Deciphering the Role of miR-30a-5p and DLGAP1 Gene in Non-small Cell Lung Cancer.

BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is the deadliest form of cancer worldwide. Understanding the mechanisms of lung cancer development is vital for targeted therapy advancements. This article explores the little-known role of the guanylate kinase-associated protein (GKAP), encoded by the Disks large-associated protein 1 (DLGAP1) gene, in NSCLC along with assessing microRNA-30a-5p's influence on DLGAP1 gene expression in the A549 cell line. MATERIALS AND METHODS: Experiments were conducted on A549 cells transfected with synthetic oligonucleotides. The luciferase assay was employed to confirm the binding site of miR-30a-5p to the 3'UTR of DLGAP1 mRNA. The role of miRNA-30a-5p mimic in regulating potential target gene expression at the protein and mRNA levels was studied by performing RT-qPCR and western blot analyses. The effects of DLGAP1 knockdown and miRNA-30a-5p mimic on cell viability and the cell cycle were evaluated using the MTT test and flow cytometry with annexin/iodide cell staining. RESULTS: The luciferase assay indicated that miR-30a-5p has the ability to bind to the 3'UTR of DLGAP1 mRNA. RT-qPCR revealed that the overexpression of miR-30a-5p down-regulates DLGAP1 mRNA. Western blot analysis indicated that miR-30a-5p slightly reduces the level of the GKAP protein. Knockdown of DLGAP1 with synthetic oligonucleotides, as well as transfection with a miR-30a-5p mimic, significantly attenuates cell proliferation and increases the number of cells in the early and late stages of apoptosis. CONCLUSION: Our findings reveal the antiproliferative effect of miR-30a-5p and DLGAP1 gene knockdown on A549 cancer cells, implying that these elements could be considered as therapeutic targets for personalized medicine in NSCLC patients.

Drop impact dynamics on the hydrophobic leaf surface of an aquatic plant: a case study of Pistia stratiotes.

Pistia stratiotes is an aquatic plant with a complex structure that allows it to stay afloat. It grows quickly, and in large numbers becomes an undesirable plant as an invasive species. Describing the dynamics of a water drop splash on P. stratiotes leaves can contribute to increasing knowledge of its behavior and finding alternative methods for eradicating it or using it for the benefit of the environment. The non-wettable surface of P. stratiotes presents a complex structure-simple uniseriate trichomes and also ridges and veins. We analyzed the drop impact on a leaf placed on the water surface and recorded it by high-speed cameras. Based on the recordings, quantitative and qualitative analyses were performed. After impacting the leaf, the water drop spread until it reached its maximum surface area accompanied by the ejection of early droplets in the initial stage. Thereafter, three scenarios of water behavior were observed: (i) drop receding and stabilization; (ii) drop receding and ejection of late droplets formed in the later stage as an effect of elastic deformation of the leaf; and (iii) drop breaking apart and ejection of late droplets. The results indicated that the increasing kinetic energy of the impacting drops expressed by the Weber number and the complex leaf surface have an effect on the course of the splash. The simple uniseriate trichomes of the P. stratiotes leaf and the high energy of the falling drops were responsible for the formation and characteristics of the early droplets. The presence of ridges and veins and the leaf's mechanical response had an impact on the occurrence of late droplets.

Proteolytic and Structural Changes in Rye and Triticale Roots under Aluminum Stress.

Proteolysis and structural adjustments are significant for defense against heavy metals. The purpose of this study was to evaluate whether the Al3+ stress alters protease activity and the anatomy of cereale roots. Azocaseinolytic and gelatinolytic measurements, transcript-level analysis of phytocystatins, and observations under microscopes were performed on the roots of Al3+-tolerant rye and tolerant and sensitive triticales exposed to Al3+. In rye and triticales, the azocaseinolytic activity was higher in treated roots. The gelatinolytic activity in the roots of rye was enhanced between 12 and 24 h in treated roots, and decreased at 48 h. The gelatinolytic activity in treated roots of tolerant triticale was the highest at 24 h and the lowest at 12 h, whereas in treated roots of sensitive triticale it was lowest at 12 h but was enhanced at 24 and 48 h. These changes were accompanied by increased transcript levels of phytocystatins in rye and triticale-treated roots. Light microscope analysis of rye roots revealed disintegration of rhizodermis in treated roots at 48 h and indicated the involvement of root border cells in rye defense against Al3+. The ultrastructural analysis showed vacuoles containing electron-dense precipitates. We postulate that proteolytic-antiproteolytic balance and structural acclimation reinforce the fine-tuning to Al3+.

The Leaf Wettability of Various Potato Cultivars.

