Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation.

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10-15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1-2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Methylation Status of miR-34a and miR-126 in Non-Small Cell Lung Cancer (NSCLC) Tumor Tissues.

Background: MiR-34a and miR-126 mainly act as tumor suppressors and are often downregulated in various cancers, including non-small cell lung cancer (NSCLC). We aimed to determine the methylation status of miR-34a and miR-126 in NSCLC patients. Methods: The current study included 63 paraffin-embedded NSCLC and paired adjacent normal tissues. After DNA extraction and bisulfite treatment, the methylation status of miR-34a and miR-126 were evaluated using the MSP method. Results: There was no statistically significant difference between tumor and normal tissues regarding the methylation status of miR-34a and miR-126 (p > 0.05). Moreover, we found no significant correlation between the methylation status of miR-34a and miR-126 with patients' demographic parameters, including gender, age, and pathology subtype (p > 0.05). Conclusion: Considering the low expression of mir-126 and mir-34 in NSCLC, more sensitive methods are recommended to be exploited for detecting the level of methylation or underlying mechanisms other than promoter hypermethylation in silencing these genes in NSCLC.

Polyethylene Glycol -Mediated Exosome Isolation: A Method for Exosomal RNA Analysis.

Background: ExoRNAs offer valuable insights into their cellular origin. ExoRNA studies were faced with challenges in obtaining sufficient amounts of high-quality RNA. Herein, we aimed to compare three traditional exosome isolation methods to introduce an appropriate strategy to extract RNA from cancer-derived exosomes for further RNA analysis. Methods: Exosomes were isolated through ultracentrifugation, precipitation kit, and size exclusion column chromatography, and then characterized by DLS and TEM, followed by extracting total RNA. The quality and quantity of the extracted RNAs were assessed by a NanoDrop and 2.5% agarose gel electrophoresis. Results: Extracted exosomes displayed a similar range of size and morphology. We found that PEG-precipitation method resulted in a higher RNA yield with a 260/280 ratio of 1.9. The obtained exoRNA appeared as a smear in the agarose gel, indicative of small exoRNAs. Conclusion: We provide researchers a suitable approach to isolate exosomes based on yield and purity of exoRNA.

Efficient CRISPR/Cas9-Mediated BAX Gene Ablation in CHO Cells To Impair Apoptosis and Enhance Recombinant Protein Production.

Background: Despite recent advances in recombinant biotherapeutics production using CHO cells, their productivity remains lower than industrial needs, mainly due to apoptosis. Objectives: Present study aimed to exploit CRISPR/Cas9 technology to specifically disrupt the BAX gene to attenuate apoptosis in recombinant Chinese hamster's ovary cells producing erythropoietin. Materials and Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 technique. The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein. Results: BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 µM.ml-1 Vs. 2505 µM.ml-1). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 µM oleuropein compared to the control cell line (p-value=0.0002). Conclusions: CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. Therefore, exploiting genome editing tools such as CRISPR/Cas9 has been proposed to develop host cells that result in a safe, feasible, and robust manufacturing operation with a yield that meets the industrial requirements.

Targeting Caspase-3 Gene in rCHO Cell Line by CRISPR/Cas9 Editing Tool and Its Effect on Protein Production in Manipulated Cell Line.

