scholarly journals Knock out of Specific Maternal Vitellogenins in Zebrafish (Danio Rerio) Evokes Vital Changes in Egg Proteomic Profiles that resemble the Phenotype of Poor Quality Eggs

2020 ◽  
Author(s):  
Ozlem YILMAZ ◽  
Amelie Patinote ◽  
Emmanuelle Com ◽  
Charles Pineau ◽  
Julien Bobe
Keyword(s):  
Energy Homeostasis ◽  
Molecular Mechanisms ◽  
Cellular Proliferation ◽  
Poor Quality ◽  
Developmental Competence ◽  
Type I ◽  
Knock Out ◽  
Type Iii ◽  
Proteome Database ◽  
Vertebrate Eggs

Abstract Background We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence of essentiality and disparate functioning of specific types of Vtg at different times during vertebrate development. However, the lack of knowledge on specific contributions of different types of Vtg to molecular functions related to major developmental processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to observe proteomic profiles of zebrafish eggs lacking three type-I Vtgs (Vtg 1, 4 and 5) simultaneously (vtg1-KO), or lacking type III Vtg (vtg3) only (vtg3-KO), as compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts.Results The vtg-KO in zebrafish revealed impaired proteomes of 1 hour post fertilization (hpf) zebrafish eggs which were highly resembled the proteomic phenotype of poor quality eggs of the same developmental stage reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs in protein homeostasis (synthesis and degradation) and in energy homeostasis even after zygotic genome activation. Increase in endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and human neurodegenerative disease related mechanisms in both vtg1- and vtg3-KO eggs point to severe molecular changes and consecutive dysfunctions in the abovementioned cellular activities. Distinctive increase in apoptosis and Parkinson disease pathways and decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms were unveiled to be considered as strong candidate markers to study details of these mechanisms during early embryonic development in zebrafish and potentially other vertebrates. Conclusions These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

scholarly journals Knock out of specific maternal vitellogenins in zebrafish (Danio rerio) evokes vital changes in egg proteomic profiles that resemble the phenotype of poor quality eggs

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Ozlem Yilmaz ◽  
Amelie Patinote ◽  
Emmanuelle Com ◽  
Charles Pineau ◽  
Julien Bobe
Keyword(s):  
Endoplasmic Reticulum ◽  
Embryonic Development ◽  
Molecular Mechanisms ◽  
Cellular Proliferation ◽  
Poor Quality ◽  
Developmental Competence ◽  
Type I ◽  
Knock Out ◽  
Type Iii ◽  
Mitochondrial Functions

Abstract Background We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence on essentiality and disparate functioning of Vtgs at different stages during early development. However, the specific contributions of different types of Vtg to major cellular processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to meet this deficit. Proteomic profiles of zebrafish eggs lacking three type-I Vtgs simultaneously (vtg1-KO), or lacking only type III Vtg (vtg3-KO) were compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts. Results The vtg-KO caused severe changes in the proteome of 1-cell stage zebrafish eggs. These changes were disclosed by molecular signatures that highly resembled the proteomic phenotype of poor quality zebrafish eggs reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs to protein and in energy homeostasis. The lack of this contribution appears to have a significant impact on endoplasmic reticulum and mitochondrial functions, and thus embryonic development, even after zygotic genome activation. Increased endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and in human neurodegenerative disease related activities in both vtg1- and vtg3-KO eggs were found to be indicators of the aforementioned conditions. Distinctive increase in apoptosis and Parkinson disease pathways, as well as the decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms have been identified as strong candidate markers for studying the details of these mechanisms during early embryonic development in zebrafish and possibly other vertebrates. Conclusions These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

scholarly journals Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1523
Author(s):  
Isabelle Anna Zink ◽  
Erika Wimmer ◽  
Christa Schleper
Keyword(s):  
Comparative Analysis ◽  
Molecular Mechanisms ◽  
Model Organisms ◽  
Special Focus ◽  
Natural Environments ◽  
Type I ◽  
Accessory Proteins ◽  
Type Iii

Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

Corticosterone impairs the mRNA expression and activity of 3β- and 17β-hydroxysteroid dehydrogenases in adult rat Leydig cells

