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.

Evaluation of the impact of polypeptide-p on diabetic rats upon its cloning, expression, and secretion in Saccharomyces boulardii.

This study was designed to evaluate the effectiveness of recombinant polypeptide-p derived from Momordica charantia on diabetic rats. In this research, the optimized sequence of polypeptide-p gene fused to a secretion signal tag was cloned into the expression vector and transformed into probiotic Saccharomyces boulardii. The production of recombinant secretion protein was verified by western blotting, HPLC, and mass spectrometry. To assay recombinant yeast bioactivity in the gut, diabetic rats were orally fed wild-type and recombinant S. boulardii, in short SB and rSB, respectively, at two low and high doses as well as glibenclamide as a reference drug. In untreated diabetic and treated diabetic + SB rats (low and high doses), the blood glucose increased from 461, 481, and 455 (mg/dl), respectively, to higher than 600 mg/dl on the 21st day. Whereas glibenclamide and rSB treatments showed a significant reduction in the blood glucose level. The result of this study promised a safe plant-source supplement for diabetes through probiotic orchestration.

Comparison of the Dynamic Postural Control During Lifting and Lowering an External Load in Low Back Pain: A Cross-Sectional Study.

OBJECTIVE: This study aimed to compare dynamic postural control between individuals with and without chronic low back pain (LBP) through load lifting and lowering. METHODS: This cross-sectional study included 52 male patients with chronic LBP (age: 33.37 ± 9.23 years) and 20 healthy male individuals (age: 31.75 ± 7.43 years). The postural control parameters were measured using a force plate system. The participants were instructed to stand barefoot (hip-width apart) on the force plate and lift a box (10% of the weight of the participants) from the waist height to overhead and then lower it from overhead to waist height. The interaction between the groups and tasks was determined using a 2-way repeated-measures analysis of variance. RESULTS: There was no significant interaction between the groups and tasks. Regardless of the groups, postural control parameters including amplitude (P = .001) and velocity (P < .001) in anterior-posterior (AP) direction, phase plane in medial-lateral (ML) direction (P = .001), phase plane in AP-ML direction (P = .001), and the mean total velocity (P < .001) were lesser during the lowering compared with lifting. The results indicated that, regardless of the tasks, the postural control parameters including velocity (P = .004) and phase plane in AP direction (P = .004), velocity in ML direction (P < .001), phase plane (AP-ML) (P = .028), and mean total velocity (P = .001) in LBP were lesser compared with the normal group. CONCLUSION: Different tasks affected postural control differently in patients with LBP and healthy individuals. Moreover, postural control was more challenged during the load-lowering than the load-lifting task. This may have been a result of a stiffening strategy. It may be that the load-lowering task might be considered as a more influential factor for the postural control strategy. These results may provide a novel understanding of selecting the rehabilitation programs for postural control disorders in patients.

Contribution of HLA class II genes, DRB4*01:01, DRB1*07:01, and DQB1*03:03:2 to clinical features of Vitiligo disease in Iranian population.

BACKGROUND: Vitiligo is a multifactorial depigmentation condition, which is due to skin melanocyte destruction. Increased expression of HLA class II genes in patients with pre-lesions of Vitiligo suggests a crucial role for the participation of immune response in Vitiligo development. Recent studies progressively focused on HLA-DRB1 and DQB1 genes. In this study, we have evaluated the association and role of HLA-DRB4*01:01, -DRB1*07:01, and -DQB1*03:03:2 genes in different clinical subtypes of Vitiligo in the Iranian population. METHODS: First, Genomic DNA from peripheral blood of 125 unrelated Vitiligo patients and 100 unrelated healthy controls were extracted through the salting-out method. Then, HLA class II genotyping was performed using the sequence-specific primer PCR method. Finally, the clinical relevance of the testing for these genotypes was evaluated by applying the PcPPV (prevalence-corrected positive predictive value) formula. RESULTS: Our results indicated the positive associations of DRB4*01:01 and DRB1*07:01 allelic genes with early-onset Vitiligo (p = 0.024 and 0.022, respectively). DRB4*01:01 also showed strong protection against late-onset Vitiligo (p = 0.0016, RR = 0.360). Moreover, our data revealed that the DRB1*07:01 increases the susceptibility to Sporadic Vitiligo (p = 0.030, RR = 1.702). Furthermore, our findings proposed that elevated vulnerability of Vitiligo patients due to DRB4*01:01 and DRB1*07:01 alleles maybe is correlated with the presence of amino acid Arginine at position 71 at pocket 4 on the antigen-binding site of the HLA-DRB1 receptor. CONCLUSION: Our findings on different subtypes of Vitiligo suggest that, despite a more apparent autoimmune involvement, a non-autoimmune nature for the etiology of Vitiligo should also be considered.

Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.

CDC25A pathway toward tumorigenesis: Molecular targets of CDC25A in cell-cycle regulation.

The cell division cycle 25 (CDC25) phosphatases regulate key transitions between cell-cycle phases during normal cell division, and in the case of DNA damage, they are key targets of the checkpoint machinery that ensure genetic stability. Little is known about the mechanisms underlying dysregulation and downstream targets of CDC25. To understand these mechanisms, we silenced the CDC25A gene in breast cancer cell line MDA-MB-231 and studied downstream targets of CDC25A gene. MDA-MB-231 breast cancer cells were transfected and silenced by CDC25A small interfering RNA. Total messenger RNA (mRNA) was extracted and analyzed by quantitative real-time polymerase chain reaction. CDC25A phosphatase level was visualized by Western blot analysis and was analyzed by 2D electrophoresis and LC-ESI-MS/MS. After CDC25A silencing, cell proliferation reduced, and the expression of 12 proteins changed. These proteins are involved in cell-cycle regulation, programmed cell death, cell differentiation, regulation of gene expression, mRNA editing, protein folding, and cell signaling pathways. Five of these proteins, including ribosomal protein lateral stalk subunit P0, growth factor receptor bound protein 2, pyruvate kinase muscle 2, eukaryotic translation elongation factor 2, and calpain small subunit 1 increase the activity of cyclin D1. Our results suggest that CDC25A controls the cell proliferation and tumorigenesis by a change in expression of proteins involved in cyclin D1 regulation and G1/S transition.

Association study of M235T and A-6G polymorphisms in angiotensinogen gene with risk of developing preeclampsia in Iranian population.

OBJECTIVE: Preeclampsia (PE) is a life-threatening complication of pregnancy that accounts for 12% of all maternal deaths worldwide. The aim of this study is to investigate the relationships between the polymorphisms of angiotensinogen (AGT) gene and preeclampsia. MATERIAL AND METHODS: In this study, 240 unrelated preeclampsia patients and 178 normotensive women were examined. Genomic DNA was extracted then we assessed M235T(C/T) and A-6G polymorphisms of the AGT gene. Genotyping of M235T and A-6G polymorphisms were performed using SSP-PCR and MS-PCR, respectively. RESULTS: A significant protective association was observed between A-6G G allele, A-6G A/G heterozygote genotype (OR = 0.6, p = 0.007 and OR = 0.6, p = 0.04) against PE. Furthermore, it was shown that two copies of A-6G A allele would increase PE risk (OR: 0.62, p = 0.04). Our results did not show a significant association for M235T polymorphism and PE. However, the combinations of A-6G A/A genotype and M235T T/C genotype (OR = 0.4, p = 0.02) and also A-6G A/G genotype and M235T T/C genotype (OR = 0.5, p = 0.04) in controls represented a significant protective association against PE. CONCLUSION: According to the existence of significant correlation between two candidate polymorphisms, A-6G and M235T polymorphisms, with PE disease in our study, they may be considered as valuable factors in susceptibility to PE disease in Iranian women.

Design and construction of a recombinant lentiviral vector with specific tropism to human epidermal growth factor-overexpressed cancer cells: Developing a new retargeting system for lentivirus vectors.

