Enhancing the productivity and proliferation of CHO-K1 cells by oncoprotein YAP (Yes-associated protein).

CHO cells are extensively employed in biological drug industry to manufacture therapeutic proteins. Nevertheless, production of biopharmaceuticals faces obstacles such as limited growth and inadequate productivity. Employing host cell engineering techniques for CHO cells serves as a valuable approach to address the constraints encountered in biologics manufacturing. Despite advancements, most techniques focus on specific genes to address individual cellular challenges. The significance of YAP, transcriptional co-activator, cannot be overstated due to its involvement in regulating organ size and tumor formation. YAP's influence extends to various cellular processes and is regulated by kinase cascade in the Hippo pathway, which phosphorylates serine residues in specific LATS recognition motifs. Activation of YAP has been observed to impact both the size and quantity of cells. This research investigates the effects of YAP5SA on proliferation, apoptosis, and productivity in CHO-K1 cells. YAP5SA, with mutations in all five LATS-target sites, is selected for its heightened activity and resistance to repression through the Hippo-LATS1/2 kinase signaling pathway. Plasmid harboring YAP5SA was transfected into EPO-CHO and the influence of YAP5SA overexpression was investigated. According to our findings, transfection of EPO-CHO cells with YAP5SA exhibited a substantial enhancement in CHO cell productivity, resulting in a 3-fold increase in total protein and EPO, as well as a 1.5-fold increase in specific productivity. Additionally, it significantly contributes in augmenting viability, size, and proliferation. Overall, the findings of this study exemplify the potential of utilizing YAP5SA to impact particular cellular mechanisms, thereby presenting an avenue for customizing cells to fulfill production demands. KEY POINTS: • YAP5SA in CHO cells boosts growth, reduces apoptosis, and significantly improves productivity. • YAP5SA regulates genes involved in proliferation, survival, and mTOR activation. • YAP5SA increases productivity by improving cell cycle, c-MYC expression, and mTOR pathway.

Enhancing protein production and growth in chinese hamster ovary cells through miR-107 overexpression.

Chinese Hamster Ovary (CHO) cells are widely employed as host cells for biopharmaceutical production. The manufacturing of biopharmaceuticals poses several challenges, including restricted growth potential and inadequate productivity of the host cells. MicroRNAs play a crucial role in regulating gene expression and are considered highly promising tools for cell engineering to enhance protein production. Our study aimed to evaluate the effects of miR-107, which is recognized as an onco-miR, on erythropoietin-producing CHO cells (CHO-hEPO). To assess the impact of miR-107 on CHO cells, a DNA plasmid containing miR-107 was introduced to CHO-hEPO cells through transfection. Cell proliferation and viability were assessed using the trypan blue dye exclusion method. Cell cycle analysis was conducted by utilizing propidium iodide (PI) staining. The quantification of EPO was determined using an immunoassay test. Moreover, the impact of miR-107 on the expression of downstream target genes was evaluated using qRT-PCR. Our findings highlight and underscore the substantial impact of transient miR-107 overexpression, which led to a remarkable 2.7-fold increase in EPO titers and a significant 1.6-fold increase in the specific productivity of CHO cells (p < 0.01). Furthermore, this intervention resulted in significant enhancements in cell viability and growth rate (p < 0.05). Intriguingly, the overexpression of miR­107 was linked to the downregulation of LATS2, PTEN, and TSC1 genes while concurrently driving upregulation in transcript levels of MYC, YAP, mTOR, and S6K genes within transgenic CHO cells. In conclusion, this study collectively underscores the feasibility of utilizing cancer-associated miRNAs as a powerful tool for CHO cell engineering. However, more in-depth exploration is warranted to unravel the precise molecular intricacies of miR-107's effects in the context of CHO cells.

Fibroblast Growth Factor-2 Levels are Elevated in The Serum of Patients with Multiple Sclerosis

BACKGROUND: Various factors contribute to the pathogenesis of Multiple Sclerosis (MS), one of which is Fibroblast Growth Factor 2 (FGF2). The function of FGF2 is pleiotropic. The investigation of the role of this factor in the myelination has produced conflicting results. OBJECTIVE: To investigate the serum levels of FGF2 in patients with MS. SUBJECTS AND METHODS: Eighty patients with MS and eighty healthy volunteers with no history of inflammation or demyelinating disorders were included, and serum samples were collected to evaluate serum levels of FGF2 using the ELISA technique. Both groups had the same age and gender distribution. For analysis, the Mann-Whitney U test was used. RESULTS: Patients with MS had considerably greater serum FGF2 levels than the control group (p = 0.005). There was no difference between the FGF2 level in men and women. CONCLUSION: Our data indicate that FGF2 levels may be related to the susceptibility of Iranian patients with MS. Further studies are required to analyze the involvement of FGF2 in enhancing the inflammatory process in MS.

Cloning and Expression of Poly 3-Hydroxybutyrate Operon Into Escherichia coli.

BACKGROUND: Poly 3-Hydroxybutyrate (PHB), a class of Poly Hydroxyalkanoates (PHAs), is a group of bacterial storage polymers, produced by various microorganisms in response to nutrient limitation. PHAs are biodegradable polymers which could be a good substitute for current petrochemical plastics. PHB has been synthesized during three enzymatic steps including three genes. OBJECTIVES: Our aim was PHB production from recombinant bacteria. MATERIALS AND METHODS: Ralstonia eutropha was cultured and its genomic DNA was extracted. The phbCAB operon was amplified using designed primers. The fragment was cloned into pET-28a expression vector and then transformed into Escherichia coli BL21. Sudan black staining was used to show the production of PHB. RESULTS: The extracted recombinant plasmid was digested with restriction enzymes. Separation of the desired fragment from the vector was performed to prove the correct insertion of the PCR products into the vector. The colony PCR and sequencing results confirmed the successful transformation. The production of PHB was confirmed by Sudan Black B staining under a light microscope. CONCLUSIONS: Various metabolic and fermentation methods have been used in some bacterial strains for PHB production. The use of a recombinant system harboring PHB synthesis genes can produce PHB in higher concentrations compare to natural PHA-producing bacteria. The present study was one of the most important and basic steps of designing a recombinant E. coli that can produce PHB.

Genetic analysis of two STR loci (VWA and TPOX) in the Iranian province of Khuzestan.

OBJECTIVES: Short tandem repeat (STR) loci are the most informative DNA genetic markers for attempting to individualize biological material for application in paternity and forensic cases. MATERIALS AND METHODS: Blood samples were collected and the total genomic DNA was extracted. The DNA samples were used for genotyping VWA and TPOX STR loci using PCR and polyacrylamide gel electrophoresis. RESULTS: This report presents allele frequency data and parameters of biological or legal interest, such as heterozygosity value, polymorphic information content (PIC), genetic diversity index (GD) and population parameter (θ) in Arab and non-Arab population of Khuzestan province (Iran) for the loci VWA and TPOX. Blood samples (N= 392 for VWA and N=308 for TPOX) were collected from individuals unrelated throughout Khuzestan province. The loci were genotyped using the polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis (PAGE) and silver staining. Chi-square test showed that neither STR loci were in agreement with the Hardy-Weinberg equilibrium. CONCLUSION: The examined STR loci in this study have proven a relatively high genetic variation in the Iranian population. The data could be used for construction of a forensic genetic database for the Iranian population.