Increase recombinant antibody yields through optimizing vector design and production process in CHO cells.

Chinese hamster ovary (CHO) cells are the most commonly used host cells for the production of recombinant monoclonal antibodies (mAbs) due to their several advantages. Although the yields of recombinant mAbs can be greatly increased by some strategies, such as medium formulation, culture conditions, and cell engineering, most studies focused on either upstream design or downstream processes. In the present study, we first expressed recombinant adalimumab through combination of the vector design and production process optimization in CHO cells. Bicistronic vector, monocistronic vector, and dual promoter vector were constructed, and the production process was optimized using low-temperature and fed-batch culture. The results showed that the dual promoter vector exhibited the highest yield under the transient and stable transfected cells among three different vector systems in CHO cells. In addition, low-temperature and fed-batch culture could further improve the yields of adalimumab. The purified antibody displayed tumor necrosis factor-α (TNF-α) binding activity. In conclusion, combination of expression vector design and production process optimization can achieve higher expression of recombinant mAbs in CHO cells. KEY POINTS: • The dual promoter vector is more effective for expressing recombinant antibodies. • The yields of antibodies are related to the LC chain expression level. • Low-temperature and feed addition can promote antibody production.

Surface values, volumetric measurements and radiomics of structural MRI for the diagnosis and subtyping of attention-deficit/hyperactivity disorder.

Attention-deficit/hyperactivity disorder (ADHD) is diagnosed subjectively based on an individual's behaviour and performance. The clinical community has no objective biomarker to inform the diagnosis and subtyping of ADHD. This study aimed to explore the potential diagnostic biomarkers of ADHD among surface values, volumetric metrics and radiomic features that were extracted from structural MRI images. Public data of New York University and Peking University were downloaded from the ADHD-200 Consortium. MRI T1-weighted images were pre-processed using CAT12. We calculated surface values based on the Desikan-Killiany atlas. The volumetric metrics (mean grey matter volume and mean white matter volume) and radiomic features within each automated anatomical labelling (AAL) brain area were calculated using DPABI and IBEX, respectively. The differences among three groups of participants were tested using ANOVA or Kruskal-Wallis test depending on the normality of the data. We selected discriminative features and classified typically developing controls (TDCs) and ADHD patients as well as two ADHD subtypes using least absolute shrinkage and selection operator and support vector machine algorithms. Our results showed that the radiomics-based model outperformed the others in discriminating ADHD from TDC and classifying ADHD subtypes (area under the curve [AUC]: 0.78 and 0.94 in training test; 0.79 and 0.85 in testing set). Combining grey matter volumes, surface values and clinical factors with radiomic features can improve the performance for classifying ADHD patients and TDCs with training and testing AUCs of 0.82 and 0.83, respectively. This study demonstrates that MRI T1-weighted features, especially radiomic features, are potential diagnostic biomarkers of ADHD.

Identification and Validation of Diagnostic Model Based on Angiogenesis- and Epithelial Mesenchymal Transition-Related Genes in Myocardial Infarction.

Background: Myocardial infarction (MI) is a chronic cardiovascular disease. This study aims to discern potentially angiogenesis- and epithelial mesenchymal transition (EMT)-related genes as biomarkers for MI diagnosis through bioinformatics. Methods: All datasets and angiogenesis- and EMT-related genes were collected from the public database. The differentially expressed genes (DEGs) of MI and MI-related genes were acquired. DEGs, MI-related genes, and angiogenesis- and EMT-related genes were intersected to obtain hub genes. Functional enrichment, immune microenvironment, and transcription factors (TFs)-hub genes regulatory network analysis were performed. The diagnostic markers and models were developed and validated. Drug prediction and molecular docking were performed. Finally, diagnostic markers expressions were validated using RT-qPCR. Results: A total of 224 angiogenesis- and EMT-related genes, 2,897 DEGs, 1,217 MI-related genes, and 9 hub genes were acquired. The immune infiltration levels of plasma cells, T cells CD4 memory activated, monocytes, macrophages M0, mast cells resting, and neutrophils were higher in patients with MI. LRPAP1, COLGALT1, QSOX1, THBD, VCAN, PLOD1, and PLAUR as the diagnostic markers were identified and used to construct diagnostic models, which can distinguish MI from controls well. Then, 9 drugs were screened, and the binding energies ranged from -7.08 to -5.21 kcal/mol. RT-qPCR results showed that the expression of LRPAP1, PLAUR, and PLOD1 was significantly increased in the MI group. Conclusion: The 7 diagnostic markers may play potential roles in MI and could contribute to improved future diagnostics.