BACKGROUND: Targeting of specific tissues and cells by viruses is one of the challenges faced by researchers. Lentiviral vectors (LVs) are one of the most promising gene delivery systems in cancer gene therapy. Therefore, we aimed to design a novel lentiviral delivery system that expresses anti- human epidermal growth factor 2 (HER2) designed anykrin repeat protein (DARPin) on the vector envelope to create a pseudotyped lentivirus for targeting HER2-positive cancer cells. METHODS: A helper plasmid producing the viral vector envelope containing anti-HER2 DARPin-G3 was constructed. LV was produced by transfer vector containing green fluorescent protein (GFP) gene and helper plasmids in human embryonic kidney 293 cells. The human breast cancer cell lines SKBR3 (normal and with inhibited endocytosis) (HER2-positive) and MDA-MB-231 (HER2-negative) were transduced by the recombinant viral vector. The GFP-based transduction rate was determined by flow cytometry and fluorescence microscopy. RESULTS: The anti-HER2 DARPin concentration in DARPin-LVs was significantly higher than the envelope G glycoprotein of the vesicular stomatitis virus-LVs (non-anti-HER2 control) (p < 0.0001). In flow cytometry assays, the percentage of transduction by recombinant LV was significantly higher in SKBR3 cells than in SKBR3 cells with inhibited endocytosis (p = 0.0074) and MDA-MB-231 cells (p = 0.0037). In fluorescence microscopy assays, the percentage of transduction by new LV was significantly higher in SKBR3 cells than in SKBR3 cells with inhibited endocytosis (p = 0.0026) and MDA-MB-231 cells (p = 0.0014). CONCLUSIONS: We constructed a new recombinant LV with a defect in cell entry directly, containing an anti-HER2 DARPin on the vector envelope with specific tropism to HER2 receptor on HER2-positive cancer cells. We assumed that this viral vector transduces cells via an endocytosis-dependent process.

Interleukin-1 gene cluster Haplotype analysis in the chronic outcome prediction of the Hepatitis B virus infection.

Hepatitis B virus (HBV) infection is well known as an important cause of the chronic liver disease. The screening of the genotype of certain cytokines might be helpful to predict the clinical outcome of an HBV infection. The present study investigates the relationship between the polymorphism and haplotypes of the Interleukin-1 (IL-1) gene family, including IL-1-alpha (IL-1A), IL-1-beta (IL-1B,) and IL-1 receptor antagonist (IL-1RN), with chronic HBV infection. A total of 297 chronic HBV and 333 matched on sex and age control individuals were genotyped using the standard sequence-specific-polymerase chain reaction primer (SSP-PCR) method. Four different haplotype analysis software packages were applied for data interpretation. The results showed excess genotype A1/A1 and A2/A2 at IL-1RN (40.2%, 39.9%), C/T at IL-1A-889 (55.6%), and C/C at IL-1B-511 (41.1%) in controls while A1/A1 at IL-1RN (59.3%), T/T at IL-1B-31 (46.5%), C/T at IL-1B + 3953 (65%), in chronic HBV infection cases. A total of 148 haplotypes were observed overall (96 in the case group and 89 in the control group). The haplotype combination of genotype A1/A1 at IL1-RN along with a C/T for all three IL-1B polymorphic positions and either C/T or T/T at the IL-1A-899 position may increase the probability of the chronic outcome for the HBV infection.

Determination of pegfilgrastim aggregates by size-exclusion high-performance liquid chromatography on a methacrylate-based column.

A size-exclusion high-performance liquid chromatographic method using a methacrylate-based column was developed, validated and implemented for the determination of pegfilgrastim aggregates. The samples were directly injected into a TSKgel G4000PWXL column (7.5 mm × 300 mm, 10 µm, <500 A°) with a mobile phase of 100 mM phosphate, pH 2.5. Detection was made at 215 nm and analyses were run at a flow-rate of 0.6 ml/min at 10 °C. Vortex-mixing of samples produced oligomers, however, very high molecular weight aggregates were formed at high temperatures. The method exhibited linearity over the concentration range of 0.1-14 mg/ml for pegfilgrastim monomer and high molecular weight aggregates with a correlation coefficient of greater than 0.99. The method was specific and sensitive, with a lower quantification limit of 0.1 mg/ml and a detection limit of 0.02 mg/ml. Over 1200 samples were analyzed by the present method without significant change in the column performance.

Combination of interleukin-10 gene promoter polymorphisms with HLA-DRB1*15 allele is associated with multiple sclerosis.

