scholarly journals 7UTILIZATION OF CELL PROFILING TO EVALUATE BOVINE SPERMATOZOA IN NORMAL AND SIMULATED MICROGRAVITY

2004 ◽  
Vol 16 (2) ◽  
pp. 126
Author(s):  
J.C. Andrews ◽  
S. Winters-Hilt
Keyword(s):  
Time Course ◽  
Simulated Microgravity ◽  
Single Cells ◽  
Mrna Translation ◽  
Cell Types ◽  
Support Vector ◽  
Mitochondrial Activity ◽  
C Programs ◽  
Mammalian Spermatozoon ◽  
Bovine Spermatozoa

We developed a method to evaluate bovine sperm membranes in normal (1G) and simulated microgravity (Sim-μG). Bovine spermatozoa are used as a model system because they have cellular membranes analogous to those of other cell types, and yet are much simpler because they have no cytoplasm and do not participate in DNA transcription or mRNA translation. They can be cultured as single cells and are easily evaluated for membrane characteristics using flow cytometry. These features make the mammalian spermatozoon a useful model for exploring the principles of membrane structure/function in the presence of a variety of environmental challenges such as simulated microgravity. Cryopreserved, washed beef bull sperm (4–8×106mL−1)were incubated under non-capacitating conditions (modified glucose-free Tyrode’s medium containing low bicarbonate, HEPES buffer, pyruvate and 3mgmL−1 BSA V; 23°C in air), and these spermatozoa remained alive for 24–48h at 1G. To simulate μG, spermatozoa were incubated under the same conditions, in a HARV 10 rotating wall vessel (RWV, Synthecon, Inc, Houston, TX, USA) at 9rpms. Spermatozoa were incubated in 1G and Sim-μG environments for 2.5–4.5h and subsequently exposed to 0, 60 or 80μgmL−1 LC for 0, 4, 8, 12, 16 and 20min. Three fluorochrome combinations were used as probes at each [LC]/time point: (1) propidium Iodide (dead status)+SYBR 14 (live status); (2) PI+FITC-PSA (acrosome reactions [ARs]); (3) PI+MitoTracker Deep Red (mitochondrial activity). Approximately 1million spermatozoa from 3 bulls were evaluated over 4 days. Data were acquired on a FACSVantage SE flow cytometer, and initially analyzed (quality control) using the bundled FACSVantage SE software package (Cell Quest, BD BioSciences, San Jose, CA, USA). This provided graphics of simple cell relations (fluorescence v. LC exposure time). For further statistical analysis, and incorporation of non-parametric statistical tools (including pattern recognition using Support Vector Machines), the data were processed using a collection of Perl scripts and C programs. Results: Live/dead status: When Sim-μG+60μgmL−1 LC sperm were compared to 1G+60μgmL−1LC, and 80μgmL−1 LC sperm, their profiles were more similar to the 1G 80μgmL−1 LC profiles. AR status: the Sim-μG+60μgmL−1 LC profiles were similar to the 1G+60μgmL−1 LC profiles. Mitochondrial Status: the Sim-μG+60μgmL−1LC profiles were more similar to 1G+80μgmL−1 LC profiles. Summary: although Sim-μG sperm lost their motility within 3h, they were alive. Cell profiles indicate that Sim-μG sperm nuclear membranes are less stable and their mitochondria are less functional than the 1G controls, but their acrosomes are intact indicating that fertilizing potential may remain. Additional experiments are needed to determine the time course for Sim-μG, induced changes, and whether Sim-μG sperm can penetrate eggs. Funding: NASA (2002)-Stennis-24 and The University of New Orleans.

scholarly journals Mouse intestinal organoid time-course experiments from single cells

2019 ◽  
Author(s):  
Denise Serra ◽  
Urs Mayr ◽  
Andrea Boni ◽  
Ilya Lukonin ◽  
Markus Rempfler ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
Time Course ◽  
Single Cells ◽  
Rna Extraction ◽  
Cell Types ◽  
The Self ◽  
Organoid Culture ◽  
Tissue Organization ◽  
Grown Structure

Abstract Organoids recapitulate the self-organizing capacity of stem cells and the tissue organization of the original organ in a controlled and trackable environment. Intestinal organoids, in particular, can develop from a single cell into a fully-grown structure that contains most of the cell types, patterns, and morphogenetic properties of the adult intestine. Here we present a protocol for high-throughput organoid culture in multi-well plate format combined with high content immunofluorescence imaging and RNA extraction. Our protocol allows recording and analysis of thousands of organoids during several days of development.