Optimization of extended Kozak elements enhances recombinant proteins expression in CHO cells.

In eukaryotes, the localization of small ribosomal subunits to mRNA transcripts requires the translation of Kozak elements at the starting site. The sequence of Kozak elements affects the translation efficiency of protein synthesis. However, whether the upstream nucleotide of Kozak sequence affects the expression of recombinant proteins in Chinese hamster ovary (CHO) cells remains unclear. In order to find the optimal sequence to enhance recombinant proteins expression in CHO cells, -10 to +4 sequences around ATG in 100 CHO genes were compared, and the extended Kozak elements with different translation intensities were constructed. Using the classic Kozak element as control, the effects of optimized extended Kozak elements on the secreted alkaline phosphatase (SEAP) and human serum albumin (HSA) gene were studied. The results showed that the optimized extended Kozak sequence can enhance the stable expression level of recombinant proteins in CHO cells. Furthermore, it was found that the increased expression level of the recombinant protein was not related with higher transcription level. In summary, optimizing extended Kozak elements can enhance the expression of recombinant proteins in CHO cells, which contributes to the construction of an efficient expression system for CHO cells.

Factors Affecting the Expression of Recombinant Protein and Improvement Strategies in Chinese Hamster Ovary Cells.

Recombinant therapeutic proteins (RTPs) are important parts of biopharmaceuticals. Chinese hamster ovary cells (CHO) have become the main cell hosts for the production of most RTPs approved for marketing because of their high-density suspension growth characteristics, and similar human post-translational modification patterns et al. In recent years, many studies have been performed on CHO cell expression systems, and the yields and quality of recombinant protein expression have been greatly improved. However, the expression levels of some proteins are still low or even difficult-to express in CHO cells. It is urgent further to increase the yields and to express successfully the "difficult-to express" protein in CHO cells. The process of recombinant protein expression of is a complex, involving multiple steps such as transcription, translation, folding processing and secretion. In addition, the inherent characteristics of molecular will also affect the production of protein. Here, we reviewed the factors affecting the expression of recombinant protein and improvement strategies in CHO cells.

Identification of potentially relevant genes for myocardial infarction using RNA sequencing data analysis.

Myocardial infarction (MI) is a heart disease with high morbidity and mortality rates, thus it is critical to identify genes that serve roles during its pathogenesis. The objective of the present study was to identify potentially relevant genes during the progression of the disease. Blood samples from patients with MI and normal controls (n=3/group) were obtained, the RNA was extracted and cDNA libraries were established. RNA sequencing (RNA-seq) was performed on a HiSeq 2500 platform and fragments per kilobase of exon per million fragments mapped was utilized to calculate the gene expression value following preprocessing of the RNA-seq data. Electronic validation of several identified differentially expressed genes (DEGs) was performed on a Gene Expression Omnibus (GEO) dataset GSE59867 (390 cases and 46 healthy controls). Functional enrichment and protein-protein interaction network analysis was conducted for DEGs. A total of 977 DEGs, including 817 upregulated and 160 downregulated genes were identified in patients with MI. These DEGs were significantly enriched for 'positive regulation of the immune system process,' 'inflammatory response,' 'regulation of I-kappaB-kinase/NF-kappaB signaling' and 'TNF signaling pathway'. A protein-protein interaction network of the top 40 DEGs was used to identify high degree genes, including interferon induced protein with tetratricopeptide repeats 3 (IFIT3), MX dynamin like GTPase 1 (MX1), major histocompatibility complex, class II, DQ α1 (HLA-DQA1), RAR related orphan receptor A (RORA), prostaglandin D2 synthase (PTGDS), cysteine rich protein 2 (CRIP2), collagen type VI α 2 chain (COL6A2) and S100 calcium binding protein P (S100P). The results of validation in the GEO dataset were consistent with the sequencing analysis. A total of eight genes, including IFIT3, MX1, HLA-DQA1, RORA, PTGDS, CRIP2, COL6A2 and S100P may therefore be considered as potentially relevant genes in the pathology of MI.

