scholarly journals Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals

2009 ◽  
Vol 297 (6) ◽  
pp. C1347-C1357 ◽  
Author(s):  
Monika Sramkova ◽  
Andrius Masedunskas ◽  
Laura Parente ◽  
Alfredo Molinolo ◽  
Roberto Weigert
Keyword(s):  
Plasmid Dna ◽  
Target Genes ◽  
Specific Cell ◽  
Experimental Conditions ◽  
Live Animal ◽  
Cellular Processes ◽  
Submandibular Salivary Glands ◽  
Powerful Approach ◽  
Approaches To Study ◽  
Granular Ducts

The ability to dynamically image cellular and subcellular structures in a live animal and to target genes to a specific cell population in a living tissue provides a unique tool to address many biological questions in the proper physiological context. Here, we describe a powerful approach that is based on the use of rat submandibular salivary glands, which offer the possibility to easily perform intravital imaging and deliver molecules from the oral cavity, and plasmid DNA, which offers the advantage of rapid manipulations. We show that, under different experimental conditions, a reporter molecule can be rapidly expressed in specific compartments of the glands: 1) in the intercalated ducts, when plasmid DNA is administered alone, and 2) in granular ducts, striated ducts, and, to a lesser extent, acini, when plasmid DNA is mixed with replication-deficient adenovirus subtype 5 particles. Remarkably, we also found that gene expression can be directed to acinar cells when plasmid DNA is administered during isoproterenol-stimulated exocytosis, suggesting a novel mechanism of plasmid internalization regulated by compensatory endocytosis. Finally, as a practical application of these strategies, we show how the expression of fluorescently tagged molecules enables the study of the dynamics of various organelles in live animals at a resolution comparable to that achieved in cell cultures.

MiRNA-145 and Its Direct Downstream Targets in Digestive System Cancers: A Promising Therapeutic Target

2020 ◽  
Vol 26 ◽  
Author(s):  
Yini Ma ◽  
Xiu Cao ◽  
Guojuan Shi ◽  
Tianlu Shi
Keyword(s):  
Digestive System ◽  
Target Genes ◽  
Malignant Neoplasm ◽  
Vital Role ◽  
Diagnostic Biomarkers ◽  
Cellular Processes ◽  
Promising Therapeutic Target ◽  
Direct Target ◽  
Potential Strategy

: MicroRNAs (miRNAs) play a vital role in the onset and development of many diseases, including cancers. Emerging evidence shows that numerous miRNAs have the potential to be used as diagnostic biomarkers for cancers, and miRNA-based therapy may be a promising therapy for the treatment of malignant neoplasm. MicroRNA-145 (miR-145) has been considered to play certain roles in various cellular processes, such as proliferation, differentiation and apoptosis, via modulating expression of direct target genes. Recent reports show that miR-145 participates in the progression of digestive system cancers, and plays crucial and novel roles for cancer treatment. In this review, we summarize the recent knowledge concerning the function of miR-145 and its direct targets in digestive system cancers. We discuss the potential role of miR-145 as valuable biomarkers for digestive system cancers and how miR-145 regulates these digestive system cancers via different targets to explore the potential strategy of targeting miR-145.

scholarly journals A practical solution to pseudoreplication bias in single-cell studies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kip D. Zimmerman ◽  
Mark A. Espeland ◽  
Carl D. Langefeld
Keyword(s):  
Single Cell ◽  
Mixed Models ◽  
Random Effect ◽  
Cell Types ◽  
Error Rates ◽  
Specific Cell ◽  
Experimental Conditions ◽  
Type 1 Error ◽  
Sample Correlation ◽  
Inflated Type