Mechanisms of intracellular calcium release during hormone and neurotransmitter action investigated with flash photolysis

1993 ◽  
Vol 184 (1) ◽  
pp. 105-127
Author(s):  
D. C. Ogden ◽  
K. Khodakhah ◽  
T. D. Carter ◽  
P. T. Gray ◽  
T. Capiod
Keyword(s):  
Time Course ◽  
Calcium Release ◽  
Flash Photolysis ◽  
Single Cells ◽  
Cell Types ◽  
Peripheral Tissues ◽  
Perfusion Studies ◽  
Kinetics Of ◽  
Purkinje Neurones ◽  
Fast Perfusion

To understand the complex time course of cytosolic Ca2+ signalling evoked by hormones and neurotransmitters, it is necessary to know the kinetics of steps in the second-messenger cascade, particularly cooperative and inhibitory interactions between components that might give rise to periodic fluctuations. In the case of inositol trisphosphate (InsP3)-evoked Ca2+ release, fast perfusion studies with subcellular fractions or permeabilised cells can be made if sufficient homogeneous tissue is available. Single-cell studies can be made by combining whole-cell patch-clamp techniques and microspectrofluorimetry with flash photolytic release of InsP3 to give quantitative, time-resolved data of Ca2+ release from stores. A technical description is given here of flash photolysis of caged InsP3, and the results of fast perfusion and flash photolytic experiments are reviewed. Studies of kinetics of Ca2+ release have shown that the InsP3 receptor/channel is regulated first by positive and then by negative feedback by free cytosolic Ca2+ concentration, producing a pulse of Ca2+ release having properties that may be important in the spatial propagation of Ca2+ signals within and between cells. The properties of InsP3-evoked Ca2+ release in single cells differ between peripheral tissues, such as the liver, and Purkinje neurones of the cerebellum. Purkinje neurones need 20–50 times higher InsP3 concentrations and release Ca2+ to change the free cytosolic concentration 30 times faster and to higher peak concentrations than in liver. The InsP3 receptors in the two cell types appear to differ in apparent affinity, and the greater Ca2+ efflux from stores in Purkinje cells is probably due to a high receptor density.

scholarly journals Taste-Specific Cell Assemblies in a Biologically Informed Model of the Nucleus of the Solitary Tract

2010 ◽  
Vol 104 (1) ◽  
pp. 4-17 ◽  
Author(s):  
Andrew M. Rosen ◽  
Heike Sichtig ◽  
J. David Schaffer ◽  
Patricia M. Di Lorenzo
Keyword(s):  
Time Course ◽  
Single Cells ◽  
Cell Types ◽  
Specific Cell ◽  
Solitary Tract ◽  
Cell Assemblies ◽  
Taste Response ◽  
Electrophysiological Recordings ◽  
Interactive Network ◽  
Time Courses

Although the cellular organization of many primary sensory nuclei has been well characterized, questions remain about the functional architecture of the first central relay for gustation, the rostral nucleus of the solitary tract (NTS). Here we used electrophysiological data recorded from single cells in the NTS to inform a network model of taste processing. Previous studies showed that electrical stimulation of the chorda tympani (CT) nerve initiates two types of inhibitory influences with different time courses in separate groups of NTS cells. Each type of inhibition targeted cells with distinct taste response properties. Further analyses of these data identified three NTS cell types differentiated by their latency of evoked response, time course of CT evoked inhibition, and degree of selectivity across taste qualities. Based on these results, we designed a model of the NTS consisting of discrete, reciprocally connected, stimulus-specific “cell” assemblies. Input to the network of integrate-and-fire model neurons was based on electrophysiological recordings from the CT nerve. Following successful simulation of paired-pulse CT stimulation, the network was tested for its ability to discriminate between two “taste” stimuli. Network dynamics of the model produced biologically plausible responses from each unit type and enhanced discrimination between taste qualities. We propose that an interactive network of taste quality specific cell assemblies, similar to our model, may account for the coherence in across-neuron patterns of NTS responses between similar tastants.

