scholarly journals The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids

2021 ◽  
Author(s):  
Ana Carolina Monteleone-Cassiano ◽  
Janaina A Dernowsek ◽  
Romario S Mascarenhas ◽  
Amanda F Assis ◽  
Dimitrius Pitol ◽  
...  
Keyword(s):  
3D Structure ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Thymic Epithelial Cells ◽  
Thymic Epithelial Cell ◽  
Autoimmune Regulator ◽  
Aire Gene ◽  
Genes Encoding ◽  
Thymic Medulla

Besides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown that AIRE knockout (KO) mice have a small and disorganized thymic medulla; however, whether Aire influences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed an in vitro model system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. The effect of Aire on mTEC-mTEC adhesion was evaluated by comparing AIRE wild type (AIREWT) versus Aire KO (AIRE-/-) mTECs. Considering the 3D spheroid model evaluated, we reported that the absence of AIRE disorganizes the 3D structure of mTEC spheroids, promotes a differential regulation of mTEC classical surface markers, and modulates genes encoding adhesion and other molecules.

scholarly journals A New Bioreactor to Promote Follicular Growth With or Without Activin A

2021 ◽  
Author(s):  
Cynthia Jovet ◽  
Eloise Fraison ◽  
Jacqueline Lornage ◽  
Nicolas Morel-journel ◽  
Antoine Gavoille ◽  
...  
Keyword(s):  
3D Structure ◽  
Three Dimensional ◽  
Activin A ◽  
Structure Group ◽  
Dimensional Structure ◽  
Follicular Growth ◽  
Primordial Follicles ◽  
Estradiol Secretion ◽  
Follicular Density

Abstract Background: The aim of the present study was to evaluate the effect of Activin A on the activation of in vitro folliculogenesis of human ovarian tissues with or without our new three-dimensional structure (3D-structure). Methods: Five fresh ovarian human tissues were cultured in vitro in 4 groups with 100ng/mL Activin A or without Activine A and within or without a 3D-structure for 20 or 22 days of in vitro culture. Follicular density and quality were evaluated, and follicular diameters were measured. Estradiol secretion was quantified. Proliferation and apoptosis through immunostaining were performed.Results: The proportion of primordial follicles was significantly reduced, and the proportion of primary and secondary follicles was significantly increased in all four groups (p<0.001). Antral cavities were observed in the four culture groups. Activin A supplementation did not significantly affect the follicular density, follicular quality, follicular growth, or estradiol secretion (p>0.05). The 3D-structure increased the density of primary follicles and decreased the estradiol secretion (p<0.001). Tissular proliferation was significantly lower in the 3D-structure group compared to the non-3D-structure group (p=0.008). Regarding tissular apoptosis, it was significantly higher in the Activin group compared to the non-Activin group (0.006). Conclusion: The presence of Activin A did not seem to play a key role in in vitro folliculogenesis activation. However, the results may indicate that the 3D-structure could be more physiological and could prevent a pejorative in vitro folliculogenesis flare-up.

scholarly journals miR-155 exerts posttranscriptional control of autoimmune regulator (Aire) and tissue-restricted antigen genes in medullary thymic epithelial cells

2021 ◽  
Author(s):  
Pedro P Tanaka ◽  
Ernna H Oliveira ◽  
Mayara C Machado ◽  
Max J Duarte ◽  
Amanda F Assis ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Large Scale ◽  
Interaction Analysis ◽  
Luciferase Reporter ◽  
Thymic Epithelial Cells ◽  
Posttranscriptional Control ◽  
Autoimmune Regulator ◽  
Aire Gene ◽  
Medullary Thymic Epithelial Cells

