CâDâ˛. (C) Higher magnification image of the box shown in the top right of Panel Bâ˛. Note that the weakly-EGFP-positive progenitors co-label with EdU (arrowheads), but strongly-EGFP-positive rod nuclei (arrow) are EdU-negative. (DâDâ˛) Higher magnification image of the box shown in the left of Panel Bâ˛, showing EGFP and EdU immunolocalization, respectively, in a cluster of INL progenitors. (E) Schematic representation of a single EdU injection prior starting the light treatment in order to label a subset of the progenitors. (FâFâ˛). High magnification confocal microscopy showing EGFP and EGFP/EdU co-labeling in the ONL at 96 hours after light onset. An individual EdU-positive cell in the ONL (arrowhead) co-labels with weak EGFP expression. The strongly-EGFP-positive cell, in contrast, is EdU-negative (arrow).</p
High magnification images of retinal sections from adult Tg(<i>nrd:egfp</i>)<i>/albino</i> zebrafish over a time course of light treatment.
<p>Sections were immunolabeled with EGFP (green) to visualize the <i>nrd:egfp</i> transgene and Zpr-3 (red) to visualize rod photoreceptors. (A) Prior to light treatment (0 hr), EGFP co-labels with Zpr-3 and is observed in rod photoreceptor soma, rod inner segments (RIS) and rod outer segments (ROS). (B) At 48 hours after light onset, the ROS and RIS are almost completely destroyed and only a few EGFP-positive cells remain in the ONL. (C) At 72 hours after light onset, newly-formed rod progenitor cells are present in the ONL. These could be readily discerned from existing rod photoreceptors due to their comparably weak expression of the transgene (inset shows new rod progenitor on the left). (D) At 96 hours after light onset, a greater number of new regenerated cells are present in the ONL, although it still somewhat disorganized. (E) At 7 days after light onset, newly differentiated rod photoreceptors appear more organized and greater in abundance. (F) At 11 days after light onset, EGFP is expressed in the newly formed rod photoreceptors and co-labels with Zpr-3-positive and newly-formed RIS and R0S. Scale bar: 50 microns (AâF).</p