Iranian Journal of Biotechnology
Volume:22 Issue: 1, Winter 2024

CHO cells are preferred for producing biopharmaceuticals, and genome editing technologies offeropportunities to enhance recombinant protein production. Targeting apoptosis-related genes, such as Caspases 8-Associated Protein 2 (CASP8AP2), improves CHO cell viability and productivity. Integrating robust strategies with the CRISPR-Cas9 system enables its application in CHO cell engineering. This study was performed to develop a cost-effective protocol using the CRISPR-Cas9 system combinedwith the HITI strategy for simultaneous CASP8AP2 gene deletion/insertion in CHO cells and to assess its impact on cellviability and protein expression. We developed an efficient protocol for CHO cell engineering by combining CRISPR/Cas9with the HITI strategy. Two distinct sgRNA sequences were designed to target the 3’ UTR region of the CASP8AP2 geneusing CHOPCHOP software. The gRNAs were cloned into PX459 and PX460-1 vectors and transfected into CHO cellsusing the cost-effective PEI reagent. A manual selection system was employed to streamline the process of single-cellcloning. MTT assays assessed gene silencing and cell viability at 24, 48, and 72 hours. Flow cytometry evaluated proteinexpression in CASP8AP2-silenced CHO cells. The study confirmed the robustness of combining CRISPR-Cas9 with the HITI strategy, achieving a high60% efficiency in generating knockout clones. PEI transfection successfully delivered the constructs to nearly 65%of the clones, with the majority being homozygous. The protocol proved feasible for resource-limited labs, requiringonly an inverted fluorescent microscope. CASP8AP2 knockout (CHO-KO) cells exhibited significantly extended cellviability compared to CHO-K1 cells when treated with NaBu, with IC50 values of 7.28 mM and 14.25 mM at 48 hours,respectively (P-value: 24 hours = < 0.0001, 48 hours = < 0.0001, P-value: 72 hours = 0.0007). CHO CASP8AP2-silencedcells showed a 1.3-fold increase in JRed expression compared to native cells. CRISPR-Cas9 and HITI strategy was used to efficiently engineer CHO cells for simultaneous CASP8AP2gene deletion/insertion, which improved cell viability and protein expression.

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. lycopersici (Fol) (Sacc.) W.C. Snyder andH.N. Hans is one of the most prevalent and devastating diseases of tomato plants (Solanum lycoprsicum L.) that leads toa severe reduction in crop yield almost worldwide. Evaluation of biocontrol potential of Bacillus thuringiensis (Bt) isolate IBRC-M11096, against Fol in tomatothrough priming  qRT-PCR technique was applied to analyze the effect of the strain on the hormonal defensivepathways; transcriptional responses of jasmonic acid (COI1, Pin2) and salicylic acid (NRP1 and PR1) pathway genes inBt-treated plants following inoculation of Fol as compared to the plants only challenged with Fol. Also, the potential ofthe bacterial strain as a biocontrol agent was studied by evaluating growth indices and area under disease progress curve(AUDPC). The transcription of both defensive hormonal pathway genes (COI1, Pin2, NPR1, PR1) increased due to bacterialpriming. The bacterial priming reduced the AUDPC compared to the inoculation with only Fol. The strain reduced thedisease symptoms, and compared to the plants only challenged with the fungus, the bacterial strain significantly raisedshoot dry and fresh weights and root dry weight. Priming with the Bt strain led to improved shoot and root growth indices, reduced AUDPC, and fortifiedresponses of both JA and SA hormonal pathways. However, further full-span studies are required to judge the efficacy ofthe bacterial strain in the biological control of tomato fusarium wilt under field conditions.