AbstractCells from the same individual share common genetic and environmental backgrounds and are not statistically independent; therefore, they are subsamples or pseudoreplicates. Thus, single-cell data have a hierarchical structure that many current single-cell methods do not address, leading to biased inference, highly inflated type 1 error rates, and reduced robustness and reproducibility. This includes methods that use a batch effect correction for individual as a means of accounting for within-sample correlation. Here, we document this dependence across a range of cell types and show that pseudo-bulk aggregation methods are conservative and underpowered relative to mixed models. To compute differential expression within a specific cell type across treatment groups, we propose applying generalized linear mixed models with a random effect for individual, to properly account for both zero inflation and the correlation structure among measures from cells within an individual. Finally, we provide power estimates across a range of experimental conditions to assist researchers in designing appropriately powered studies.

scholarly journals MiR-200c-3p Contrasts PD-L1 Induction by Combinatorial Therapies and Slows Proliferation of Epithelial Ovarian Cancer through Downregulation of β-Catenin and c-Myc

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 519
Author(s):  
Eleni Anastasiadou ◽  
Elena Messina ◽  
Tiziana Sanavia ◽  
Lucia Mundo ◽  
Federica Farinella ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Epithelial Ovarian Cancer ◽  
Therapeutic Strategy ◽  
Target Genes ◽  
Ovarian Cancer Cells ◽  
Oncogenic Signaling ◽  
Experimental Conditions ◽  
Skov3 Cells ◽  
In Silico Analyses ◽  
Post Therapy

Conventional/targeted chemotherapies and ionizing radiation (IR) are being used both as monotherapies and in combination for the treatment of epithelial ovarian cancer (EOC). Several studies show that these therapies might favor oncogenic signaling and impede anti-tumor responses. MiR-200c is considered a master regulator of EOC-related oncogenes. In this study, we sought to investigate if chemotherapy and IR could influence the expression of miR-200c-3p and its target genes, like the immune checkpoint PD-L1 and other oncogenes in a cohort of EOC patients’ biopsies. Indeed, PD-L1 expression was induced, while miR-200c-3p was significantly reduced in these biopsies post-therapy. The effect of miR-200c-3p target genes was assessed in miR-200c transfected SKOV3 cells untreated and treated with olaparib and IR alone. Under all experimental conditions, miR-200c-3p concomitantly reduced PD-L1, c-Myc and β-catenin expression and sensitized ovarian cancer cells to olaparib and irradiation. In silico analyses further confirmed the anti-correlation between miR-200c-3p with c-Myc and β-catenin in 46 OC cell lines and showed that a higher miR-200c-3p expression associates with a less tumorigenic microenvironment. These findings provide new insights into how miR-200c-3p could be used to hold in check the adverse effects of conventional chemotherapy, targeted therapy and radiation therapy, and offer a novel therapeutic strategy for EOC.

scholarly journals Epigenetic Methylations on N6-Adenine and N6-Adenosine with the same Input but Different Output

2019 ◽  
Vol 20 (12) ◽  
pp. 2931 ◽  
Author(s):  
Zhiqing Li ◽  
Ping Zhao ◽  
Qingyou Xia
Keyword(s):  
Genetic Information ◽  
Target Genes ◽  
Technological Development ◽  
Wide Spectrum ◽  
Functional Identification ◽  
Methyl Group ◽  
Cellular Processes ◽  
Dna And Rna ◽  
Future Challenges ◽  
Detection Strategies

Epigenetic modifications on individual bases in DNA and RNA can encode inheritable genetic information beyond the canonical bases. Among the nucleic acid modifications, DNA N6-methadenine (6mA) and RNA N6-methyladenosine (m6A) have recently been well-studied due to the technological development of detection strategies and the functional identification of modification enzymes. The current findings demonstrate a wide spectrum of 6mA and m6A distributions from prokaryotes to eukaryotes and critical roles in multiple cellular processes. It is interesting that the processes of modification in which the methyl group is added to adenine and adenosine are the same, but the outcomes of these modifications in terms of their physiological impacts in organisms are quite different. In this review, we summarize the latest progress in the study of enzymes involved in the 6mA and m6A methylation machinery, including methyltransferases and demethylases, and their functions in various biological pathways. In particular, we focus on the mechanisms by which 6mA and m6A regulate the expression of target genes, and we highlight the future challenges in epigenetic regulation.