Background: Chinese hamster ovary (CHO) cells are the widely used mammalian cell host for biopharmaceutical manufacturing. During cell cultures, CHO cells lose viability mainly from apoptosis. Inhibiting cell death is useful because prolonging cell lifespans can direct to more productive cell culture systems for biotechnology requests. Objectives: This study exploited a CRISPR/Cas9 technology to generate site-specific gene disruptions in the caspase-3 gene in the apoptosis pathway, which acts as an apoptotic regulator to extend cell viability in the CHO cell line. Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 system. The guide RNAs targeting the caspase-3 gene were designed, and vectors containing sgRNA and Cas9 were transfected into CHO cells that expressed erythropoietin as a heterologous protein. Indel formation was investigated by DNA sequencing. Caspase-3 expression was quantified by real-time PCR and western blot. The effect of editing the caspase-3 gene on the inhibition of apoptosis was also investigated by induction of apoptosis in manipulated cell lines by oleuropein. Finally, the erythropoietin production in the edited cells was compared to the control cells. Results: The caspase-3 manipulation significantly prolongation of the cell viability and decreased the caspase-3 expression level of protein in manipulated CHO cells (more than 6-fold, P-value < 0.0001). Manipulated cells displayed higher threshold tolerance to apoptosis compared to the control cells when they were induced by oleuropein. They show a higher IC50 than the control ones (7271 µM/mL Vs. 5741 µM/mL). They also show a higher proliferation rate than the control cells in the presence of an apoptosis inducer (P-value < 0.0001). Furthermore, manipulated cell lines significantly produce more recombinant protein in the presence of 2,000 µM oleuropein compared to the control ones (P-value = 0.0021). Conclusions: We understood that CRISPR/Cas9 could be effectively applied to suppress the expression of the caspase-3 gene and rescue CHO cells from apoptosis induced by cell stress and metabolites. The CRISPR/Cas9 system-assisted caspase-3 gene ablation can potentially increase erythropoietin yield in CHO cells.

Expression of miR-9 and miR-200c, ZEB1, ZEB2 and E-cadherin in Non-Small Cell Lung Cancers in Iran.

MicroRNAs (miRNAs) exert a critical influence on physiological and pathological processes through posttranscriptional modification of their mRNA targets. They play important roles in tumorigenesis and are considered to be potential diagnostic and prognostic biomarkers with various cancers. MiR-200c and miR-9 are regulatory elements that can have dual impacts as oncogenes and/or tumor suppressor genes. MiR-200c regulates two transcription factors, ZEB1 and ZEB2, while miR-9 is a regulatory factor for the E-cadherin protein which has a critical function in cell-cell junctions and is inhibited by two transcription factors ZEB1 and ZEB2. In this study, expression levels of miR-200c and miR-9, ZEB-1, ZEB-2 and E-cadherin were assessed in 30 non-small cell lung cancers (NSCLCs) by real-time qPCR. MiR-9 was down-regulated significantly in tumor tissues compared to normal adjacent tissues, while there was no significant change in expression level of miR-200c. On the other hand, ZEB1 demonstrated significant increase and ZEB2a decrease at the mRNA level. These results indicate roles for miR-9 and ZEB1 in genesis of lung cancer, although clinico-pathological associations were not evident. Further studies are necessary to assess implications for treatment of lung cancer.

Association Between Neuregulin-1 Gene Variant (rs2439272) and Schizophrenia and Its Negative Symptoms in an Iranian Population.

Objective: Although the etiology of schizophrenia is unknown, it has a significant genetic component. A number of studies have indicated that neuregulin-1 (NRG1) gene may play a role in the pathogenesis of schizophrenia. In this study, we examined whether the rs2439272 of NRG1 is associated with schizophrenia and its negative symptoms in an Iranian population. Method: Rs2439272 was genotyped in 469 participants including 276 unrelated patients with schizophrenia and 193 healthy controls. The association of genetic risk with negative symptoms (by using panss) was examined in the total, male and female samples. COCAPHASE and CLUMP22 programs were used to compare the allele and genotype frequencies, and general linear regression was used to analyze the quantitative dependent variables by the selected variant. Results: In this study, it was revealed that the G allele of rs2439272 might be an allele with the increased risk of developing schizophrenia, especially in the male participants. In addition, significant differences were found between the G allele and GG genotype frequencies, and negative symptoms in the total and male participants. Conclusion: Our results supported the association between rs2439272 in NRG1 gene and risk of schizophrenia and its negative symptoms in an Iranian population. .

A study of the effect of Bacillus thuringiensis serotype H14 (subspecies israelensis) delta endotoxin on Musca larva.

