ABSTRACT
In situ RT-PCR presents advantages over other expression analysis methods due to its rapid processing and low-cost equipment. However, this technique is not without its challenges. A protocol based on a capsule made from centrifuge tubes that offers advantages over slides is presented. This capsule protects histological sections from drying out, and its easy assembly reduces time pauses between incubations. In addition, the container size where the sample is deposited allows the addition and withdrawal of the different solutions. The capsule does not need previous sealing after each incubation, and, above all, it is a low-cost and accessible material. A guideline for tissue sectioning using a cryostat that offers advantages over other sectioning methods is also described.
Subject(s)
Centrifugation , Reverse Transcriptase Polymerase Chain Reaction , Centrifugation/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Plants/genetics , RNA, Plant/geneticsABSTRACT
Auxin is involved in almost every aspect of plant growth and development, from embryogenesis to senescence. Indole-3-acetic acid (IAA) is the main known natural auxin that is synthesized by enzymes tryptophan aminotransferase of arabidopsis (TAA) and YUCCA (YUC) of the flavin-containing monooxygenases family (FMO) from one of the tryptophan-dependent pathways. Genome-wide identification and comprehensive analysis of the YUC-protein family have been conducted in Coffea canephora in the present study. A total of 10 members CcYUC gene family were identified in C. canephora. Phylogenetic analysis revealed that the CcYUC protein family is evolutionarily conserved, and they consist of four groups. In contrast, bioinformatic analysis predicted a hydrophobic transmembrane helix (TMH) for one CcYUC (YUC10) member only. Isoelectric point (pI), molecular mass (Ms), signal peptide, subcellular localization, and phosphorylation sites were predicted for CcYUC proteins. YUC enzymes require the prosthetic group flavin adenine dinucleotide (FAD) and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) for their enzymatic activity. Therefore, we include the molecular docking for CcYUC2-FAD-NADPH-IPyA and yucasin, which is a specific inhibitor for YUC activity. The docking results showed FAD and NADPH binding at the big and small domain sites, respectively, in CcYUC2. IPyA binds very close to FAD along the big domain, and yucasin competes for the same site as IPA, blocking IAA production. Furthermore, in silico point mutations affect the stability of the CcYUC2-4 proteins.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Coffea , Yucca , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Coffea/genetics , Coffea/metabolism , Flavin-Adenine Dinucleotide/metabolism , Indoleacetic Acids/chemistry , Indoleacetic Acids/metabolism , Molecular Docking Simulation , NADP/metabolism , Phylogeny , Yucca/metabolismABSTRACT
Polyglycerol sebacate (PGS) scaffolds obtained using a leaching technique were modified with iodine-doped polypyrrole (PPy-I) in a plasma reactor in order to study the effect of exposure time on the cell viability of hDPSCs. SEM analysis showed the formation and growth of PPy-I particles as the exposure time was increased, while FTIR and XPS analysis revealed the presence of -NH- and N+ groups in the chemical composition of the surfaces, relating to the increase in the amount of PPY-I particles. The water contact angle measurements showed an increase in the scaffold's hydrophilicity with greater exposure times which was also attributed to the rising of PPy-I particles. It was also observed that PPy-I promotes the rigidity of the treated PGS scaffolds. when in direct contact with treated PGS scaffolds, cell viability improved with respect to non-treated scaffolds, however only at shorter time exposures. Extracts of plasma-treated PGS scaffolds showed high cytotoxicity as the time exposure to plasma treatment was increased.