Soil salinity is a major problem in the world that affects the growth and yield of plants. Application of newand up-to-date techniques can help plants in dealing with salinity stress. One of the approaches for reducing environmental stress is the use of rhizosphere bacteria. The aim of present study was to investigate the effect of the inoculation of Bacillus cereus on physiologicaland biochemical indicators and the expression of some key genes involved in the Artemisinin biosynthesis pathway inArtemisia absinthium under salinity stress. The results showed that with increasing salinity, root and stem length, root and stem weight, root and stemdry weight, and potassium content were decreased, although the content of sodium was increased. Rhizosphere bacteriaincreased the contents of Artemisinin, potassium, calcium, magnesium, and iron and the expression of Amorpha-4,11-diene synthase and Cytochrome P450 monooxygenase1 genes as well as the growth indicators; although decreased the sodium content. The highest ADS expression was related to co-inoculation with B. cereus isolates E and B in 150 mM salinity. The highest CYP71AV1 expression was related to co-inoculation with B. cereus isolates E and B in 150 mM salinity. These findings showed that the increase in growth indices under salinity stress was probably due to theimprovement of nutrient absorption conditions as a result of ion homeostasis, sodium ion reduction and Artemisininproduction conditions by rhizosphere B. cereus isolates E and B.

Growth-regulating factors (GRFs) are crucial in rice for controlling plant growth and development. Among the rice cultivation practices, aerobic methods are water efficient but result in significant yield reduction relative to non- aerobic cultivation. Therefore, mechanistic insights into aerobic rice cultivation are important for improving the aerobic performance of rice. Objectives This study aimed to examine the evolution of GRFs in different rice species, analyse the phenotypic differences between aerobic and non-aerobic conditions in three rice varieties, and assess the expression of GRFs in these varieties under both aerobic and non-aerobic conditions.

This study comprehensively examined the GRFs gene family in 11 rice species (Oryza barthii, Oryza brachyantha, Oryza glaberrima, Oryza glumipatula, Oryza sativa subsp. indica, Oryza longistaminata, Oryza meridionalis, Oryza nivara, Oryza punctata, Oryza rufipogon, Oryza sativa subsp. japonica) focusing on phylogenetic analysis. Additionally, the expression patterns of 12 GRFs were investigated in three distinct genotypes of O. sativa subsp. indica rice, under both non-aerobic and aerobic conditions.

 Results:

 Three major phylogenetic clades were formed based on conserved motifs in the 123 GRFs proteins in eleven rice species. Further, novel motifs were identified especially in O. longistaminata indicative of the species level evolutionary differences in rice. Among the trait performance, the number of tillers was reduced by ~ 36% under aerobic conditions, but the reduction was found to be less in CR Dhan 201, an aerobic variety. Besides, three GRFs namely GRF3, GRF4, and GRF7 were found to be distinct in expression between aerobic and non-aerobic conditions.

Conclusion:

 Three GRF genes namely GRF3, GRF4, and GRF7 could be associated with the aerobic adaptation in rice

The search for sources of industrial biocatalysts, which are non-pathogenic and can utilise cheap nutrientsources, has been a continuous endeavour in the ~ 7 billion USD enzyme industry. Beauveria bassiana, an endophyticfungal entomopathogen, is non-pathogenic and possesses the potential to secrete various bioproducts while utilisingreadily available lignocellulosic biomass.  This study investigated the optimised production of two glycosyl hydrolases, amylase and polygalacturonase,by B. bassiana while utilising readily available agricultural residues. Subsequently, the industrial potential of the enzymesin the clarification of fruit juice was evaluated.Initially, seven agro residues were screened for the concomitant production of amylase andpolygalacturonase by B. bassiana SAN01. Subsequently, statistical optimisation tools, Plackett Burman Design (PBD)and Central Composite Design (CCD), were employed for the optimisation of enzyme production. The enzyme mixturewas partially purified and applied in the clarification of pineapple juice. The production of B. bassiana SAN01 amylase and polygalacturonase was found to be maximal while utilisingwheat bran. Subsequent to PBD and CCD optimisation, the optimal conditions for enzyme production were identified tobe at 30 °C, pH 6.0 and wheat bran concentration of ~40 g.L-1. Under these optimised conditions, heightened productionlevels of 34.82 and 51.05 U.mL-1 were recorded for amylase and polygalacturonase, respectively, which were 179% and187% of the initial unoptimised levels. In addition, the most effective clarification of the juice (~90%) was observed at 35°C after an incubation time of 120 min with no significant effect on the pH and total dissolved solids. B. bassiana, a well-known biocontrol agent, was shown to produce amylase and polygalacturonase usingreadily available agricultural residues for the first time. These enzyme production levels are the highest for these enzymesfrom any known endophytic fungal entomopathogen. This study further demonstrates the potential applicability of B.bassiana in other industrial processes besides its widespread use as a biopesticide.