Bioinformatic screening for key miRNAs and genes associated with myocardial infarction.

Despite significant advances in understanding of the causes of and treatment of myocardial infarction (MI) in recent years, morbidity and mortality is still high. The aim of this study was to identify miRNA and genes potentially associated with MI. mRNA and miRNA expression datasets were downloaded from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/). Interactions between miRNA and the expression and function of target genes were analyzed, and a protein-protein interaction network was constructed. The diagnostic value of identified miRNA and genes was assessed. Quantitative RT-PCR was applied to validate the results of the bioinformatics analysis. MiR-27a, miR-31*, miR-1291, miR-139-5p, miR-204, miR-375, and target genes including CX3CR1,HSPA6, and TPM3 had potential diagnostic value. The genes TFEB,IRS2,GRB2,FASLG,LIMS1,CX3CR1,HSPA6,TPM3,LAT2,CEBPD,AQP9, and MAPKAPK2 were associated with recovery from MI. In conclusion, the identified miRNA and genes might be associated with the pathology of MI.

The Volume of Hippocampal Subfields in Relation to Decline of Memory Recall Across the Adult Lifespan.

Background: The hippocampus is an important limbic structure closely related to memory function. However, few studies have focused on the association between hippocampal subfields and age-related memory decline. We investigated the volume alterations of hippocampal subfields at different ages and assessed the correlations with Immediate and Delayed recall abilities. Materials and Methods: A total of 275 participants aged 20-89 years were classified into 4 groups: Young, 20-35 years; Middle-early, 36-50 years; Middle-late, 51-65 years; Old, 66-89 years. All data were acquired from the Dallas Lifespan Brain Study (DLBS). The volumes of hippocampal subfields were obtained using Freesurfer software. Analysis of covariance (ANCOVA) was performed to analyze alterations of subfield volumes among the 4 groups, and multiple comparisons between groups were performed using the Bonferroni method. Spearman correlation with false discovery rate correction was used to investigate the relationship between memory recall scores and hippocampal subfield volumes. Results: Apart from no significant difference in the left parasubiculum (P = 0.269) and a slight difference in the right parasubiculum (P = 0.022), the volumes of other hippocampal subfields were significantly different across the adult lifespan (P < 0.001). The hippocampal fissure volume was increased in the Old group, while volumes for other subfields decreased. In addition, Immediate recall scores were associated with volumes of the bilateral molecular layer, granule cell layer of the dentate gyrus (GC-DG), cornus ammonis (CA) 1, CA2/3, CA4, left fimbria and hippocampal amygdala transition area (HATA), and right fissure (P < 0.05). Delayed recall scores were associated with the bilateral molecular layer, GC-DG, CA2/3 and CA4; left tail, presubiculum, CA1, subiculum, fimbria and HATA (P < 0.05). Conclusion: The parasubiculum volume was not significantly different across the adult lifespan, while atrophy in dementia patients in some studies. Based on these findings, we speculate that volume changes in this region might be considered as a biomarker for dementia disorders. Additionally, several hippocampal subfield volumes were significantly associated with memory scores, further highlighting the key role of the hippocampus in age-related memory decline. These regions could be used to assess the risk of memory decline across the adult lifespan.