scholarly journals Development and optimization of an in vivo electrocardiogram recording method and analysis program for adult zebrafish

2021 ◽  
Author(s):  
ThuyVy Duong ◽  
Rebecca Rose ◽  
Adriana Blazeski ◽  
Noah Fine ◽  
Courtney E Woods ◽  
...  
Keyword(s):  
Cardiac Electrophysiology ◽  
Data Interpretation ◽  
Model Systems ◽  
Adult Zebrafish ◽  
High Quality ◽  
Qt Intervals ◽  
Flecainide Acetate ◽  
Electrocardiogram Ecg ◽  
The Impact

Clinically pertinent electrocardiogram (ECG) data from model systems, such as zebrafish, are crucial for illuminating factors contributing to human cardiac electrophysiology abnormalities and disease. Current zebrafish ECG collection strategies have not adequately addressed the consistent acquisition of high-quality traces or sources of phenotypic variation that could obscure data interpretation. Thus, we developed a novel platform to ensure high-quality recording of in vivo subdermal adult zebrafish ECGs and zERG (Zebrafish ECG Reading GUI), a program to acquire measurements from traces commercial software cannot examine due to erroneous peak calling. We evaluate normal ECG trait variation, revealing the intervals are highly reproducible while wave amplitude variation appears largely driven by recording artifacts, and identify sex and body size as potential confounders to PR, QRS, and QT intervals. With this framework, we characterize the effect of the class I anti-arrhythmic drug flecainide acetate on adults, provide support for the impact of a Long QT syndrome model, and establish power calculations for this and other studies. These results highlight our pipeline as a robust approach to evaluate zebrafish models of human cardiac electrophysiologic phenotypes.

scholarly journals Latent Antigen Vaccination in a Model Gammaherpesvirus Infection

2001 ◽  
Vol 75 (17) ◽  
pp. 8283-8288 ◽  
Author(s):  
Edward J. Usherwood ◽  
Kimberley A. Ward ◽  
Marcia A. Blackman ◽  
James P. Stewart ◽  
David L. Woodland
Keyword(s):  
Latent Infection ◽  
Model Systems ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Associated Proteins ◽  
Infected Cells ◽  
The Impact ◽  
First Time

ABSTRACT Vaccines that can reduce the load of latent gammaherpesvirus infections are eagerly sought. One attractive strategy is vaccination against latency-associated proteins, which may increase the efficiency with which T cells recognize and eliminate latently infected cells. However, due to the lack of tractable animal model systems, the effect of latent-antigen vaccination on gammaherpesvirus latency is not known. Here we use the murine gammaherpesvirus model to investigate the impact of vaccination with the latency-associated M2 antigen. As expected, vaccination had no effect on the acute lung infection. However, there was a significant reduction in the load of latently infected cells in the initial stages of the latent infection, when M2 is expressed. These data show for the first time that latent-antigen vaccination can reduce the level of latency in vivo and suggest that vaccination strategies involving other latent antigens may ultimately be successfully used to reduce the long-term latent infection.

scholarly journals Unveiling Tumor Microenvironment Interactions Using Zebrafish Models

2021 ◽  
Vol 7 ◽  
Author(s):  
Reid Loveless ◽  
Chloe Shay ◽  
Yong Teng
Keyword(s):  
Tumor Microenvironment ◽  
Molecular Mechanisms ◽  
Immune Cell ◽  
Genetic Manipulation ◽  
Innate Immune ◽  
Model Systems ◽  
Cytokine Signaling ◽  
Adult Zebrafish ◽  
Metastatic Cascade

The tumor microenvironment (TME) is a rich and active arena that is strategically evolved overtime by tumors to promote their survival and dissemination. Over the years, attention has been focused to characterize and identify the tumor-supporting roles and subsequent targeting potentials of TME components. Nevertheless, recapitulating the human TME has proved inherently challenging, leaving much to be explored. In this regard, in vivo model systems like zebrafish, with its optical clarity, ease of genetic manipulation, and high engraftment, have proven to be indispensable for TME modeling and investigation. In this review, we discuss the recent ways by which zebrafish models have lent their utility to provide new insights into the various cellular and molecular mechanisms driving TME dynamics and tumor support. Specifically, we report on innate immune cell interactions, cytokine signaling, metastatic plasticity, and other processes within the metastatic cascade. In addition, we reflect on the arrival of adult zebrafish models and the potential of patient-derived xenografts.

