BioTech

Human embryonic kidney (HEK293) cells are a widespread choice for recombinant protein expression. To optimise yields, the hydrolysate Tryptone N1 (TN1) is commonly added post-transfection. TN1 is obtained by controlled enzymatic digestion of casein. As an animal by-product, TN1 faces stricter regulations during cross-country shipments than plant-based products. This raises the question of whether plant-derived peptides are a suitable alternative to TN1. Using polyethyleneimine (PEI) as a cationic polymer, we transfected HEK293-6E cells grown in suspension in serum-free medium and divided the transfectants into four groups (each in triplicate). Two plant-based hydrolysates each derived from pea and broad bean were compared with TN1 and a no-hydrolysate control group. We monitored the cultures for total cell numbers and viability at days 1, 4, and 5 post-transfection. Both plant-based hydrolysates and TN1 showed similar live cell percentages, in contrast to the no-hydrolysate control, which showed lower viability. Five days post-transfection, the expressed His-tagged protein, a tegumental antigen from the eukaryotic parasite Echinococcus granulosus, was retrieved from the serum-free culture supernatant, and the expressed recombinant protein was quantified. The linear ranges for the protein load on the stain-free blot and for the use of the fluorescent anti-His-Tag Alexa₄₈₈ antibody were determined. Using these parameters, stain-free Western blotting and total protein normalization were performed. The plant-derived pea and broad bean hydrolysates reproducibly resulted in similar expression levels as animal-derived TN1; all three hydrolysates were better than no hydrolysate. We conclude that plant-derived hydrolysates are a suitable, more sustainable replacement for TN1.

This study investigates the functional role of OsHSBP1, a heat shock factor-binding protein, in regulating abiotic stress tolerance in rice, with the aim of enhancing climate resilience in the elite indica cultivar Bacthom 7 (BT7). Using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing, we generated transgene-free homozygous knockout lines targeting OsHSBP1 and evaluated their physiological, biochemical, and agronomic responses under heat stress. Mutant lines exhibited markedly improved tolerance to both stresses, with survival rates reaching 43–46% under heat stress, compared to near-zero in wildtype plants. Enhanced tolerance was associated with significantly increased catalase and peroxidase activities and reduced oxidative damage, including lower malondialdehyde content and decreased superoxide accumulation. Despite these stress-related advantages, the knockout lines showed minimal differences in key agronomic traits under normal growing conditions, with comparable plant height, tillering ability, grain yield, and amylose content relative to the wildtype. These results demonstrate that OsHSBP1 functions as a negative regulator of abiotic stress tolerance in rice, and its knockout enhances resilience without compromising yield potential. The study highlights OsHSBP1 as a promising target for precision breeding of climate-resilient rice cultivars.

Prostate cancer (PCa) is a leading cause of cancer-related mortality in men and is often characterized by aggressive growth and bone metastasis. Angiogenesis plays a central role in tumor progression and dissemination. This study aimed to explore the regulatory roles of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in angiogenesis and metastasis during PCa progression. Publicly available RNA-seq datasets were analyzed to identify differentially expressed miRNAs between metastatic (N1) and nonmetastatic (N0) PCa. Bioinformatic tools were used to reconstruct co-regulatory networks involving miRNAs, lncRNAs, and angiogenesis-related mRNAs. RT-qPCR was performed on serum-derived liquid biopsies from N0 and N1 patients and healthy controls to validate the key regulatory axes. Transcriptomic analysis revealed that miRNAs such as hsa-miR-183-5p and hsa-miR-216a-5p were upregulated in N1 PCa and associated with pro-angiogenic signaling, whereas hsa-miR-206 and hsa-miR-184, known for their anti-angiogenic functions, were downregulated. Network analysis identified the LINC00261–miR-206–HIF1A axis as the central regulatory module. RT-qPCR validation confirmed the significant downregulation of LINC00261 and miR-206, along with HIF1A overexpression in N1 samples compared to N0 and controls (p < 0.001), supporting in silico predictions. These findings highlight the role of ncRNA-mediated regulation of PCa angiogenesis and metastasis. The LINC00261–miR-206–HIF1A axis may serve as a promising noninvasive biomarker and potential therapeutic target. The integration of computational and experimental data provides a strong rationale for the further functional validation of advanced PCa.