2006 ◽  
Vol 84 (5) ◽  
pp. 745-754 ◽  
Author(s):  
R. Badrinarayanan ◽  
S. Rengarajan ◽  
P. Nithya ◽  
K. Balasubramanian
Keyword(s):  
Mrna Expression ◽  
Leydig Cells ◽  
Molecular Mechanisms ◽  
Experimental Studies ◽  
In Vitro Studies ◽  
Type I ◽  
Total Rna ◽  
Type Iii ◽  
Hydroxysteroid Dehydrogenases

Clinical and experimental studies, including our own observations, have shown the adverse effects of excess glucocorticoids on testicular steroid hormone production. The present study was designed to gain insight into the molecular mechanisms by which excess corticosterone impairs Leydig cell steroidogenesis. To achieve this, adult rats were administered with corticosterone-21-acetate (2 mg/100 g body weight) twice daily for 15 days. After the treatment period, rats were killed by decapitation. The testes were removed, decapsulated aseptically and used for the isolation of Leydig cells. Purified Leydig cells were used for assessing the activity of 3β- and 17β-hydroxysteroid dehydrogenases (HSDs) and total RNA isolation. For in vitro studies, purified Leydig cells (7.5 × 106 cells) of control rats were plated in culture flasks and exposed to different concentrations (50, 100, 200, 400, and 800 nmol/L) of corticosterone for 24 h. At the end of incubation, total RNA was isolated from cultured Leydig cells, and the mRNA of 3β- and 17β-HSDs was quantified by RT–PCR. A significant reduction in the activities and levels of 3β-HSD type-I and 17β-HSD type-III mRNAs in Leydig cells were observed. In vitro studies demonstrated a dose-dependent significant impairment in both the activity and mRNA expression of these enzymes. These results suggest that corticosterone might have a direct effect on the transcription of the genes of 3β- and 17β-HSD. It is inferred from the present in vivo and in vitro studies that one of the molecular mechanisms by which excess corticosterone decreases the steroidogenic potency of Leydig cells is by suppressing the mRNA expression of 3β-HSD type-I and 17β-HSD type-III enzymes.

scholarly journals Microscopic Colitis. Common Features and Differences

2019 ◽  
Vol 26 (5) ◽  
pp. 65-76
Author(s):  
Galina M. Mogil’naya ◽  
Vladimir M. Durleshter ◽  
Vera L. Mogil’naya ◽  
Lida K. Kovaleva ◽  
Lyudmila G. Dryaeva
Keyword(s):  
Lamina Propria ◽  
Molecular Mechanisms ◽  
Microscopic Colitis ◽  
Lymphocytic Colitis ◽  
Surface Epithelium ◽  
Synthesis Rate ◽  
Type Iii Collagen ◽  
Type I ◽  
Type Iii ◽  
Irritable Bowel

Aim. To study the morpho-molecular mechanisms underlying the formation of microscopic colitis (MC), as well as to identify features of its two forms – collagenous and lymphocytic.Material and methods. We studied biopsy samples from 23 patients exhibiting a clinical picture of irritable bowel syndrome; the material was obtained at the Endoscopic Department of the Region Clinic Hospital No. 2. The material was sampled from the five zones of the large intestine specified in the guidelines (Clinical Guidelines — Diagnosis and treatment of patients with digestive diseases, Appendix No. 3). The material was fixed in 10 % formalin, processed and embedded in paraffin. Sections were stained with hematoxylin and eosin (according to Mallory and Masson), as well as with picrosirius red, followed by the examination of these sections in polarised light. The immunohistochemical study was performed in line with the guidelines using monoclonal antibodies. Abcam antibodies (England) were used to detect type I and type III collagen; Cell Marque antibodies to CD4+ T and CD8+ T-lymphocytes (USA) were used to characterise lymphocytes.Results. It has been established that fi broblasts in the lamina propria play a key role in the pathogenesis of collagenous MC. This cell population synthesises extracellular matrix and forms layers of collagen fibres in the area under the surface epithelium. Pericryptal fibroblasts are also activated. Their differentiation occurs simultaneously with the migration of epithelial cells to the surface of the crypts with a possible change in their cellular composition. Intercryptal fi broblasts provide an increase in the synthesis rate of type III collagen. In the case of lymphocytic colitis, the pathogenetic mechanism is based on the relationship between lymphocytes and the cells in the lamina propria. The outcome is determined by the type of activated lymphocytes. CD8+ lymphocytes infiltrate the epithelial lining, causing a reaction to the luminal component, whereas CD4+ lymphocytes act as helpers and populate the lamina propria in the area under the epithelium.Conclusion. The pathogenesis of collagenous MC is based on the mechanism exhibited by the fibroblasts in the colon lamina propria, whereas the pathogenesis of lymphocytic colitis is determined by the dynamics of CD4+ T and CD8+ T-lymphocyte subpopulations.