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 Positional motif analysis reveals the extent of specificity of protein-RNA interactions observed by CLIP

2021 ◽  
Author(s):  
Klara Kuret ◽  
Aram Gustav Amalietti ◽  
Jernej Ule
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Data Interpretation ◽  
Low Complexity ◽  
Motif Analysis ◽  
Link Type ◽  
Motif Enrichment ◽  
The Impact

AbstractBackgroundCrosslinking and immunoprecipitation (CLIP) is a method used to identify in vivo RNA– protein binding sites on a transcriptome-wide scale. With the increasing amounts of available data for RNA-binding proteins (RBPs), it is important to understand to what degree the enriched motifs specify the RNA binding profiles of RBPs in cells.ResultsWe develop positionally-enriched k-mer analysis (PEKA), a computational tool for efficient analysis of enriched motifs from individual CLIP datasets, which minimises the impact of technical and regional genomic biases by internal data normalisation. We cross-validate PEKA with mCross, and show that background correction by size-matched input doesn’t generally improve the specificity of detected motifs. We identify motif classes with common enrichment patterns across eCLIP datasets and across RNA regions, while also observing variations in the specificity and the extent of motif enrichment across eCLIP datasets, between variant CLIP protocols, and between CLIP and in vitro binding data. Thereby we gain insights into the contributions of technical and regional genomic biases to the enriched motifs, and find how motif enrichment features relate to the domain composition and low-complexity regions (LCRs) of the studied proteins.ConclusionsOur study provides insights into the overall contributions of regional binding preferences, protein domains and LCRs to the specificity of protein-RNA interactions, and shows the value of cross-motif and cross-RBP comparison for data interpretation. Our results are presented for exploratory analysis via an online platform in an RBP-centric and motif-centric manner (https://imaps.goodwright.com/apps/peka/). PEKA is available from https://github.com/ulelab/peka.

Modeling the CNS Tropism of Diffuse Large B-Cell Lymphomas in Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 576-576
Author(s):  
Maurice Reimann ◽  
Sven Masswig ◽  
Kolja Schleich ◽  
Andrea Herrmann ◽  
Philipp Lohneis ◽  
...  
Keyword(s):  
B Cell ◽  
B Cell Lymphoma ◽  
Gene Set Enrichment Analysis ◽  
Model Systems ◽  
B Cell Lymphomas ◽  
Cns Involvement ◽  
The Impact ◽  
The Brain ◽  
Large B Cell

Abstract Introduction: Central nervous system (CNS) manifestation accounts for dismal prognosis in patients diagnosed with systemic diffuse large B-cell lymphoma (DLBCL). Myc rearrangement, deletions encompassing the CDKN2A (a.k.a. INK4a/ARF) or ATM gene loci as well as NF-kB-hyperactivating mutations are frequently detected in primary and secondary CNS-tropic lymphoma, however, investigations that functionally link these lesions to CNS involvement in adequate in vivo model systems are missing. Methods: We generated primary Eµ-myc transgenic mouse lymphomas with and without distinct naturally occurring NF-kB mutations (within genes encoding for MyD88, CD79B, A20, IkBζ, IkBε or BIRC3) or deletions at the INK4a/ARF and ATM loci by retroviral gene transfer and crossbreeding to the respective knockout strains. A subset of the lymphomas was subjected to gene expression profiling and whole-exome sequencing (WES). Wild-type recipient mice propagated with lymphoma cells via tail vein injection were monitored for systemic lymphoma development, the time at which the brain was isolated and examined regarding lymphoma infiltration. Results: Underlining Myc's role as a putative co-driver of CNS involvement, we found in about 40% of primary Eµ-myc lymphomas (with no additional exogenous lesions) meningeal lymphoma manifestations, and transplantation of the same individual lymphomas into numerous recipients reproduced the CNS lymphoma status. Gene set enrichment analysis of genome-wide transcriptome profiles indicated NF-kB hyperactivation in the CNS-tropic lymphoma group, suggesting that constitutive NF-kB signaling may promote CNS-prone pathogenesis in vivo. Transplantation of Eµ-myc transgenic hematopoietic stem cells expressing a variety of NF-kB-activating mutants in myeloablated recipient mice resulted in a significant acceleration of Eµ-myc -driven lymphomagenesis, with some, but not all of these mutants conferring a CNS-tropic lymphoma phenotype. Global NF-kB suppression in CNS-tropic Eµ-myc lymphomas via the NF-kB-antagonizing IkBΔN super-repressor did not fully abrogate lymphoma infiltration of the brain, suggesting that additional factor(s) must contribute. Accordingly, targeted ablation of the INK4a/ARF and ATM loci robustly enhanced CNS tropism of Eµ-myc lymphomas. Conclusions: The Eµ-myc mouse lymphoma model is well-suited to genetically dissect and rebuild components of DLBCL-like CNS tropism. We identified CDKN2A or ATM deletions as critical determinants of CNS tropism in vivo. Our systematic analyses of different NF-kB mutants - so far rather recognized as functionally interchangeable - indicated that only distinct NF-kB mutants contribute to CNS tropism in B-cell lymphomas. WES data, results from compound genotypes (e.g. combining ATM deletions with an NF-kB-activating mutation), and the impact of the host's cellular immune status will be reported at the meeting. Our findings underscore the need for functional analyses of oncogenic network contexts, and provide important insights into candidate target lesions for personalized CNS-directed therapies in DLBCL patients in the future. Disclosures No relevant conflicts of interest to declare.

scholarly journals Zebrafish as a Screening Model to Study the Single and Joint Effects of Antibiotics