BACKGROUND & OBJECTIVES: Multiple sclerosis (MS) is common in some ethnic groups. Interleukin-10 (IL-10) is a potent anti-inflammatory and immunosuppressive cytokine that may be an important regulator in MS disease pathogenesis. IL-10 promoter includes several single nucleotide polymorphisms and the level of IL-10 expression is related to these polymorphisms. Furthermore, loci within the histocompatibility regions are responsible for susceptibility to MS. The aim of this study was to investigate the association of IL-10 gene promoter polymorphisms and HLA-DRB1*15 allele frequencies with MS susceptibility in an Iranian population. METHODS: In this study 336 MS patients and 454 healthy controls were included. Genomic DNA was purified from peripheral blood samples by a standard protocol. Genotyping was performed by the sequence-specific primer polymerase chain reaction method. RESULTS: IL-10 -1082 G/G and IL-10 -819 C/C genotypes were more frequent in MS patients than healthy individuals. DRB1*15 allele showed a higher frequency among MS patients compared to controls. INTERPRETATION & CONCLUSIONS: The IL-10 and HLA-DRB1*15 polymorphisms were associated with the susceptibility to MS in Iranian patients. Our results suggest that gene-gene interaction of IL-10 polymorphisms and HLA-DRB1*15 alleles may be important factors in the development of MS.

The Effect of an Overhead External Load Lifting and Lowering on Dynamic Postural Control in Subgroups of Low Back Pain.

BACKGROUND: Understanding postural control in low back pain (LBP) subgroups can help develop targeted interventions to improve postural control. The studies on this topic are limited. Therefore, the primary purpose of this study was to compare the postural control of LBP subgroups with healthy individuals during overhead load lifting and lowering. METHODS: In this cross-sectional study, the participants were 52 with LBP and 20 healthy. The LBP patients were classified based on the O'Sullivan classification system into 21 flexion patterns and 31 active extension patterns. The participants lifted the box from their waists to their overheads and lowered it to their waists. Changes in postural control parameters were measured with a force plate system. RESULTS: The results of the analysis of variance showed that during load lifting, the mediolateral phase plane (p = .044) and the mean total velocity (p = .029) had significant differences between flexion patterns and healthy. Also, the load-lowering results showed that active extension patterns, compared with healthy, had significant differences in the anteroposterior-mediolateral phase plane (p = .042). The patients showed less postural sway than the healthy. CONCLUSIONS: The results in this work highlight the importance of identifying the homogenous subgroups in LBP and support the classification of heterogeneous LBP. Different subgroups exhibit different postural control behaviors. These behaviors can be due to the loading of various tissues during different tasks.

Harnessing the TP53INP1/TP53I3 axis for inhibition of colorectal cancer cell proliferation through MEG3 and Linc-ROR Co-expression.

Dysregulation of long noncoding RNAs (lncRNAs), such as maternally expressed gene 3 (MEG3) and long intergenic noncoding RNA regulator of reprogramming (linc-ROR), plays a crucial role in colorectal cancer progression. We aimed to assess linc-ROR silencing and MEG3 activation on the colorectal cancer cell proliferation simultaneously; and explore the underlying mechanisms in the TP53-associated Pathway. The MEG3 and linc-ROR shRNA were cloned under the bidirectional CEA promoter (UM1). Subsequently, additional vectors were constructed to express linc-ROR shRNA (UM2) and MEG3 (UM3). After transfecting colorectal cancer cell lines with these recombinant vectors, experiments on cell viability, apoptosis, and cell cycle analysis were conducted. Furthermore, TP53's transcriptional activity and associated genes were assessed using quantitative real-time polymerase chain reaction (qRT-PCR). Interestingly, UM1 significantly inhibited the proliferation of both cell lines than UM2 and UM3. In response to UM1, TP53 transcript remarkably increased in HCT116 cells (10.46) than SW480 cells (6.16); which resulted in up-regulation of TP53INP1, TP53I3, GDF15, CCKN1A and BAX, and down-regulation of G1 cyclins (D1, E1). The rate of apoptosis increased in HCT116 (36.35 %) and SW480 (16.64 %) cells than control. Moreover, UM1-transfected HCT116 cells exhibited a notable arrest in the G0/G1 phase, accompanied by a reduction in the G2/M cell population. Compared to unidirectional vectors, the concurrent targeting approach enhanced TP53 activation at the transcription level. The cell response to UM1 resulted in rapid upregulation of TP53, leading to inhibition of cell proliferation, increased apoptosis, and cell cycle arrest. These findings suggest that the synergistic effect of targeting both MEG3 and linc-ROR could serve as a promising therapeutic strategy for TP53-associated colon cancer.