BACKGROUND/AIM: House flies (Musca domestica) are of major public health concern in areas with poor sanitation and hygiene conditions. Biological control through the use of parasitoids and pathogens is one of the alternatives to the use of chemical pesticides for control of insects of public health importance. MATERIALS AND METHODS: The effects of the delta endotoxin of Bacillus thuringiensis on house fly larval mortality were studied. Gel filtration and SDS-PAGE methods were used for separation and purification of proteins. Delta endotoxin was incubated with larvae in concentrations of 0.43 mg/mL and 0.27 mg/mL in bioassay tests. RESULTS: The results of this study indicated protein crystal toxicity against larvae of the house fly. A concentration of 0.43 mg/mL of this toxin caused 100% mortality in house fly larvae. The LD50 amount of these toxins was calculated as 125 µg/g. CONCLUSION: The results of this study suggest that the use of the protein crystal including delta endotoxin of Bacillus thuringiensis serotype H14 is an effective weapon in the biological fight against the house fly.

Drying of a plasmid containing formulation: chitosan as a protecting agent.

BACKGROUND: The purpose of the study. Along with research on development of more efficient gene delivery systems, it is necessary to search on stabilization processes to extend their active life span. Chitosan is a nontoxic, biocompatible and available gene delivery carrier. The aim of this study was to assess the ability of this polymer to preserve transfection efficiency during spray-drying and a modified freeze-drying process in the presence of commonly used excipients. METHOD: Molecular weight of chitosan was reduced by a chemical reaction and achieved low molecular weight chitosan (LMWC) was complexed with pDNA. Obtained nanocomplex suspensions were diluted by solutions of lactose and leucine, and these formulations were spray dried or freeze dried using a modified technique. Size, polydispersity index, zeta potential, intensity of supercoiled DNA band on gel electrophoresis, and transfection efficiency of reconstituted nanocomplexes were compared with freshly prepared ones. RESULTS AND MAJOR CONCLUSION: Size distribution profiles of both freeze dried, and 13 out of 16 spray-dried nanocomplexes remained identical to freshly prepared ones. LMWC protected up to 100% of supercoiled structure of pDNA in both processes, although DNA degradation was higher in spray-drying of the nanocomplexes prepared with low N/P ratios. Both techniques preserved transfection efficiency similarly even in lower N/P ratios, where supercoiled DNA content of spray dried formulations was lower than freeze-dried ones. Leucine did not show a significant effect on properties of the processed nanocomplexes. It can be concluded that LMWC can protect DNA structure and transfection efficiency in both processes even in the presence of leucine.

Drying of a plasmid containing formulation: chitosan as a protecting agent.

BACKGROUND: Along with research on development of more efficient gene delivery systems, it is necessary to search on stabilization processes to extend their active life span. Chitosan is a nontoxic, biocompatible and available gene delivery carrier. The aim of this study was to assess the ability of this polymer to preserve transfection efficiency during spray-drying and a modified freeze-drying process in the presence of commonly used excipients. METHODS: Molecular weight of chitosan was reduced by a chemical reaction and achieved low molecular weight chitosan (LMWC) was complexed with pDNA. Obtained nanocomplex suspensions were diluted by solutions of lactose and leucine, and these formulations were spray dried or freeze dried using a modified technique. Size, polydispersity index, zeta potential, intensity of supercoiled DNA band on gel electrophoresis, and transfection efficiency of reconstituted nanocomplexes were compared with freshly prepared ones. RESULTS AND CONCLUSION: Size distribution profiles of both freeze dried, and 13 out of 16 spray-dried nanocomplexes remained identical to freshly prepared ones. LMWC protected up to 100% of supercoiled structure of pDNA in both processes, although DNA degradation was higher in spray-drying of the nanocomplexes prepared with low N/P ratios. Both techniques preserved transfection efficiency similarly even in lower N/P ratios, where supercoiled DNA content of spray dried formulations was lower than freeze-dried ones. Leucine did not show a significant effect on properties of the processed nanocomplexes. It can be concluded that LMWC can protect DNA structure and transfection efficiency in both processes even in the presence of leucine.