scholarly journals ELeFHAnt: A supervised machine learning approach for label harmonization and annotation of single cell RNA-seq data

2021 ◽  
Author(s):  
Konrad Thorner ◽  
Aaron M. Zorn ◽  
Praneet Chaturvedi
Keyword(s):  
Machine Learning ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
A Priori ◽  
Cell Types ◽  
R Package ◽  
Supervised Machine Learning ◽  
Support Vector ◽  
Rna Seq

AbstractAnnotation of single cells has become an important step in the single cell analysis framework. With advances in sequencing technology thousands to millions of cells can be processed to understand the intricacies of the biological system in question. Annotation through manual curation of markers based on a priori knowledge is cumbersome given this exponential growth. There are currently ~200 computational tools available to help researchers automatically annotate single cells using supervised/unsupervised machine learning, cell type markers, or tissue-based markers from bulk RNA-seq. But with the expansion of publicly available data there is also a need for a tool which can help integrate multiple references into a unified atlas and understand how annotations between datasets compare. Here we present ELeFHAnt: Ensemble learning for harmonization and annotation of single cells. ELeFHAnt is an easy-to-use R package that employs support vector machine and random forest algorithms together to perform three main functions: 1) CelltypeAnnotation 2) LabelHarmonization 3) DeduceRelationship. CelltypeAnnotation is a function to annotate cells in a query Seurat object using a reference Seurat object with annotated cell types. LabelHarmonization can be utilized to integrate multiple cell atlases (references) into a unified cellular atlas with harmonized cell types. Finally, DeduceRelationship is a function that compares cell types between two scRNA-seq datasets. ELeFHAnt can be accessed from GitHub at https://github.com/praneet1988/ELeFHAnt.

The Effects of Load on Transmural Differences in Contraction of Isolated Mouse Ventricular Cardiomyocytes

2016 ◽  
Author(s):  
Anastasia Khokhlova ◽  
Gentaro Iribe ◽  
Leonid Katsnelson ◽  
Keiji Naruse ◽  
Olga Solovyova
Keyword(s):  
Mathematical Models ◽  
Cellular Response ◽  
Time Course ◽  
Single Cells ◽  
Cell Types ◽  
Left Ventricular ◽  
Time To Peak ◽  
Response To Change ◽  
Transmural Dispersion ◽  
Transmural Gradient

Mechanical properties of cardiomyocytes from different transmural regions are heterogeneous in the left ventricular wall. The cardiomyocyte mechanical environment affects this heterogeneity because of mechano-electric feedback mechanisms. In the present study, we investigated the effects of load upon transmural differences in contraction of subendocardial (ENDO) and subepicardial (EPI) single cells isolated from the murine left ventricle. Various loads were applied to the cells using carbon fiber techniques for single myocytes. To simulate experimentally obtained results and to predict mechanisms underlying the cellular response to change in load, our mathematical models of the ENDO and EPI cells were used. Extent of the transmural gradient in the time course of contractions was independent of the loading conditions where unloaded and heavy loaded (isometric) contractions were examined, but the regional gradient of the relaxation time characteristics tended to decrease when the load decreased. Under auxotonic contractions, time to peak contraction (Tmax) was significantly longer in ENDO cells than in EPI cells at low preload. An increase in preload (axial stretch) prolonged Tmax in both cell types; however, the prolongation was greater in EPI cells, resulting in a decrease in transmural gradient in Tmax at high preload. The [Ca2+]i transient decay time constant was consistent with the greater preload dependency in Tmax of EPI cells. Our modified mathematical models reproduced experimental results, suggesting that differences in cooperativity of cross bridges and calcium troponin C complex interactions between the ENDO and EPI cardiomyocytes may contribute to the different cellular responses to stretch, which may provide a decrease in transmural dispersion of cellular shortening in the intact heart.

