scholarly journals Optical flow analysis reveals that Kinesin-mediated advection impacts on the orientation of microtubules in the Drosophila oocyte

2019 ◽  
Author(s):  
Maik Drechsler ◽  
Lukas F. Lang ◽  
Layla Al-Khatib ◽  
Hendrik Dirks ◽  
Martin Burger ◽  
...  
Keyword(s):  
Optical Flow ◽  
Cytoplasmic Streaming ◽  
Flow Analysis ◽  
Growth Direction ◽  
Model Systems ◽  
Microtubule Orientation ◽  
A Cell ◽  
The Impact

ABSTRACTThe orientation of microtubule networks is exploited by motors to deliver cargoes to specific intracellular destinations, and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of microtubule filaments. In cells however, microtubules are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. Here we implement variational optical flow analysis as a new approach to analyze the polarity of microtubules in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing microtubule orientation from confocal movies, we find that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, we find that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of microtubule plus ends is also altered. These findings lead us to propose that cytoplasmic streaming - and thus motion by advection – contributes to the correct orientation of MTs in vivo. Finally, we propose a possible mechanism for a specialised cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds; to counteract the recruitment of Kinesin to microtubules.HIGHLIGHT SUMMARYCytoskeletal networks do not exist in isolation, but experience crowded and dynamic intracellular environments. However, microtubule-environment interactions are not well understood, and such system-environment interactions are an unresolved question in biology that demands bridging across disciplines. Here we introduce an optical flow motion estimation approach to study microtubule orientation in the Drosophila oocyte, a cell displaying substantial cytoplasmic streaming. We show that microtubule polarity is affected by the regime of these flows, and furthermore, that the presence of flows is necessary for MTs to adopt their proper polarity. With these findings we are contributing to further understanding how microtubules organize in their impacting natural environment.

scholarly journals Optical flow analysis reveals that Kinesin-mediated advection impacts the orientation of microtubules in the Drosophila oocyte

2020 ◽  
Vol 31 (12) ◽  
pp. 1246-1258 ◽  
Author(s):  
Maik Drechsler ◽  
Lukas F. Lang ◽  
Layla Al-Khatib ◽  
Hendrik Dirks ◽  
Martin Burger ◽  
...  
Keyword(s):  
Motion Estimation ◽  
Optical Flow ◽  
Cytoplasmic Streaming ◽  
Flow Analysis ◽  
Flow Motion ◽  
A Cell

Here we introduce an optical flow motion estimation approach to study microtubule (MT) orientation in the Drosophila oocyte, a cell displaying substantial cytoplasmic streaming. We show that MT polarity is affected by the regime of these flows and, furthermore, that the presence of flows is necessary for MTs to adopt their proper polarity.

scholarly journals A Mouse Model Of Binge Alcohol Consumption andBurkholderiaInfection

2018 ◽  
Author(s):  
Victor Jimenez ◽  
Ryan Moreno ◽  
Erik Settles ◽  
Bart J Currie ◽  
Paul Keim ◽  
...  
Keyword(s):  
Alcohol Consumption ◽  
Binge Drinking ◽  
Burkholderia Pseudomallei ◽  
Alcohol Exposure ◽  
Model Systems ◽  
Phagocytic Cells ◽  
Intracellular Invasion ◽  
The Impact