Association of rs5051 and rs699 polymorphisms in angiotensinogen with coronary artery disease in Iranian population: A case-control study.

Coronary artery disease (CAD) is the third most common cause of mortality globally (with 17.8 million deaths annually). Angiotensinogen (AGT) and polymorphisms in this gene can be considered as susceptibility factors for CAD. We performed a retrospective case-control study to determine the correlation of AGT rs5051 and rs699 polymorphisms with CAD in an Iranian population. We genotyped 310 CAD patients and 310 healthy subjects using polymerase chain reaction-based methods. To confirm the accuracy of the screening approach, 10% of genotyped subjects were validated using gold-standard Sanger Sequencing. To evaluate the effect of the candidate polymorphisms, white blood cells were randomly purified from the subjects and AGT expression was measured by quantitative reverse transcriptase-polymerase chain reaction. Sex stratification indicated a significant correlation between CAD and male sex (P = .0101). We found a significant association between the rs5051 A allele (P = .002) and the rs699 C allele, and CAD (P = .0122) in recessive and dominant models. Moreover, our findings showed a significant association of the haplotype, including the rs5051 A/A and rs699 T/C genotypes, with CAD (P = .0405). Finally, AGT mRNA levels were significantly decreased in patients harboring the candidate polymorphisms (P = .03). According to our findings The AGT rs5051 A and AGT rs699 C alleles are predisposing variants of CAD risk and severity in the Iranian population.

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.

Evaluation of the safety and efficacy of biosimilar recombinant growth hormone in children with growth hormone deficiency: non-inferiority, randomized, parallel, multicentric and Phase III trial.

OBJECTIVES: This study is designed in order to compare the efficacy and safety of recombinant human growth hormone (rhGH) with the reference brand. METHODS: According to the inclusion criteria, 85 people in 13 Iranian centers were randomly selected to receive biosimilar Somatropin (Somatin®) (44 people) and reference Somatropin (Norditropin®) (41 people) at a dose of 35 µg/kg/d, seven days/week for 12 months. The primary outcomes included height velocity (HV) was measured during 12 months of treatment. RESULTS: The two intervention groups' Height changes were similar. The mean HV was 10.96 cm/year in the biosimilar group and 10.05 cm/year in the reference groups after 12 months. Estimates of the lower bounds of 95% CI for mean height differences in the biosimilar intervention group compared to the reference intervention group did not exceed the 2 cm margin. Therefore, the non-inferiority of biosimilar intervention compared to the brand product is verified. Common ADRs in both groups were nausea in two patients (2.4%), diarrhea in two patients (2.4%), increased body temperature in one patient (1.2%), and headache in one patient (1.2%). CONCLUSIONS: The finding of this study indicated that Somatin® and Norditropin® have comparable efficacy and safety profiles. CLINICAL TRIAL REGISTRATION: www.IRCT.irIRCT20171122037571N1.

Lenalidomide improves NKG2D-based CAR-T cell activity against colorectal cancer cells invitro.

Introduction: Although CAR-based immunotherapy is viewed as a promising treatment for tumors, particularly hematological malignancies, solid tumors can pose challenges. It has been suggested that the immunomodulatory medication Lenalidomide (LEN) may increase the effectiveness of CAR T cells in the treatment of solid tumors. The purpose of our study was to investigate the effect of NKG2D-based CAR T cell therapy on colorectal cancer cell lines, and then we assessed combinatorial therapy using NKG2D CAR T cells and lenalidomide in vitro. Methods and results: To prepare NKG2D CAR T cells, a second-generation NKG2D-CAR construct was designed and transfected into the T cells using a lentiviral system. The NKG2D CAR T cells showed significantly higher cytotoxic activity against colorectal cancer cell lines, HCT116 and SW480, compared to untransduced T cells. In addition, our data demonstrated that the cytotoxicity and cytokine secretion of NKG2D CAR T cells significantly increased in the presence of higher doses of lenalidomide. Conclusions: The study findings suggest that combinational therapy, utilizing NKG2D-based CAR T cells and lenalidomide, has a high potential for effectively eliminating tumor cells in vitro.