Neuronal mRNA Translation in Addiction

2018 ◽  
Author(s):  
Emma Puighermanal ◽  
Emmanuel Valjent
Keyword(s):  
Structural Changes ◽  
Drugs Of Abuse ◽  
Mrna Translation ◽  
Cell Types ◽  
Behavioral Changes ◽  
Translational Machinery ◽  
Addictive Drugs ◽  
Specific Mrnas ◽  
Cell Type Specific ◽  
Future Work

Addictive drugs trigger persistent synaptic and structural changes in the neuronal reward circuits that are thought to underlie the development of drug-adaptive behavior. While transcriptional and epigenetic modifications are known to contribute to these circuit changes, accumulating evidence indicates that altered mRNA translation is also a key molecular mechanism. This chapter reviews recent studies demonstrating how addictive drugs alter protein synthesis and/or the translational machinery and how this leads to neuronal circuit remodeling and behavioral changes. Future work will determine precisely which neuronal circuits and cell types participate in these changes. The chapter summarizes current methodologies for identifying cell type-specific mRNAs whose translation is affected by drugs of abuse and gives recent examples of the mechanistic insights into addiction they provide.

scholarly journals MiR-7 in Cancer Development

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 325
Author(s):  
Petra Korać ◽  
Mariastefania Antica ◽  
Maja Matulić
Keyword(s):  
Cell Fate ◽  
Dominant Role ◽  
Tumor Development ◽  
Mrna Translation ◽  
Cell Types ◽  
Fine Tuning ◽  
Non Coding Rna ◽  
Tumor Types ◽  
Different Cell Types ◽  
The Brain

MicroRNAs (miRNAs) are short non-coding RNA involved in the regulation of specific mRNA translation. They participate in cellular signaling circuits and can act as oncogenes in tumor development, so-called oncomirs, as well as tumor suppressors. miR-7 is an ancient miRNA involved in the fine-tuning of several signaling pathways, acting mainly as tumor suppressor. Through downregulation of PI3K and MAPK pathways, its dominant role is the suppression of proliferation and survival, stimulation of apoptosis and inhibition of migration. Besides these functions, it has numerous additional roles in the differentiation process of different cell types, protection from stress and chromatin remodulation. One of the most investigated tissues is the brain, where its downregulation is linked with glioblastoma cell proliferation. Its deregulation is found also in other tumor types, such as in liver, lung and pancreas. In some types of lung and oral carcinoma, it can act as oncomir. miR-7 roles in cell fate determination and maintenance of cell homeostasis are still to be discovered, as well as the possibilities of its use as a specific biotherapeutic.

scholarly journals The landscape of alternative polyadenylation in single cells of the developing mouse embryo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vikram Agarwal ◽  
Sereno Lopez-Darwin ◽  
David R. Kelley ◽  
Jay Shendure
Keyword(s):  
Rna Binding ◽  
Developmental Stages ◽  
Single Cells ◽  
Neuronal Cell ◽  
Alternative Polyadenylation ◽  
Cell Types ◽  
Untranslated Regions ◽  
Mammalian Development ◽  
Translation Rate ◽  
Dynamic Landscape

Abstract3′ untranslated regions (3′ UTRs) post-transcriptionally regulate mRNA stability, localization, and translation rate. While 3′-UTR isoforms have been globally quantified in limited cell types using bulk measurements, their differential usage among cell types during mammalian development remains poorly characterized. In this study, we examine a dataset comprising ~2 million nuclei spanning E9.5–E13.5 of mouse embryonic development to quantify transcriptome-wide changes in alternative polyadenylation (APA). We observe a global lengthening of 3′ UTRs across embryonic stages in all cell types, although we detect shorter 3′ UTRs in hematopoietic lineages and longer 3′ UTRs in neuronal cell types within each stage. An analysis of RNA-binding protein (RBP) dynamics identifies ELAV-like family members, which are concomitantly induced in neuronal lineages and developmental stages experiencing 3′-UTR lengthening, as putative regulators of APA. By measuring 3′-UTR isoforms in an expansive single cell dataset, our work provides a transcriptome-wide and organism-wide map of the dynamic landscape of alternative polyadenylation during mammalian organogenesis.