AbstractBackgroundBinge drinking, a common form of alcohol consumption, is associated with increased mortality and morbidity; yet, its effects on the immune system’s ability to defend against infectious agents are poorly understood.Burkholderia pseudomallei, the causative agent of melioidosis can occur in healthy humans, yet binge alcohol use is progressively being recognized as a major risk factor. Although our previous studies demonstrated that binge alcohol exposure results in reduced alveolar macrophage function and increasedBurkholderiavirulencein vitro, no experimental studies have investigated the outcomes of binge alcohol onBurkholderiaspp. infectionin vivo.Principal FindingsWe used the close genetic relatives ofB. pseudomallei, B. thailandensisE264 andB. vietnamiensis, as useful BSL-2 model systems. Eight-week-old female C57BL/6 mice were administered alcohol comparable to human binge drinking episodes (4.4 g/kg) or PBS intraperitoneally 30 min before a non-lethal intranasal infection. In an initialB. thailandensisinfection (3 x 105), bacteria accumulated in the lungs and disseminated to the spleen in alcohol administered mice only, compared with PBS treated mice at 24 h post-infection (PI). The greatest bacterial load occurred withB. vietnamiensis(1 x 106) in lungs, spleen, and brain tissue by 72 h PI. Pulmonary cytokine expression (TNF-α, GM-CSF) decreased, while splenic cytokine (IL-10) increased in binge drunk mice. Increased lung and brain permeability was observed as early as 2 h post alcohol administrationin vivo.Trans-epithelial electrical resistance (TEER) was significantly decreased, while intracellular invasion of non-phagocytic cells increased with 0.2% v/v alcohol exposurein vitro.ConclusionsOur results indicate that a single binge alcohol dose suppressed innate immune functions and increased the ability of less virulentBurkholderiastrains to disseminate through increased barrier permeability and intracellular invasion of non-phagocytic cells.Author SummaryBurkholderia pseudomalleicauses the disease melioidosis, which occurs in most tropical regions across the globe. Exposure rarely evolves to significant disease in the absence of specific comorbidities, such as binge alcohol intoxication. In susceptible hosts, the disease is primarily manifested as pneumonic melioidosis and can be rapidly fatal if untreated. In this study, we utilizedB. thailandensis, a genetically similar strain toB. pseudomallei, and opportunisticB. vietnamiensis, a known human pathogen that utilizes similar virulence strategies asB. pseudomalleiin immunocompromised and cystic fibrosis patients. The study investigates the impact of a single binge alcohol episode on infectivity and immune responsein vivo. We show that a single binge alcohol episode prior to inhalingBurkholderiaspecies increases bacterial spread to the lungs and brain. We also identify alcohol-induced tissue permeability and epithelial cell invasion as modes of action for greater bacterial spread and survival inside the host. Our results support the public health responses being developed in melioidosis-endemic regions that emphasize the nature of binge drinking as a prime concern, especially around potential times of exposure to environmentalB. pseudomallei.

scholarly journals Angptl2 is a Marker of Cellular Senescence: The Physiological and Pathophysiological Impact of Angptl2-Related Senescence

2021 ◽  
Vol 22 (22) ◽  
pp. 12232
Author(s):  
Nathalie Thorin-Trescases ◽  
Pauline Labbé ◽  
Pauline Mury ◽  
Mélanie Lambert ◽  
Eric Thorin
Keyword(s):  
Cell Fate ◽  
Cellular Senescence ◽  
Cellular Response ◽  
Inflammatory Diseases ◽  
Age Related ◽  
A Cell ◽  
Future Work ◽  
The Impact

Cellular senescence is a cell fate primarily induced by DNA damage, characterized by irreversible growth arrest in an attempt to stop the damage. Senescence is a cellular response to a stressor and is observed with aging, but also during wound healing and in embryogenic developmental processes. Senescent cells are metabolically active and secrete a multitude of molecules gathered in the senescence-associated secretory phenotype (SASP). The SASP includes inflammatory cytokines, chemokines, growth factors and metalloproteinases, with autocrine and paracrine activities. Among hundreds of molecules, angiopoietin-like 2 (angptl2) is an interesting, although understudied, SASP member identified in various types of senescent cells. Angptl2 is a circulatory protein, and plasma angptl2 levels increase with age and with various chronic inflammatory diseases such as cancer, atherosclerosis, diabetes, heart failure and a multitude of age-related diseases. In this review, we will examine in which context angptl2 was identified as a SASP factor, describe the experimental evidence showing that angptl2 is a marker of senescence in vitro and in vivo, and discuss the impact of angptl2-related senescence in both physiological and pathological conditions. Future work is needed to demonstrate whether the senescence marker angptl2 is a potential clinical biomarker of age-related diseases.

scholarly journals Influence of Microgravity on Apoptosis in Cells, Tissues, and Other Systems In Vivo and In Vitro