scholarly journals Two-photon activated precision molecular photosensitizer targeting mitochondria

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Inês F. A. Mariz ◽  
Sandra N. Pinto ◽  
Ana M. Santiago ◽  
José M. G. Martinho ◽  
Javier Recio ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Near Infrared ◽  
Therapeutic Potential ◽  
Blue Shift ◽  
Specific Cell ◽  
Animal Cells ◽  
Mitochondrial Potential ◽  
Live Animal ◽  
Two Photon ◽  
Mitochondria Targeting

AbstractMitochondria metabolism is an emergent target for the development of novel anticancer agents. It is amply recognized that strategies that allow for modulation of mitochondrial function in specific cell populations need to be developed for the therapeutic potential of mitochondria-targeting agents to become a reality in the clinic. In this work, we report dipolar and quadrupolar quinolizinium and benzimidazolium cations that show mitochondria targeting ability and localized light-induced mitochondria damage in live animal cells. Some of the dyes induce a very efficient disruption of mitochondrial potential and subsequent cell death under two-photon excitation in the Near-infrared (NIR) opening up possible applications of azonia/azolium aromatic heterocycles as precision photosensitizers. The dipolar compounds could be excited in the NIR due to a high two-photon brightness while exhibiting emission in the red part of the visible spectra (600–700 nm). Interaction with the mitochondria leads to an unexpected blue-shift of the emission of the far-red emitting compounds, which we assign to emission from the locally excited state. Interaction and possibly aggregation at the mitochondria prevents access to the intramolecular charge transfer state responsible for far-red emission.

scholarly journals Predicting cell-cycle expressed genes identifies canonical and non-canonical regulators of time-specific expression in Saccharomyces cerevisiae

2018 ◽  
Author(s):  
Nicholas L Panchy ◽  
John P. Lloyd ◽  
Shin-Han Shiu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Target Genes ◽  
Specific Cell ◽  
Data Sets ◽  
Cell Cycle Regulators ◽  
Specific Expression ◽  
Regulatory Data ◽  
Time Specific ◽  
Cycle Regulation

AbstractThe collection all TFs, target genes and their interactions in an organism form a gene regulatory network (GRN), which underly complex patterns of transcription even in unicellular species. However, identifying which interactions regulate expression in a specific temporal context remains a challenging task. With multiple experimental and computational approaches to characterize GRNs, we predicted general and phase-specific cell-cycle expression in Saccharomyces cerevisiae using four regulatory data sets: chromatin immunoprecipitation (ChIP), TF deletion data (Deletion), protein binding microarrays (PBMs), and position weight matrices (PWMs). Our results indicate that the source of regulatory interaction information significantly impacts our ability to predict cell-cycle expression where the best model was constructed by combining selected TF features from ChIP and Deletion data as well as TF-TF interaction features in the form of feed-forward loops. The TFs that were the best predictors of cell-cycle expression were enriched for known cell-cycle regulators but also include regulators not implicated in cell-cycle regulation previously. In addition, ChIP and Deletion datasets led to the identification different subsets of TFs important for predicting cell-cycle expression. Finally, analysis of important TF-TF interaction features suggests that the GRN regulating cell cycle expression is highly interconnected and clustered around four groups of genes, two of which represent known cell-cycle regulatory complexes, while the other two contain TFs that are not known cell-cycle regulators (Ste12-Tex1 and Rap1-Hap1-Msn4), but are nonetheless important to regulating the timing of expression. Thus, not only do our models accurately reflect what is known about the regulation of the S. cerevisiae cell cycle, they can be used to discover regulatory factors which play a role in controlling expression during the cell cycle as well as other contexts with discrete temporal patterns of expression.