Background: The autoimmune regulator (Aire) gene is critical for the appropriate establishment of central immune tolerance. As one of the main controllers of promiscuous gene expression in the thymus, Aire promotes the expression of thousands of downstream tissue-restricted antigen (TRA) genes, cell adhesion genes and transcription factor genes in medullary thymic epithelial cells (mTECs). Despite the increasing knowledge about the role of Aire as an upstream transcriptional controller, little is known about the mechanisms by which this gene could be regulated. Results: Here, we assessed the posttranscriptional control of Aire by miRNAs. The in silico miRNA-mRNA interaction analysis predicted thermodynamically stable hybridization between the 3UTR of Aire mRNA and miR-155, which was confirmed to occur within the cellular milieu through a luciferase reporter assay. This finding enabled us to hypothesize that miR-155 might play a role as an intracellular posttranscriptional regulator of Aire mRNA. To test this hypothesis, we transfected a murine mTEC cell line with a miR-155 mimic in vitro, which reduced the mRNA and protein levels of Aire. Moreover, large-scale transcriptome analysis showed the modulation of 311 downstream mRNAs, which included 58 TRA mRNAs. Moreover, miR-155 mimic-transfected cells exhibited a decrease in their chemotaxis property compared with control thymocytes. Conclusion: Overall, the results indicate that miR-155 may posttranscriptionally control Aire mRNA as well as a crucial process by which mTECs allow migration of thymocytes through chemotaxis.

scholarly journals Nanofiber-Mâché Hollow Ball Mimicking the Three-Dimensional Structure of a Cyst

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2273
Author(s):  
Wan-Ying Huang ◽  
Norichika Hashimoto ◽  
Ryuhei Kitai ◽  
Shin-ichiro Suye ◽  
Satoshi Fujita
Keyword(s):  
Three Dimensional ◽  
Hollow Structure ◽  
Dimensional Structure ◽  
The Novel ◽  
Fibrous Scaffold ◽  
Hydrogel Beads ◽  
Inner Surface ◽  
Intracranial Epidermoid ◽  
Hollow Ball

The occasional malignant transformation of intracranial epidermoid cysts into squamous cell carcinomas remains poorly understood; the development of an in vitro cyst model is urgently needed. For this purpose, we designed a hollow nanofiber sphere, the “nanofiber-mâché ball.” This hollow structure was fabricated by electrospinning nanofiber onto alginate hydrogel beads followed by dissolving the beads. A ball with approximately 230 mm3 inner volume provided a fibrous geometry mimicking the topography of the extracellular matrix. Two ducts located on opposite sides provided a route to exchange nutrients and waste. This resulted in a concentration gradient that induced oriented migration, in which seeded cells adhered randomly to the inner surface, formed a highly oriented structure, and then secreted a dense web of collagen fibrils. Circumferentially aligned fibers on the internal interface between the duct and hollow ball inhibited cells from migrating out of the interior, similar to a fish bottle trap. This structure helped to form an adepithelial layer on the inner surface. The novel nanofiber-mâché technique, using a millimeter-sized hollow fibrous scaffold, is excellently suited to investigating cyst physiology.

scholarly journals Protein Folding: Search for Basic Physical Models

2003 ◽  
Vol 3 ◽  
pp. 623-635 ◽  
Author(s):  
Ivan Y. Torshin ◽  
Robert W. Harrison
Keyword(s):  
Protein Folding ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Physical Models ◽  
Dimensional Structure ◽  
Computational Techniques ◽  
Linear Sequence ◽  
Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

scholarly journals Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

2008 ◽  
Vol 190 (6) ◽  
pp. 2056-2064 ◽  
Author(s):  
Jonathan E. Ulmer ◽  
Yap Boum ◽  
Christopher D. Thouvenel ◽  
Hannu Myllykallio ◽  
Carol Hopkins Sibley
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Thymidylate Synthase ◽  
Three Dimensional ◽  
Pyrimidine Ring ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Catalytic Residue ◽  
Amino Acid Residues ◽  
Insight Into

ABSTRACT A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX7-8S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ΔthyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X24 H69X25R95HRX7 S105XRYX90R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

3D Structure of Striations in the North Pacific

2021 ◽  
Author(s):  
YU ZHANG ◽  
YU PING GUAN ◽  
RUI XIN HUANG
Keyword(s):  
North Pacific ◽  
3D Structure ◽  
Three Dimensional ◽  
Vertical Plane ◽  
Dimensional Structure ◽  
The North ◽  
Layer Analysis ◽  
Fluid Potential ◽  
The North Pacific