Leaf wettability has an impact on a plant's ability to retain water on its leaf surface, which in turn has many environmental consequences. In the case of the potato leaf (Solanum tuberosum L.), water on the leaf surface may contribute to the development of a fungal disease. If fungal disease is caused, this may reduce the size of potato harvests, which contribute significantly to meeting global food demand. The aim of this study was to assess the leaf wettability of five potato cultivars (i.e., Bryza, Lady Claire, Rudawa, Russet Burbank, Sweet Caroline) in the context of its direct and indirect impact on potato yield. Leaf wettability was assessed on the basis of contact angle measurements using a sessile drop method with an optical goniometer. For Bryza and Rudawa cultivars, which showed, respectively, the highest and the lowest contact angle values, light microscopy as well as scanning electron microscopy analyses were performed. The results of the contact angle measurements and microscopic image analyses of the potato leaf surfaces indicated that the level of wettability was closely related to the type of trichomes on the leaf and their density. Therefore, higher resistance of the Rudawa cultivar to biotic stress conditions could be the result of the presence of two glandular trichome types (VI and VII), which produce and secrete metabolites containing various sticky and/or toxic chemicals that may poison or repel herbivores.

Protease activity and phytocystatin expression in Arabidopsis thaliana upon Heterodera schachtii infection.

The activity of plant proteases is important for amino acids recycling, removal of damaged proteins as well as defence responses. The second-stage juvenile of the beet cyst nematode Heterodera schachtii penetrates host roots and induces the feeding site called a syncytium. To determine whether infection by H. schachtii affects proteolysis, the protease activity was studied in Arabidopsis roots and shoots at the day of inoculation and 3, 7 and 15 days post inoculation (dpi). Nematode infection caused a decrease of protease activities in infected roots over the entire examination period at all studied pH values. In contrast, the activities of the low molecular mass as well as Ca2+-dependent cysteine proteases were found to be stimulated. In shoots of infected plants, the protease activity was diminished only at 15 dpi at all tested pH values. It was accompanied by changes in total soluble protein content, a higher protein carbonylation and a total polyphenol content. To go deeper into proteolysis regulation, the expression of phytocystatin genes, endogenous inhibitors of cysteine proteases, was examined in syncytia, roots and shoots. Expression of AtCYS1, AtCYS5 and AtCYS6 genes was enhanced upon cyst nematode infection. Our results suggest that changes in protease activities in roots and shoots and altered cystatin expression patterns in syncytia, roots and shoots are important for protein metabolism during cyst nematode infection.

The roles of cysteine proteases and phytocystatins in development and germination of cereal seeds.

Proteolysis is an important process for development and germination of cereal seeds. Among the many types of proteases identified in plants are the cysteine proteases (CPs) of the papain and legumain families, which play a crucial role in hydrolysing storage proteins during seed germination as well as in processing the precursors of these proteins and the inactive forms of other proteases. Moreover, all of the tissues of cereal seeds undergo progressive degradation via programed cell death, which is integral to their growth. In view of the important roles played by proteases, their uncontrolled activity could be harmful to the development of seeds and young seedlings. Thus, the activities of these enzymes are regulated by intracellular inhibitors called phytocystatins (PhyCys). The phytocystatins inhibit the activity of proteases of the papain family, and the presence of an additional motif in their C-termini allows them to also regulate the activity of members of the legumain family. A balance between the levels of cysteine proteases and phytocystatins is necessary for proper cereal seed development, and this is maintained through the antagonistic activities of gibberellins (GAs) and abscisic acid (ABA), which regulate the expression of the corresponding genes. Transcriptional regulation of cysteine proteases and phytocystatins is determined by cis-acting elements located in the promoters of these genes and by the expression of their corresponding transcription factors (TFs) and the interactions between different TFs.

A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro.

Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions.

Molecular Cloning and Expression Analysis of Triticale Phytocystatins During Development and Germination of Seeds.

Three triticale cDNAs encoding inhibitors of cysteine endopeptidases, belonging to phytocystatins, have been identified and designated as TrcC-1, TrcC-4 and TrcC-5. Full-length cDNAs of TrcC-1 (617 bp) and TrcC-4 (940 bp), as well as a fragment of TrcC-5 cDNA (369 bp), were obtained. A high-level identity of the deduced amino acid sequence of TrcCs with other known phytocystatins, especially with wheat and barley, has been observed. Moreover, the presence of conserved domain, containing the G and W residues, the sequence of QxVxG and the sequence of LARFAV, characteristic for plant cysteine endopeptidase inhibitors, has been noted. The profiles of TrcC-1 and TrcC-5 mRNA levels in the developing seeds of two triticale cultivars that differ in their resistance to preharvest sprouting (Zorro and Disco) were similar. However, the expression of TrcC-4 was, higher in the developing seeds, and in the scutellum of germinating seeds of a cultivar more resistant to preharvest sprouting (Zorro) than in the less resistant (Disco). Additionally, the expression of TrcC-4 remained longer in developing seeds of Zorro as compared to Disco. The performed studies suggest that TrcC-4 might have an influence on the higher resistance of Zorro cultivar to preharvest sprouting.