Bacterial infection remains the most frequent complication of burn injury, which can lead to sepsis, evenif antibiotics are used topically and systemically. Pseudomonas aeruginosa (P. aeruginosa) is the main causative agent inmany cases. The emergence of antibiotic-resistant strains in recent years has increased the need to find novel alternativetherapies, such as probiotics. Therefore, this study aimed to examine the antimicrobial properties of probiotic cell-freesupernatant (CFS), along with the potential use of a chitosan scaffold both as an antimicrobial agent and as a carrier forthe delivery of these complexes

Evaluation of the antimicrobial properties of cell-free soluble factors of probiotic bacteria both alone and incombination with chitosan scaffolds

Nine isolates of P. aeruginosa previously identified by standard diagnostic tests were investigated.The antimicrobial effects of probiotics in the form of Pedilact® oral drop which contained three probiotic strains,Kidilact® sachet, which contained seven probiotic strains, and strains of Lactobacillus casei (L. casei) and Lactobacillusacidophilus (L. acidophilus) isolated from yogurt were studied by an agar well diffusion assay and by using CFS harvestedat various growth stages, without pH neutralization. Chitosan with different concentrations of glutaraldehyde (GA) as acrosslinking agent was fabricated to produce a suitable scaffold for loading cell-free supernatants of probiotic strains. The scaffolds were then characterized using scanning electron microscopy. The antimicrobial properties of the CFS, chitosan, and chitosan scaffolds loaded with CFS were analyzed against MDR P. aeruginosa.

In the agar well diffusion assay, CFS obtained from probiotic strains effectively inhibited the growth of a clinicalstrain of P. aeruginosa. This effect was observed when CFS was assessed without pH neutralization. Kidilact® was the most promising synbiotic formulation based on its inhibitory activity. The chitosan scaffold was successfully fabricated, as shown by SEM, and its structure was not affected by acidic CFS. The fabricated scaffolds were able to deliver CFS and, interestingly, antibacterial activity against P. aeruginosa when CFS was loaded on the chitosan scaffold was enhanced significantly.

The results of this study showed chitosan scaffold loaded with cell-free probiotics metabolites can beconsidered to be a promising antimicrobial dressing in wound healing applications

Uricase or urate oxidase, as a therapeutic enzyme, is extensively applied to oxidize accumulated uric acidin the body to soluble form to treat related illnesses.

This study was conducted with the aim of searching for potential sources of uricase-producing Streptomycesfrom Eshtehard salt desert in Alborz province, Iran and heterologous expression, purification and functional assay of theenzyme.

Main screening was conducted by cultivation of the strains on a medium enriched with 0.3 percent (w/v) uric acid. The uricase gene from the most potent strain was then recombinantly expressed in E. coli BL21 (DL3)

Out of the tested strains, only seven showed uricase activity. The highest level of native uricase activity (11.5735U.mL-1) belonged to strain 17-1, which had the closest similarity to Streptomyces nigra. A recombinant uricase witha molecular mass of approximately 38 kDa was produced. The purified uricase exhibited a specific activity of about28.29±0.59 U.mg-1, which is among the highest level of uricase activity reported by other studies

This enzyme is a promising candidate for further applicable investigations and large-scale production interms of its large volume of soluble expression and selective competitive activity.