2020 ◽  
Vol 21 (24) ◽  
pp. 9373
Author(s):  
Binod Prasad ◽  
Daniela Grimm ◽  
Sebastian M. Strauch ◽  
Gilmar Sidnei Erzinger ◽  
Thomas J. Corydon ◽  
...  
Keyword(s):  
Cosmic Radiation ◽  
Current Knowledge ◽  
Life Forms ◽  
Model Systems ◽  
Space Environment ◽  
Cellular Damage ◽  
The Impact ◽  
In Cells

All life forms have evolved under the constant force of gravity on Earth and developed ways to counterbalance acceleration load. In space, shear forces, buoyance-driven convection, and hydrostatic pressure are nullified or strongly reduced. When subjected to microgravity in space, the equilibrium between cell architecture and the external force is disturbed, resulting in changes at the cellular and sub-cellular levels (e.g., cytoskeleton, signal transduction, membrane permeability, etc.). Cosmic radiation also poses great health risks to astronauts because it has high linear energy transfer values that evoke complex DNA and other cellular damage. Space environmental conditions have been shown to influence apoptosis in various cell types. Apoptosis has important functions in morphogenesis, organ development, and wound healing. This review provides an overview of microgravity research platforms and apoptosis. The sections summarize the current knowledge of the impact of microgravity and cosmic radiation on cells with respect to apoptosis. Apoptosis-related microgravity experiments conducted with different mammalian model systems are presented. Recent findings in cells of the immune system, cardiovascular system, brain, eyes, cartilage, bone, gastrointestinal tract, liver, and pancreas, as well as cancer cells investigated under real and simulated microgravity conditions, are discussed. This comprehensive review indicates the potential of the space environment in biomedical research.

scholarly journals The Impact of CRISPR/Cas9 Technology on Cardiac Research: From Disease Modelling to Therapeutic Approaches

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Benedetta M. Motta ◽  
Peter P. Pramstaller ◽  
Andrew A. Hicks ◽  
Alessandra Rossini
Keyword(s):  
Genome Editing ◽  
Ethical Issues ◽  
Therapeutic Potential ◽  
Gene Knockout ◽  
Model Systems ◽  
Large Deletions ◽  
Induced Pluripotent ◽  
The Impact

Genome-editing technology has emerged as a powerful method that enables the generation of genetically modified cells and organisms necessary to elucidate gene function and mechanisms of human diseases. The clustered regularly interspaced short palindromic repeats- (CRISPR-) associated 9 (Cas9) system has rapidly become one of the most popular approaches for genome editing in basic biomedical research over recent years because of its simplicity and adaptability. CRISPR/Cas9 genome editing has been used to correct DNA mutations ranging from a single base pair to large deletions in both in vitro and in vivo model systems. CRISPR/Cas9 has been used to increase the understanding of many aspects of cardiovascular disorders, including lipid metabolism, electrophysiology and genetic inheritance. The CRISPR/Cas9 technology has been proven to be effective in creating gene knockout (KO) or knockin in human cells and is particularly useful for editing induced pluripotent stem cells (iPSCs). Despite these progresses, some biological, technical, and ethical issues are limiting the therapeutic potential of genome editing in cardiovascular diseases. This review will focus on various applications of CRISPR/Cas9 genome editing in the cardiovascular field, for both disease research and the prospect of in vivo genome-editing therapies in the future.

Ectopic HOXB4 Overcomes the Inhibitory Effect of TNFα on Hematopoietic Stem and Progenitor Cells in a Murine Fanconi Anemia Model.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2041-2041
Author(s):  
Michael D. Milsom ◽  
Bernhard Schiedlmeier ◽  
Jeff Bailey ◽  
Abdullah Ali ◽  
MiOk Kim ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Flow Analysis ◽  
Significant Inhibition ◽  
Transcriptional Level ◽  
Inhibitory Effects ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact

Abstract Ectopic delivery of HOXB4 elicits the in vitro and in vivo expansion of hematopoietic stem cells (HSC) although the mechanism is still unknown. We have previously shown that overexpression of HOXB4 attenuates the TNFα signaling pathway at the transcriptional level (Schiedlmeier et al., 2007, PNAS). TNFα is expression is induced at high levels in vivo after irradiation used in bone marrow (BM) transplantation preparative regimens. Since HSC and progenitors (P) derived from Fanconi anemia (FA) knockout mice are hypersensitive to the action of inhibitory cytokines such as TNFα, we chose the Fancc−/− mouse as a model to study the physiologic effects of HOXB4 on TNFα sensitivity of HSC/P and the relationship of these effects to the engraftment defect of FA HSC. Competitive repopulating assays were used to evaluate the effect of HOXB4 overexpression upon engraftment of Fancc−/− BM. Control (CON, expressing eGFP only) transduced Fancc−/− BM demonstrated 80-fold lower engraftment compared with wild type (WT) eGFP+ BM at 26 weeks post-transplant (Table 1). In marked contrast, HOXB4 transduced (HOXB4+) Fancc−/− BM showed a 26-fold higher level of engraftment compared with CON transduced Fancc−/− BM and a 2-fold higher level of engraftment compared with Fancc−/− cells corrected with a FANCC-expressing vector. Fancc−/− BM co-transduced with both vectors (expressing HOXB4 and FANCC) further increased the level of engraftment to that of WT eGFP+ BM suggesting a synergistic correction of the FA HSC engraftment defect. To determine the potential role of TNFα signaling in these effects, we directly assessed the impact of HOXB4 expression on the response of Fancc−/− BM to treatment with TNFα. Fancc−/− BM cells transduced with the CON eGFP vector demonstrated >70% reduction in colony formation upon treatment with 10ng/ml TNFα (Table 2), while Fancc−/− BM overexpressing HOXB4 showed no significant inhibition of CFU at 10ng/ml TNFα compared to either untreated cells or to treated WT BM transduced with eGFP CON vector. Additionally, in vitro treatment of eGFP CON transduced Fancc−/− BM with 100ng/ml TNFα resulted in a 21±5% decrease in lineage-, Sca-1+, c-Kit+ (LSK) cells within 24 hrs. In contrast, similarly treated HOXB4+Fancc−/− demonstrated a 6±9% increase in LSK cells (p<0.01 compared to Fancc−/− eGFP CON). Hence HOXB4 completely protects Fancc−/− HSC/P against the inhibitory effects of TNFα. In order to further define the mechanism through which HOXB4 attenuates TNFα signaling, we determined the level of expression of the TNFα receptors, TNFR1 and TNFR2, on transduced Fancc−/− BM by flow analysis. Fancc−/− LSK BM transduced with eGFP CON or FANCC expressing vectors had equivalent expression of TNFR1 and TNFR2 compared to WT eGFP CON transduced BM (Table 3). In contrast, Fancc−/− LSK cells transduced HOXB4 demonstrated a >25% reduction in the number of cells that stained positive for either TNFR1 or TNFR2. In addition, there was a >35% decrease in the MFI of staining for TNFR1 in HOXB4+Fancc−/− LSK compared to other groups. In summary, ectopic HOXB4 protects Fancc−/− and WT HSC/P from the inhibitory effects of TNFα. Since HOXB4 expression also protects WT cells from TNFα treatment (data not shown), we propose that HOXB4 enhances engraftment by protecting HSC from the elevated TNFα levels, which is a result of conditioning regimens applied to transplant recipients. We suggest that this mechanism reveals a novel target for the pharmacologic manipulation of HSC during engraftment, thus avoiding the potential adverse effects of constitutive HOXB4 overexpression. Table 1: % peripheral blood chimerism at 26 weeks post-transplant Fancc−/− + eGFP 0.5±0.2 Fancc−/− + FANCC 7.8±6.5 Fancc−/− + HOXB4 18.1±6.0** Fancc−/− + FANCC + HOXB4 34.1±7.1** WT + eGFP 44.3±5.3** Table 2: CFU as % non-treated control ± SEM TNFα dose 1ng/ml 10ng/ml Fancc−/− + eGFP 47±7 20±7 Fancc−/− + FANCC 81±7** 57±7** Fancc−/− + HOXB4 99±5** 92±5** WT + eGFP 90±7** 77±7** Table 3: % (and MFI) of BM positive for TNFR1 and TNFR2 expression ± SEM TNFR1 TNFR2 **p<0.01 compared to Fancc-/- + eGFP Fancc−/− + eGFP 75±5 (740±133) 67±8 (676±160) Fancc−/− + FANCC 69±5 (808±133) 64±8 (673±160) Fancc−/− + HOXB4 46±5** (464±133) 37±8** (425±160) WT + eGFP 70±5 (689±133) 67±8 (636±160)