scholarly journals RNA Regulated Toxin-Antitoxin Systems in Pathogenic Bacteria

2021 ◽  
Vol 11 ◽  
Author(s):  
David D. Sarpong ◽  
Erin R. Murphy
Keyword(s):  
Small Rnas ◽  
Pathogenic Bacteria ◽  
Molecular Mechanisms ◽  
Environmental Changes ◽  
Bacterial Toxin ◽  
Type I ◽  
Bacterial Physiology ◽  
Type Iii ◽  
Two Factors

The dynamic host environment presents a significant hurdle that pathogenic bacteria must overcome to survive and cause diseases. Consequently, these organisms have evolved molecular mechanisms to facilitate adaptation to environmental changes within the infected host. Small RNAs (sRNAs) have been implicated as critical regulators of numerous pathways and systems in pathogenic bacteria, including that of bacterial Toxin-Antitoxin (TA) systems. TA systems are typically composed of two factors, a stable toxin, and a labile antitoxin which functions to protect against the potentially deleterious activity of the associated toxin. Of the six classes of bacterial TA systems characterized to date, the toxin component is always a protein. Type I and Type III TA systems are unique in that the antitoxin in these systems is an RNA molecule, whereas the antitoxin in all other TA systems is a protein. Though hotly debated, the involvement of TA systems in bacterial physiology is recognized by several studies, with the Type II TA system being the most extensively studied to date. This review focuses on RNA-regulated TA systems, highlighting the role of Type I and Type III TA systems in several pathogenic bacteria.

Methionine-Specific Staining of Collagen

1982 ◽  
Vol 40 ◽  
pp. 294-295
Author(s):  
E.M. Kuhn ◽  
K.D. Marenus ◽  
M. Beer
Keyword(s):  
Amino Acids ◽  
Collagen Fibers ◽  
Type Iii Collagen ◽  
Type I ◽  
Electron Microscopic ◽  
Good Correspondence ◽  
Specific Staining ◽  
Type Iii ◽  
Different Types ◽  
Sulfur Containing

Fibers composed of different types of collagen cannot be differentiated by conventional electron microscopic stains. We are developing staining procedures aimed at identifying collagen fibers of different types.Pt(Gly-L-Met)Cl binds specifically to sulfur-containing amino acids. Different collagens have methionine (met) residues at somewhat different positions. A good correspondence has been reported between known met positions and Pt(GLM) bands in rat Type I SLS (collagen aggregates in which molecules lie adjacent to each other in exact register). We have confirmed this relationship in Type III collagen SLS (Fig. 1).

Labelling of non-A, non-B hepatitis infected chimpanzee hepatocytes with nuclease-gold conjugates

1984 ◽  
Vol 42 ◽  
pp. 250-251
Author(s):  
G. D. Gagne ◽  
M. F. Miller ◽  
D. A. Peterson
Keyword(s):  
Endoplasmic Reticulum ◽  
Experimental Infection ◽  
Uranyl Acetate ◽  
Type I ◽  
Cellular Injury ◽  
Type Ii ◽  
Tubular Structures ◽  
Type Iii ◽  
Type Iv ◽  
Infected Chimpanzee

Experimental infection of chimpanzees with non-A, non-B hepatitis (NANB) or with delta agent hepatitis results in the appearance of characteristic cytoplasmic alterations in the hepatocytes. These alterations include spongelike inclusions (Type I), attached convoluted membranes (Type II), tubular structures (Type III), and microtubular aggregates (Type IV) (Fig. 1). Type I, II and III structures are, by association, believed to be derived from endoplasmic reticulum and may be morphogenetically related. Type IV structures are generally observed free in the cytoplasm but sometimes in the vicinity of type III structures. It is not known whether these structures are somehow involved in the replication and/or assembly of the putative NANB virus or whether they are simply nonspecific responses to cellular injury. When treated with uranyl acetate, type I, II and III structures stain intensely as if they might contain nucleic acids. If these structures do correspond to intermediates in the replication of a virus, one might expect them to contain DNA or RNA and the present study was undertaken to explore this possibility.