Our knowledge of Type 1 Diabetes Mellitus (T1DM) etiology is incomplete; however, the pathogenesis ofthe disease includes T-cell-mediated destruction of β-cells.  The present study aimed to investigate the key gene pathways and co-expression networks in T1DM disease.  TIDM-associated genes were identified from 13 databases, enrichment of pathways annotated with functional annotations, and analysis of protein-protein network interactions. Next, functional modules and transcription factor networks were constructed. The analysis of gene co-expression networks was conducted to discover associated pivotal modules A total of 172 expressed genes and four variants (SNP) were filtered in the of T1DM disease; pathway enrichment analysis identified key pathways, such as inflammatory bowel disease, type I diabetes mellitus, cytokine-cytokine receptor interaction, Th17 cell differentiation, JAK-STAT signaling pathway, and graft-versus-host disease. A weighted correlation network analysis revealed one module that was strongly correlated with T1DM. Functional annotation revealed that the module was mainly enriched in pathways such as T cell activation, regulation of immune system process, and response to the organic substance. IRF2, IRF4, IRF8, and CDX2 were regulated in the module at a significant level.  The study identified IL-2 as a significant T1DM hotspot and highlighted the role of hub genes and transcription factors in the autoimmune disease, offering potentials for treatment and prevention.

Due to the heterogeneity of breast cancer, most advanced-stage patients are resistant to therapy. Disruption of SUMOylation, a post-translational modification, is linked to breast cancer.
This study aimed to assess the impact of thymoquinone nanoparticles (Liposomal-TQ), an anti-cancerdrug, combined with doxorubicin (DXR), the most effective chemotherapeutic drug used to treat breast cancer, onthe expression of SENP2 and SENP6, two major components involved in the SUMOylation process, in normal andcancerous breast cell lines.
 The MCF7 cell line, a breast cancer cell line, and MCF10, a non-tumor epithelial cell line, wereseparately treated with Liposomal-TQ and DXR. Cell viability and cell migration were assessed using MTT and scratchtests. Apoptosis analysis was performed using annexin-V/PI staining. Gene expression analysis of SENP2 and SENP6 wasconducted using quantitative real-time PCR (RT-qPCR). Additionally, the scratch test evaluated the anti-cell migratoryeffect of Liposomal-TQ. The findings obtained from RT-qPCR analysis indicated a significant increase in the expression of SENP2 andSENP6 genes in the TQ and DXR treatment groups compared to the control group in MCF7 but not in MCF10 cell lines(p-value < 0.05). Also, after 24 hours of treatment of MCF7 and MCF10 cells with liposomal-TQ, late apoptotic cellswere significantly increased compared to the control and liposome groups (p-value < 0.0001) and compared to the control group, both DXR and Liposomal-TQ dramatically reduced the migratory ability of breast cancer cells (p-value = 0.001and p-value = 0.001, respectively)  Our study indicated that Liposomal-TQ promotes apoptosis in breast cancer cells and inhibits cell migrationability. These findings enhance our understanding of the role of Liposomal-TQ in the carcinogenic activities of SENP2and SENP6 in the SUMOylation pathway of breast cancer.

The use of nanomaterial-based radiosensitizers to improve the therapeutic ratio has gained attraction inradiotherapy. Increased radiotoxicity applied to the tumor region may result in adverse impact on the unexposed normalcells to the radiation, a phenomenon known as radiation-induced bystander effect (RIBE). This study aimed to investigate the effect of Bi2S3@BSA nanoparticles (NPs) as radiosensitizers on theenhancement of bystander response in non-irradiated cells. Lung carcinoma epithelial cells were exposed to 6 MV x-ray photons at different doses of 2 and8 Gy, with and without Bi2S3@BSA NPs. The irradiated-cell’s conditioned medium (ICCM) was collected and incubatedwith MCR-5 human fetal lung fibroblasts. This study showed that ICCM collected from 2-Gy-irradiated A549 cells in the presence of Bi2S3@BSA NPsreduced the cell viability of MCR-5 bystander cells more than ICCM collected from irradiated cells without NPs (P<0.05),whereas such a difference was not observed after 8-Gy radiation. The mRNA expression of the BAX and XPA genes, aswell as the cell death rate in MCR-5 bystander cells, revealed that the Bi2S3@BSA NPs significantly improved bystanderresponse at 2-Gy (P<0.05), but the efficacy was not statistically significant after 8-Gy Irradiation. The results indicated that the presence of NPs did not affect bystander response enhancement at higherconcentrations. These findings highlighted the potential use of radiation-enhancing agents and their benefits in radiotherapy techniques with high doses per fraction.