Transcriptome Profiling of Cisplatin Resistance in Triple-negative Breast Cancer: New Insight into the Role of PI3k/Akt Pathway.

BACKGROUND: Aggressive nature of triple negative breast cancer (TNBC) is associated with poor prognosis compared with other breast cancer types. Current guidelines recommend the use of Cisplatin for the management of TNBC. However, the development of resistance to cisplatin is the primary cause of chemotherapy failure. OBJECTIVE: In the present study, we aimed to develop a stable cisplatin-resistant TNBC cell line to investigate the key pathways and genes involved in cisplatin-resistant TNBC. METHODS: The MDA-MB-231 cell was exposed to different concentrations of cisplatin. After 33 generations, cells showed a resistant phenotype. Then, RNA-sequencing analysis was performed in cisplatin-resistant and parent cell lines. The RNA-sequencing data was verified by quantitative PCR (qPCR). RESULTS: The IC50 of the resistant cell increased to 10-fold of a parental cell (p<0.001). Also, cisplatin-resistant cells show cross-resistance to other drugs, including 5- fluorouracil, paclitaxel, and doxorubicin. Resistant cells demonstrated reduced drug accumulation compared to the parental cells. Results showed there were 116 differentially expression genes (DEGs) (p<0.01). Gene ontology analysis revealed that the DEGs have several molecular functions, including binding and transporter activity. Functional annotation showed that the DEGs were enriched in the drug resistancerelated pathways, especially the PI3K-Akt signaling pathway. The most important genes identified in the protein-protein interaction network were heme oxygenase 1 (HMOX1) and TIMP metallopeptidase inhibitor 3 (TIMP3). CONCLUSION: We have identified several pathways and DEGs associated with the PI3KAkt pathway, which provides new insights into the mechanism of cisplatin resistance, and potential drug targets in TNBC.

Analytical chromatography approaches during the synthesis and conjugation of methoxypolyethylene glycol-succinimidyl butanoate (mPEG-SBA) to epoetin beta.

Conjugation of epoetin beta (EPO) with methoxypolyethylene glycol-succinimidyl butanoate (mPEG-SBA) was studied. The compound mPEG-SBA was synthesized from mPEG, and the obtained intermediates and final product were analyzed by a reversed-phase chromatographic system equipped with an evaporative light scattering detector. Labeling the hydroxyl group in PEGs with benzoyl chloride and succinimide with benzylamine was applied to resolve and characterize different PEGs. The synthesized mPEG-SBA was used for the PEGylation of EPO. A size-exclusion chromatographic method monitored the reaction, simultaneously determining the PEGylated and unreacted EPO and protein aggregates. A borate buffer (0.1 M, pH 7.8) and PEG/protein molar ratio of 3:1 produced a maximum amount of monoPEGylated EPO with the minimum amount of polyPEGylated EPO variants. Although EPO is considered a stable glycoprotein hormone that remains monomeric when refrigerated, PEGylation of EPO with mPEG-SBA resulted in the significant formation of EPO dimer. The formation of EPO dimer and polyPEGylated EPO was pH-dependent, showing higher amounts of aggregates and lower amounts of polyPEGylated forms in lower pH values. Accordingly, aggregated EPO should be considered a major PEGylation-related impurity. In conclusion, the present study highlighted the importance of having suitable analytical approaches in controlling mPEG-SBA synthesis and conjugation to EPO.

Enhancing productivity of Chinese hamster ovary (CHO) cells: synergistic strategies combining low-temperature culture and mTORC1 signaling engineering.

Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature. Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry. Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, "pB513-b CMV-P/4shRNAs/GFP" plasmid, "pB513-bHSP90-P/4sh-RNAs/GFP" and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days. Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.