Inhibition of ribosome assembly factor PNO1 by CRISPR/Cas9 technique suppresses lung adenocarcinoma and Notch pathway: Clinical application

Growth is crucially controlled by the functional ribosomes available in cells. To meet the enhanced energy demand, cancer cells re-wire and increase their ribosome biogenesis. The RNA-binding protein PNO1, a ribosome assembly factor, plays an essential role in ribosome biogenesis. The purpose of this study was to examine whether PNO1 can be used as a biomarker for lung adenocarcinoma and also examine the molecular mechanisms by which PNO1 knockdown by CRISPR/Cas9 inhibited growth and epithelial–mesenchymal transition (EMT). The expression of PNO1 was significantly higher in lung adenocarcinoma compared to normal lung tissues. PNO1 expression in lung adenocarcinoma patients increased with stage, nodal metastasis, and smoking. Lung adenocarcinoma tissues from males expressed higher PNO1 than those from females. Furthermore, lung adenocarcinoma tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53, suggesting the influence of Tp53 status on PNO1 expression. PNO1 knockdown inhibited cell viability, colony formation, and EMT, and induced apoptosis. Since dysregulated signalling through the Notch receptors promotes lung adenocarcinoma, we measured the effects of PNO1 inhibition on the Notch pathway. PNO1 knockdown inhibited Notch signalling by suppressing the expression of Notch receptors, their ligands, and downstream targets. PNO1 knockdown also suppressed CCND1, p21, PTGS-2, IL-1α, IL-8, and CXCL-8 genes. Overall, our data suggest that PNO1 can be used as a diagnostic biomarker, and also can be an attractive therapeutic target for the treatment of lung adenocarcinoma.     

Isolation of 4-hydroxy 3-methyl 2-butenyl 4-diphosphate reductase (ApHDR) gene of methyl erythritol diphosphate (MEP) pathway,in silico analysis and differential tissue specific ApHDR expression in Andrographis paniculata (Burm. f) Nees

The full length Andrographis paniculate 4-hydroxy 3-methyl 2-butenyl 4-diphosphate reductase (ApHDR) gene of MEP pathway was isolated for the first time. The ApHDR ORF with 1404 bp flanked by 100 bp 5′UTR and 235 bp 3′UTR encoding 467 amino acids (NCBI accession number: {"type":"entrez-nucleotide","attrs":{"text":"MK503970","term_id":"1732262198"}}MK503970) and cloned in pET 102, transformed and expressed in E. coli BL21. The ApHDR protein physico-chemical properties, secondary and tertiary structure were analyzed. The Ramachandran plot showed 93.8% amino acids in the allowed regions, suggesting high reliability. The cluster of 16 ligands for binding site in ApHDR involved six amino acid residues having 5–8 ligands. The Fe-S cluster binding site was formed with three conserved residues of cysteine at positions C123, C214, C251 of ApHDR. The substrate HMBPP and inhibitors clomazone, paraquat, benzyl viologen’s interactions with ApHDR were also assessed using docking. The affinity of Fe-S cluster binding to the cleft was found similar to HMBPP. The HPLC analysis of different type of tissue (plant parts) revealed highest andrographolide content in young leaves followed by mature leaves, stems and roots. The differential expression profile of ApHDR suggested a significant variation in the expression pattern among different tissues such as mature leaves, young leaves, stem and roots. A 16-fold higher expression of ApHDR was observed in the mature leaves of A. paniculata as compared to roots. The young leaves and stem showed 5.5 fold and fourfold higher expression than roots of A. paniculata. Our result generated new genomic information on ApHDR which may open up prospects of manipulation for enhanced diterpene lactone andrographolide production in A. paniculata.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00952-0.     

Antagonistic Activity of Cellular Components of Potential Probiotic Bacteria, Isolated from the Gut of Labeo rohita, Against Aeromonas hydrophila

The objective of this study was to characterise the antagonistic activity of cellular components of potential probiotic bacteria isolated from the gut of healthy rohu (Labeo rohita), a tropical freshwater fish, against the fish pathogen, Aeromonas hydrophila. Three potential probiotic strains (referred to as R1, R2, and R5) were screened using a well diffusion, and their antagonistic activity against A. hydrophila was determined. Biochemical tests and 16S rRNA gene analysis confirmed that R1, R2, and R5 were Lactobacillus plantarum VSG3, Pseudomonas aeruginosa VSG2, and Bacillus subtilis VSG1, respectively. Four different fractions of cellular components (i.e. the whole-cell product, heat-killed whole-cell product [HKWCP], intracellular product [ICP], and extracellular product) of these selected strains were effective in an in vitro sensitivity test against 6 A. hydrophila strains. Among the cellular components, the ICP of R1, HKWCP of R2, and ICP of R5 exhibited the strongest antagonistic activities, as evidenced by their inhibition zones. The antimicrobial compounds from these selected cellular components were partially purified by thin-layer and high-performance liquid chromatography, and their properties were analysed. The ranges of pH stability of the purified compounds were wide (3.0–10.0), and compounds were thermally stable up to 90 °C. Considering these results, isolated probiotic strains may find potential applications in the prevention and treatment of aquatic aeromonosis.     

Hepatoma derived growth factor (HDGF) dynamics in ovarian cancer cells

As a leading cause of cancer death among women, identification of pathophysiologically-relevant biomarkers for ovarian cancer is important. The heparin binding, hepatoma-derived growth factor (HDGF) is overexpressed in ovarian cancer cell lines and may have prognostic value, but the mechanism by which this predominantly nuclear protein is secreted or functions is unknown. In this study, we focused on the circumstances under which HDGF is released by cells and the functional relevance of extracellular HDGF in the context of ovarian cancer. Immunofluorescence imaging showed nuclear localization of HDGF in ovarian cells, but unlike what is reported for other cell types, HDGF was minimally secreted into the media. However, HDGF was passively released by necrotic and late apoptotic cells. Extracellular HDGF was functionally relevant as it stimulated phosphorylation of ERK 1/2 and P38 in both non-cancer and ovarian cancer cells, and enhanced cellular migration. Overall, our study uncovers a novel function of HDGF as a messenger of cellular condition (alarmin) which in-turn modulates cellular function-aspects that could be used as a biomarker for ovarian cancer.