scholarly journals Relationship between the Level of Acquired Resistance to Gentamicin and Synergism with Amoxicillin in Enterococcus faecalis

2005 ◽  
Vol 49 (10) ◽  
pp. 4144-4148 ◽  
Author(s):  
Elisabeth Aslangul ◽  
Raymond Ruimy ◽  
Françoise Chau ◽  
Louis Garry ◽  
Antoine Andremont ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Acquired Resistance ◽  
Bactericidal Effect ◽  
A Cell ◽  
Enzymatic Inactivation ◽  
High Level ◽  
The Impact ◽  
Level Resistance

ABSTRACT In enterococci, intrinsic low-level resistance to gentamicin does not abolish synergism with a cell wall-active antibiotic while high-level resistance due to acquired aminoglycoside-modifying enzymes does. To study the impact of intermediate levels of resistance to gentamicin (64 < MIC < 500 μg/ml), we selected in vitro three consecutive generations of mutants of Enterococcus faecalis JH2-2 with MICs of gentamicin at 128 μg/ml for G1-1477, 256 μg/ml for G2-1573, and 512 μg/ml for G3-1688. E. faecalis 102, which is highly resistant to gentamicin by enzymatic inactivation was used as control. In in vitro killing curves experiments, gentamicin concentrations allowing bactericidal activity and synergism in combination with amoxicillin increased from 4 μg/ml (1/16th the MIC), 16 μg/ml (one-eighth the MIC), 64 μg/ml (one-quarter the MIC), and 256 μg/ml (one-half the MIC) for strains JH2-2, G1-1477, G2-1573 and G3-1688, respectively. As expected, no bactericidal effect of the combination or synergism could be obtained with strain 102. In rabbits with aortic endocarditis caused by strain G1-1477 or G2-1573, combination therapy with amoxicillin and gentamicin was significantly more active than amoxicillin alone (P < 0.05) but not in those infected with the strains G3-1688 and 102. Thus, intermediate levels of resistance to gentamicin was not associated with a loss of a beneficial effect of the gentamicin-amoxicillin combination in vivo even though higher concentrations of gentamicin were necessary to achieve in vitro synergism. Therefore, the use of an MIC of 500 μg/ml as a clinical cutoff limit to predict in vivo benefit of the combination remains a simple and effective tool.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

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.

Analysis of the Dynamics of the Electric Capacitance of a Cell Monolayer at the Late Stages of Caco-2 cell Differentiation

2019 ◽  
Vol 35 (6) ◽  
pp. 87-90
Author(s):  
S.V. Nikulin ◽  
V.A. Petrov ◽  
D.A. Sakharov
Keyword(s):  
Impedance Spectroscopy ◽  
Cell Monolayer ◽  
Microfluidic Chips ◽  
Russian Science ◽  
Electric Capacitance ◽  
A Cell ◽  
Static Conditions ◽  
Late Stages

The real-time monitoring of electric capacitance (impedance spectroscopy) allowed obtaining evidence that structures which look like intestinal villi can be formed during the cultivation under static conditions as well as during the cultivation in microfluidic chips. It was shown in this work via transcriptome analysis that the Hh signaling pathway is involved in the formation of villus-like structures in vitro, which was previously shown for their formation in vivo. impedance spectroscopy, intestine, villi, electric capacitance, Hh The study was funded by the Russian Science Foundation (Project 16-19-10597).

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