scholarly journals Mitochondrial Retrograde Signaling Contributes to Metabolic Differentiation in Yeast Colonies

2021 ◽  
Vol 22 (11) ◽  
pp. 5597
Author(s):  
Vítězslav Plocek ◽  
Kristýna Fadrhonc ◽  
Jana Maršíková ◽  
Libuše Váchová ◽  
Alexandra Pokorná ◽  
...  
Keyword(s):  
Cell Types ◽  
Colony Development ◽  
Mitochondrial Activity ◽  
Mitochondrial Translation ◽  
Metabolic Intermediates ◽  
Nutritional Conditions ◽  
Mitochondrial Ribosome ◽  
Development And Differentiation ◽  
Cell Subpopulations ◽  
Different Cell Types

During development of yeast colonies, various cell subpopulations form, which differ in their properties and specifically localize within the structure. Three branches of mitochondrial retrograde (RTG) signaling play a role in colony development and differentiation, each of them activating the production of specific markers in different cell types. Here, aiming to identify proteins and processes controlled by the RTG pathway, we analyzed proteomes of individual cell subpopulations from colonies of strains, mutated in genes of the RTG pathway. Resulting data, along with microscopic analyses revealed that the RTG pathway predominantly regulates processes in U cells, long-lived cells with unique properties, which are localized in upper colony regions. Rtg proteins therein activate processes leading to amino acid biosynthesis, including transport of metabolic intermediates between compartments, but also repress expression of mitochondrial ribosome components, thus possibly contributing to reduced mitochondrial translation in U cells. The results reveal the RTG pathway’s role in activating metabolic processes, important in U cell adaptation to altered nutritional conditions. They also point to the important role of Rtg regulators in repressing mitochondrial activity in U cells.

Abstract 160: Pim-1 Preserves Cardiac Stem Cell Proliferation with Age

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Michael McGregor ◽  
Shabana Din ◽  
Natalie Gude ◽  
Mark A Sussman
Keyword(s):  
Cell Proliferation ◽  
Stem Cell ◽  
Time Course ◽  
Protein A ◽  
Cell Types ◽  
Tissue Homeostasis ◽  
Dominant Negative ◽  
Repair Capacity ◽  
Tissue Samples ◽  
The Impact

Rationale Cardiac stem cells (CSC) regulate cardiomyogenesis and support regenerative processes in the heart, but aging adversely affects stem cell repair capacity. Aging is a primary cause of impaired cardiac function characterized by accumulation of senescent cells. CSC senescence is associated with permanent growth arrest that decreases survival signaling and cellular replacement, inevitably diminishing the capacity of the heart to maintain tissue homeostasis. Therefore, promoting CSC growth may improve cardiac performance with age. Pim-1 kinase exhibits protective and proliferative effects in the myocardium but the role of Pim-1 in cardiac aging has not been thoroughly studied. Objective Demonstrate that Pim-1 promotes stem cell growth in the aged myocardium correlating with increased expression of centromere protein A (CENP-A), a kinetochore-associated protein known to support cell proliferation in numerous species and cell types. Methods & Results CENP-A expression levels were evaluated from murine myocardial tissue samples ranging in age from 11 days post coitum to 4 months of age with analysis by immunoblot as well as quantitative PCR. CENP-A expression was colocalized with c-kit as a marker of CSC by immunohistochemical labeling, revealing a decline in CENP-A expression over the time course of postnatal myocardial maturation. The impact of Pim-1 upon CENP-A level was assessed by comparative analysis of non-transgenic mice versus genetically modified transgenic mouse lines expressing either Pim-1 (wild type) or a dominant negative functionally dead Pim-1 mutant. Pim-1 overexpression increases persistence of CENP-A in CSCs with age, as well as the prevalence of cycling CSCs as marked by phosph-H3 expression, while the functionally dead mutant accelerates CENP-A diminution and decreases CSC proliferation. Conclusion CENP-A decline in c-kit positive cells with age provides intriguing evidence of a potential mechanism for the diminished capacity of CSCs to maintain tissue homeostasis. Pim-1 mitigates CENP-A diminution, demonstrating the promising potential of Pim-1 to promote cardiac growth and repair with age.

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