Decision letter: Computer assisted detection of axonal bouton structural plasticity in in vivo time-lapse images

The ability to measure minute structural changes in neural circuits is essential for long-term in vivo imaging studies. Here, we propose a methodology for detection and measurement of structural changes in axonal boutons imaged with time-lapse two-photon laser scanning microscopy (2PLSM). Correlative 2PLSM and 3D electron microscopy (EM) analysis, performed in mouse barrel cortex, showed that the proposed method has low fractions of false positive/negative bouton detections (2/0 out of 18), and that 2PLSM-based bouton weights are correlated with their volumes measured in EM (r = 0.93). Next, the method was applied to a set of axons imaged in quick succession to characterize measurement uncertainty. The results were used to construct a statistical model in which bouton addition, elimination, and size changes are described probabilistically, rather than being treated as deterministic events. Finally, we demonstrate that the model can be used to quantify significant structural changes in boutons in long-term imaging experiments.

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Integrating structural and functional imaging for computer assisted detection of prostate cancer on multi-protocol in vivo 3 Tesla MRI

10.1117/12.811899 ◽

2009 ◽

Cited By ~ 13

Author(s):

Satish Viswanath ◽

B. Nicolas Bloch ◽

Mark Rosen ◽

Jonathan Chappelow ◽

Robert Toth ◽

...

Keyword(s):

Prostate Cancer ◽

Functional Imaging ◽

3 Tesla ◽

Computer Assisted ◽

Computer Assisted Detection ◽

3 Tesla Mri

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In Vivo Time-Lapse Imaging and Analysis of Dendritic Structural Plasticity in Xenopus laevis Tadpoles

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot106781 ◽

2021 ◽

Vol 2022 (1) ◽

pp. pdb.prot106781

Author(s):

Hai-yan He ◽

Chih-Yang Lin ◽

Hollis T. Cline

Keyword(s):

Data Analysis ◽

Xenopus Laevis ◽

Dynamic Analysis ◽

Structural Changes ◽

Structural Plasticity ◽

Time Lapse ◽

High Temporal Resolution ◽

Dendritic Arbors ◽

Time Lapse Imaging

In vivo time-lapse imaging of complete dendritic arbor structures in tectal neurons of Xenopus laevis tadpoles has served as a powerful in vivo model to study activity-dependent structural plasticity in the central nervous system during early development. In addition to quantitative analysis of gross arbor structure, dynamic analysis of the four-dimensional data offers particularly valuable insights into the structural changes occurring in subcellular domains over experience/development-driven structural plasticity events. Such analysis allows not only quantifiable characterization of branch additions and retractions with high temporal resolution but also identification of the loci of action. This allows for a better understanding of the spatiotemporal association of structural changes to functional relevance. Here we describe a protocol for in vivo time-lapse imaging of complete dendritic arbors from individual neurons in the brains of anesthetized tadpoles with two-photon microscopy and data analysis of the time series of 3D dendritic arbors. For data analysis, we focus on dynamic analysis of reconstructed neuronal filaments using a customized open source computer program we developed (4D SPA), which allows aligning and matching of 3D neuronal structures across different time points with greatly improved speed and reliability. File converters are provided to convert reconstructed filament files from commercial reconstruction software to be used in 4D SPA. The program and user manual are publicly accessible and operate through a graphical user interface on both Windows and Mac OSX.

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Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084120 ◽

1978 ◽

Vol 36 (2) ◽

pp. 650-651

Author(s):

Raul I. Garcia ◽

Evelyn A. Flynn ◽

George Szabo

Keyword(s):

Direct Injection ◽

Skin Pigmentation ◽

Freeze Fracture ◽

Pigment Granule ◽

Time Lapse ◽

Ultrastructural Level ◽

Lanthanum Tracer ◽

A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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Cardiovascular Effects of Micromeria graeca (L.) Benth. ex Rchb in Normotensive and Hypertensive Rats

Endocrine Metabolic & Immune Disorders - Drug Targets ◽

10.2174/1871530319666191206163136 ◽

2020 ◽

Vol 20 (8) ◽

pp. 1253-1261

Author(s):

Mourad Akdad ◽

Mohamed Eddouks

Keyword(s):