scholarly journals Chemogenetic Tools for Causal Cellular and Neuronal Biology

2018 ◽  
Vol 98 (1) ◽  
pp. 391-418 ◽  
Author(s):  
Deniz Atasoy ◽  
Scott M. Sternson
Keyword(s):  
G Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Cellular Processes ◽  
Distinct Cell ◽  
G Protein Coupled ◽  
Pharmacological Control

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

scholarly journals Transforming Growth Factor β-Mediated Transcriptional Repression of c-myc Is Dependent on Direct Binding of Smad3 to a Novel Repressive Smad Binding Element

2004 ◽  
Vol 24 (6) ◽  
pp. 2546-2559 ◽  
Author(s):  
Joshua P. Frederick ◽  
Nicole T. Liberati ◽  
David S. Waddell ◽  
Yigong Shi ◽  
Xiao-Fan Wang
Keyword(s):  
Growth Factor ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Cell Types ◽  
Specific Cell ◽  
Smad Proteins ◽  
Smad Binding Element

ABSTRACT Smad proteins are the most well-characterized intracellular effectors of the transforming growth factor β (TGF-β) signal. The ability of the Smads to act as transcriptional activators via TGF-β-induced recruitment to Smad binding elements (SBE) within the promoters of TGF-β target genes has been firmly established. However, the elucidation of the molecular mechanisms involved in TGF-β-mediated transcriptional repression are only recently being uncovered. The proto-oncogene c-myc is repressed by TGF-β, and this repression is required for the manifestation of the TGF-β cytostatic program in specific cell types. We have shown that Smad3 is required for both TGF-β-induced repression of c-myc and subsequent growth arrest in keratinocytes. The transcriptional repression of c-myc is dependent on direct Smad3 binding to a novel Smad binding site, termed a repressive Smad binding element (RSBE), within the TGF-β inhibitory element (TIE) of the c-myc promoter. The c-myc TIE is a composite element, comprised of an overlapping RSBE and a consensus E2F site, that is capable of binding at least Smad3, Smad4, E2F-4, and p107. The RSBE is distinct from the previously defined SBE and may partially dictate, in conjunction with the promoter context of the overlapping E2F site, whether the Smad3-containing complex actively represses, as opposed to transactivates, the c-myc promoter.

Comparison of germline mosaics of genes in the Polycomb group of Drosophila melanogaster.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 231-243 ◽  
Author(s):  
M C Soto ◽  
T B Chou ◽  
W Bender
Keyword(s):  
Drosophila Melanogaster ◽  
Target Genes ◽  
Cell Types ◽  
Mitotic Recombination ◽  
Polycomb Group ◽  
Specific Cell ◽  
Independent Functions ◽  
Sex Combs ◽  
Null Phenotype ◽  
Additional Sex Combs

Abstract The genes of the Polycomb group (PcG) repress the genes of the bithorax and Antennapedia complexes, among others. To observe a null phenotype for a PcG gene, one must remove its maternal as well as zygotic contribution to the embryo. Five members of the PcG group are compared here: Enhancer of Polycomb [E(Pc)], Additional sex combs (Asx), Posterior sex combs (Psc), Suppressor of zeste 2 [Su (z) 2] and Polycomblike (Pcl). The yeast recombinase (FLP) system was used to induce mitotic recombination in the maternal germline. Mutant embryos were analyzed by staining with antibodies against six target genes of the PcG. The loss of the maternal component leads to enhanced homeotic phenotypes and to unique patterns of misexpression. E(Pc) and Su(z) 2 mutations had only subtle effects on the target genes, even when the maternal contributions were removed. Asx and Pcl mutants show derepression of the targets only in specific cell types. Psc shows unusual effects on two of the targets, Ultrabithorax and abdominal-A. These results show that the PcG genes do not act only in a common complex or pathway; they must have some independent functions.

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