2021 ◽  
Vol 14 (6) ◽  
pp. 578
Author(s):  
Roxana Jijie ◽  
Gabriela Mihalache ◽  
Ioana-Miruna Balmus ◽  
Stefan-Adrian Strungaru ◽  
Emanuel Stefan Baltag ◽  
...  
Keyword(s):  
Animal Model ◽  
Developmental Stages ◽  
Model Systems ◽  
In Vivo Studies ◽  
Research Papers ◽  
Vertebrate Model ◽  
Efficacy Assessment ◽  
Potential Impact ◽  
The Impact

The overuse of antibiotics combined with the limitation of wastewater facilities has resulted in drug residue accumulation in the natural environment. Thus, in recent years, the presence of antibiotic residues in the environment has raised concerns over the potential harmful effects on ecosystems and human health. The in vivo studies represent an essential step to study the potential impact induced by pharmaceutical exposure. Due to the limitations of traditional vertebrate model systems, zebrafish (Danio rerio) has recently emerged as a promising animal model to study the toxic effects of drugs and their therapeutic efficacy. The present review summarizes the recent advances made on the toxicity of seven representative classes of antibiotics, namely aminoglycosides, β-lactams, macrolides, quinolones, sulfonamides, tetracyclines and polyether antibiotics, in zebrafish, as well as the combined effects of antibiotic mixtures, to date. Despite a significant amount of the literature describing the impact of single antibiotic exposure, little information exists on the effects of antibiotic mixtures using zebrafish as an animal model. Most of the research papers on this topic have focused on antibiotic toxicity in zebrafish across different developmental stages rather than on their efficacy assessment.

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 The Impact of Various Culture Conditions on Human Mesenchymal Stromal Cells Metabolism

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Pavla Tonarova ◽  
Katerina Lochovska ◽  
Robert Pytlik ◽  
Marie Hubalek Kalbacova
Keyword(s):  
Human Serum ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Reciprocal Relationship ◽  
Heat Inactivation ◽  
High Quality ◽  
Native Form ◽  
The Impact

In vitro and in vivo analyses are closely connected, and the reciprocal relationship between the two comprises a key assumption with concern to the conducting of meaningful research. The primary purpose of in vitro analysis is to provide a solid background for in vivo and clinical study purposes. The fields of cell therapy, tissue engineering, and regenerative medicine depend upon the high quality and appropriate degree of the expansion of mesenchymal stromal cells (MSCs) under low-risk and well-defined conditions. Hence, it is necessary to determine suitable alternatives to fetal bovine serum (FBS—the laboratory gold standard) that comply with all the relevant clinical requirements and that provide the appropriate quantity of high-quality cells while preserving the required properties. Human serum (autologous and allogeneic) and blood platelet lysates and releasates are currently considered to offer promising and relatively well-accessible MSC cultivation alternatives. Our study compared the effect of heat-inactivated FBS on MSC metabolism as compared to its native form (both are used as the standard in laboratory practice) and to potential alternatives with concern to clinical application—human serum (allogeneic and autologous) or platelet releasate (PR-SRGF). The influence of the origin of the serum (fetal versus adult) was also determined. The results revealed the key impact of the heat inactivation of FBS on MSCs and the effectiveness of human sera and platelet releasates with respect to MSC behaviour (metabolic activity, proliferation, morphology, and cytokine production).

scholarly journals Generation of focal mutations and large genomic deletions in the pancreas using inducible in vivo genome editing

2019 ◽  
Vol 41 (3) ◽  
pp. 334-344
Author(s):  
Amrendra Mishra ◽  
Fatemeh Emamgholi ◽  
Zulrahman Erlangga ◽  
Björn Hartleben ◽  
Kristian Unger ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Genetic Alterations ◽  
Model Systems ◽  
Ductal Adenocarcinoma ◽  
In Vivo Model ◽  
Precision Oncology ◽  
Kras Mutations ◽  
Genomic Deletions ◽  
The Impact

Abstract Beyond the nearly uniform presence of KRAS mutations, pancreatic cancer is increasingly recognized as a heterogeneous disease. Preclinical in vivo model systems exist, but with the advent of precision oncology, murine models with enhanced genetic flexibility are needed to functionally annotate genetic alterations found in the human malignancy. Here, we describe the generation of focal gene disruptions and large chromosomal deletions via inducible and pancreas-specific expression of Cas9 in adult mice. Experimental mice are derived on demand directly from genetically engineered embryonic stem cells, without the need for further intercrossing. To provide initial validation of our approach, we show that disruption of the E3 ubiquitin ligase Rnf43 accelerates KrasG12D-dependent tumourigenesis. Moreover, we demonstrate that this system can be used to rapidly interrogate the impact of complex cancer-associated alleles through the generation of a previously unstudied 1.2 megabase deletion surrounding the CDKN2A and CDKN2B tumour suppressors. Thus, our approach is capable of reproducibly generating biallelic and precise loss of large chromosomal fragments that, in conjunction with mutant Kras, leads to development of pancreatic ductal adenocarcinoma with full penetrance.

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.

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