AbstractOcean striations are composed of alternating quasi-zonal band-like flows; this kind of organized structure of currents be found in all world’s oceans and seas. Previous studies have mainly been focused on the mechanisms of their generation and propagation. This study uses the spatial high-pass filtering to obtain the three-dimensional structure of ocean striations in the North Pacific in both the z-coordinate and σ-coordinate based on 10-yr averaged SODA3 data. First, we identify an ideal-fluid potential density domain where the striations are undisturbed by the surface forcing and boundary effects. Second, using the isopycnal layer analysis, we show that on isopycnal surfaces the orientations of striations nearly follow the potential vorticity (PV) contours, while in the meridional-vertical plane the central positions of striations are generally aligned with the latitude of zero gradient of the relative PV. Our analysis provides a simple dynamical interpretation and better understanding for the role of ocean striations.

scholarly journals Analysis of the three dimensional structure of the kidney glomerulus capillary network

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mark Terasaki ◽  
Jason Cory Brunson ◽  
Justin Sardi
Keyword(s):  
3D Structure ◽  
Three Dimensional ◽  
The Body ◽  
Capillary Network ◽  
Dimensional Structure ◽  
Pixel Resolution ◽  
Longitudinal Splitting ◽  
Kidney Glomerulus ◽  
Minimum Number ◽  
Glomerular Size

AbstractThe capillary network of the kidney glomerulus filters small molecules from the blood. The glomerular 3D structure should help to understand its function, but it is poorly characterized. We therefore devised a new approach in which an automated tape collecting microtome (ATUM) was used to collect 0.5 μm thick serial sections from fixed mouse kidneys. The sections were imaged by scanning electron microscopy at ~ 50 nm/pixel resolution. With this approach, 12 glomeruli were reconstructed at an x–y–z resolution ~ 10 × higher than that of paraffin sections. We found a previously undescribed no-cross zone between afferent and efferent branches on the vascular pole side; connections here would allow blood to exit without being adequately filtered. The capillary diameters throughout the glomerulus appeared to correspond with the amount of blood flow within them. The shortest path (minimum number of branches to travel from afferent to efferent arterioles) is relatively independent of glomerular size and is present primarily on the vascular pole size. This suggests that new branches and longer paths form on the urinary pole side. Network analysis indicates that the glomerular network does not form by repetitive longitudinal splitting of capillaries. Thus the 3D structure of the glomerular capillary network provides useful information with which to understand glomerular function. Other tissue structures in the body may benefit from this new three dimensional approach.

scholarly journals Molecular Cloning, Modeling, and Characterization of Type 2 Metallothionein from Plantago ovata Forsk

Sequencing ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Amitava Moulick ◽  
Debashis Mukhopadhyay ◽  
Shonima Talapatra ◽  
Nirmalya Ghoshal ◽  
Sarmistha Sen Raychaudhuri
Keyword(s):  
Molecular Cloning ◽  
Structure Prediction ◽  
3D Structure ◽  
Three Dimensional ◽  
Transcript Level ◽  
Copper Stress ◽  
Dimensional Structure ◽  
Functional Study ◽  
Plantago Ovata

Plantago ovata Forsk is a medicinally important plant. Metallothioneins are cysteine rich proteins involved in the detoxification of heavy metals. Molecular cloning and modeling of MT from P. ovata is not reported yet. The present investigation will describe the isolation, structure prediction, characterization, and expression under copper stress of type 2 metallothionein (MT2) from this species. The gene of the protein comprises three exons and two introns. The deduced protein sequence contains 81 amino acids with a calculated molecular weight of about 8.1 kDa and a theoretical pI value of 4.77. The transcript level of this protein was increased in response to copper stress. Homology modeling was used to construct a three-dimensional structure of P. ovata MT2. The 3D structure model of P. ovata MT2 will provide a significant clue for further structural and functional study of this protein.

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