Blood Pressure ◽

Cardiovascular System ◽

Arterial Blood Pressure ◽

Antihypertensive Activity ◽

Computer Assisted ◽

Hypertensive Rats ◽

Vasorelaxant Effect ◽

Arterial Blood

Aims: The present study was performed in order to analyze the antihypertensive activity of Micromeria graeca (L.) Benth. ex Rchb. Background: Micromeria graeca (L.) Benth. ex Rchb is an aromatic and medicinal plant belonging to the Lamiaceae family. This herb is used to treat various pathologies such as cardiovascular disorders. Meanwhile, its pharmacological effects on the cardiovascular system have not been studied. Objective: The present study aimed to evaluate the effect of aqueous extract of aerial parts of Micromeria graeca (AEMG) on the cardiovascular system in normotensive and hypertensive rats. Methods: In this study, the cardiovascular effect of AEMG was evaluated using in vivo and in vitro investigations. In order to assess the acute effect of AEMG on the cardiovascular system, anesthetized L-NAME-hypertensive and normotensive rats received AEMG (100 mg/kg) orally and arterial blood pressure parameters were monitored during six hours. In the sub-chronic study, rats were orally treated for one week, followed by blood pressure assessment during one week of treatment. Blood pressure was measured using a tail-cuff and a computer-assisted monitoring device. In the second experiment, isolated rat aortic ring pre-contracted with Epinephrine (EP) or KCl was used to assess the vasorelaxant effect of AEMG. Results: Oral administration of AEMG (100 mg/kg) provoked a decrease of arterial blood pressure parameters in hypertensive rats. In addition, AEMG induced a vasorelaxant effect in thoracic aortic rings pre-contracted with EP (10 μM) or KCl (80 mM). This effect was attenuated in the presence of propranolol and methylene blue. While in the presence of glibenclamide, L-NAME, nifedipine or Indomethacin, the vasorelaxant effect was not affected. Conclusion: This study showed that Micromeria graeca possesses a potent antihypertensive effect and relaxes the vascular smooth muscle through β-adrenergic and cGMP pathways.

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Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

Nature Communications ◽

10.1038/s41467-021-24486-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joonas A. Jamsen ◽

Akira Sassa ◽

Lalith Perera ◽

David D. Shock ◽

William A. Beard ◽

...

Keyword(s):

Mammalian Cells ◽

Time Lapse ◽

Strand Break ◽

Structural Basis ◽

End Joining ◽

Strand Breaks ◽

Nucleotide Pools ◽

Nucleotide Insertion ◽

Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

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Automated and Computer-Assisted Detection, Classification, and Diagnosis of Diabetic Retinopathy

Telemedicine Journal and e-Health ◽

10.1089/tmj.2020.0008 ◽

2020 ◽

Vol 26 (4) ◽

pp. 544-550 ◽

Cited By ~ 1

Author(s):

Michael D. Abràmoff ◽

Theodore Leng ◽

Daniel S.W. Ting ◽

Kyu Rhee ◽

Mark B. Horton ◽

...

Keyword(s):

Diabetic Retinopathy ◽

Computer Assisted ◽

Computer Assisted Detection

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Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽

10.1182/blood-2010-01-264382 ◽

2010 ◽

Vol 116 (6) ◽

pp. 909-914 ◽

Cited By ~ 114

Author(s):

Enid Yi Ni Lam ◽

Christopher J. Hall ◽

Philip S. Crosier ◽

Kathryn E. Crosier ◽

Maria Vega Flores

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Fluorescent Protein ◽

Living Organism ◽

Time Lapse ◽

Dorsal Aorta ◽

Transgenic Zebrafish ◽

Hematopoietic Stem ◽

Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

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A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypii

Molecular Biology of the Cell ◽

10.1091/mbc.e04-02-0104 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4622-4632 ◽

Cited By ~ 54

Author(s):

Yasmina Bauer ◽

Philipp Knechtle ◽

Jürgen Wendland ◽

Hanspeter Helfer ◽

Peter Philippsen

Keyword(s):

Fluorescent Protein ◽

Hyphal Growth ◽

Time Lapse ◽

Growth Direction ◽

Ashbya Gossypii ◽

Growth Guidance ◽

Characteristic Features ◽

Green Fluorescent ◽

Hyphal Tip

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.

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