Wnt/beta-catenin and PI3K/Akt/mTOR Signaling Pathways in Glioblastoma: Two Main Targets for Drug Design: A Review

2020 ◽  
Vol 26 (15) ◽  
pp. 1729-1741 ◽  
Author(s):  
Seyed H. Shahcheraghi ◽  
Venant Tchokonte-Nana ◽  
Marzieh Lotfi ◽  
Malihe Lotfi ◽  
Ahmad Ghorbani ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Cellular Proliferation ◽  
Chemotherapy Resistance ◽  
Wnt Signaling Pathway ◽  
Therapeutic Interventions ◽  
Beta Catenin ◽  
Clinical Prognosis ◽  
Invasion And Migration ◽  
And Migration

: Glioblastoma (GBM) is the most common and malignant astrocytic glioma, accounting for about 90% of all brain tumors with poor prognosis. Despite recent advances in understanding molecular mechanisms of oncogenesis and the improved neuroimaging technologies, surgery, and adjuvant treatments, the clinical prognosis of patients with GBM remains persistently unfavorable. The signaling pathways and the regulation of growth factors of glioblastoma cells are very abnormal. The various signaling pathways have been suggested to be involved in cellular proliferation, invasion, and glioma metastasis. The Wnt signaling pathway with its pleiotropic functions in neurogenesis and stem cell proliferation is implicated in various human cancers, including glioma. In addition, the PI3K/Akt/mTOR pathway is closely related to growth, metabolism, survival, angiogenesis, autophagy, and chemotherapy resistance of GBM. Understanding the mechanisms of GBM’s invasion, represented by invasion and migration, is an important tool in designing effective therapeutic interventions. This review will investigate two main signaling pathways in GBM: PI3K/Akt/mTOR and Wnt/beta-catenin signaling pathways.

Identification of Differentially Expressed Genes Associated with Papillary Thyroid Carcinoma

2020 ◽  
Vol 23 (6) ◽  
pp. 546-553
Author(s):  
Hongyuan Cui ◽  
Mingwei Zhu ◽  
Junhua Zhang ◽  
Wenqin Li ◽  
Lihui Zou ◽  
...  
Keyword(s):  
Papillary Thyroid Carcinoma ◽  
Thyroid Carcinoma ◽  
Differentially Expressed Genes ◽  
Molecular Mechanisms ◽  
Cellular Proliferation ◽  
Mrna Level ◽  
Thyroid Tissue ◽  
Differentially Expressed ◽  
Thyroid Tumors ◽  
Papillary Thyroid

Objective: Next-generation sequencing (NGS) was performed to identify genes that were differentially expressed between normal thyroid tissue and papillary thyroid carcinoma (PTC). Materials & Methods: Six candidate genes were selected and further confirmed with quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry in samples from 24 fresh thyroid tumors and adjacent normal tissues. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to investigate signal transduction pathways of the differentially expressed genes. Results: In total, 1690 genes were differentially expressed between samples from patients with PTC and the adjacent normal tissue. Among these, SFRP4, ZNF90, and DCN were the top three upregulated genes, whereas KIRREL3, TRIM36, and GABBR2 were downregulated with the smallest p values. Several pathways were associated with the differentially expressed genes and involved in cellular proliferation, cell migration, and endocrine system tumor progression, which may contribute to the pathogenesis of PTC. Upregulation of SFRP4, ZNF90, and DCN at the mRNA level was further validated with RT-PCR, and DCN expression was further confirmed with immunostaining of PTC samples. Conclusion: These results provide new insights into the molecular mechanisms of PTC. Identification of differentially expressed genes should not only improve the tumor signature for thyroid tumors as a diagnostic biomarker but also reveal potential targets for thyroid tumor treatment.

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