Biotechnology Journal最新文献

{"title":"Establishment and Application of Mice Models for Tracing Gene Expression and Protein Product of TNF","authors":"Fangyang Shao,&nbsp;Yi Zhou,&nbsp;Jiahao Shi,&nbsp;Mengjie Zhang,&nbsp;Hua Yang,&nbsp;Jian Fei","doi":"10.1002/biot.70080","DOIUrl":"10.1002/biot.70080","url":null,"abstract":"<div>\u0000 \u0000 <p>Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine crucial for immune function, cellular homeostasis, and disease progression, yet its complex roles in vivo remain unclear. Challenges in studying TNF-α include its widespread gene expression, variability in expression levels, diverse protein forms, and low baseline expression, which complicate traditional detection and tracking methods. To address these challenges, we constructed three distinct transgenic luciferase reporter mouse models (TNF-IRES-Luc, TNF-Nanoluc, and TNF-HiBiT) driven by the endogenous TNF-α gene, using CRISPR/Cas9 technology through homology-directed repair. The firefly luciferase gene, secreted NanoLuc gene, and HiBiT gene were individually integrated into the mouse genome under the control of the endogenous TNF-α promoter. Our results demonstrate that endogenous TNF-α expression can be effectively monitored by measuring luciferase levels in vivo and in vitro, using an in vivo imaging system and a luminometer. This was validated during inflammatory processes such as lipopolysaccharide (LPS)-induced sepsis and phorbol ester (TPA)-induced mouse ear edema. Furthermore, the anti-inflammatory drug dexamethasone (DXM) significantly inhibited TNF-α and luciferase expression in both inflammatory models. Our study demonstrates these mouse models are valuable tools for studying TNF-α expression in inflammatory responses and related diseases, as well as evaluating anti-inflammatory drug efficacy.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144635478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhanced Regioselectivity and Catalytic Activity on C7Hydroxylation of Lithocholic Acid to Produce Ursodeoxycholic Acid by Monooxygenase CYP107D1 Based on Semi-Rational Design","authors":"Ci Song,&nbsp;Xiaomin Zheng,&nbsp;Zhenru Zhou,&nbsp;Yundong Yu,&nbsp;Lei Zhang,&nbsp;Shan Li","doi":"10.1002/biot.70070","DOIUrl":"10.1002/biot.70070","url":null,"abstract":"<div>\u0000 \u0000 <p>Ursodeoxycholic acid (UDCA), a natural bile acid, is widely utilized in treating hepatobiliary disorders due to its ability to reduce bile concentration and protect liver function. While the CYP107D1 (OleP) triple-mutant F84Q/S240A/V291G is the first identified bacterial monooxygenase capable of directly converting lithocholic acid (LCA) to UDCA via C7β-hydroxylation, its industrial application has been hindered by low catalytic efficiency. The study implemented a semi-rational design strategy combined with a cell-free enzyme catalysis method, utilizing the OleP triple mutant as a template, to screen for mutants with enhanced 7β-hydroxylation activity. The superior quadruple-mutant G294A/N236H/F321W/V297A was engineered through iterative combinatorial mutagenesis, which catalyzed the production of about 0.68 mM UDCA from 1 mM substrate LCA. This variant exhibited a 50% increase in catalytic activity and a 40% improvement in regioselectivity compared to the template OleP triple-mutant. Molecular docking and kinetic simulations further demonstrated that the quadruple mutant stabilized the enzyme-substrate complex through optimized binding interactions, thereby enhancing catalytic proficiency. Our findings elucidate critical structural determinants governing C7-hydroxylation of LCA.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144614968","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advances in Fungal Promoter Engineering for Enhancing Secondary Metabolite Biosynthesis","authors":"Nan Pu,&nbsp;Huawei Zhang","doi":"10.1002/biot.70075","DOIUrl":"10.1002/biot.70075","url":null,"abstract":"<div>\u0000 \u0000 <p>In the past two decades, promoter engineering has been extensively employed as a powerful strategy for fine-tuning the regulation of gene transcription to awaken or increase the expression of secondary metabolite (SM) biosynthetic gene clusters (BGCs) in fungal genomes. This comprehensive review provides an overview of recent advances in various fungal promoter engineering techniques, including promoter replacement, optimization, and combinatorial methods, and their application in the enhancement of SM production. Additionally, current challenges and future prospects for efficient promoter engineering for precise metabolic regulation in fungi are discussed.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144614967","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigating Hepatic Glucose Metabolism in Insulin Resistance Cell Model Using 3D-Printed Open Microfluidic Chip","authors":"Meng-Meng Liu,&nbsp;Xin-Rong Li,&nbsp;Ji-Cheng Li,&nbsp;Yu Zhong,&nbsp;Yun Lei,&nbsp;Ai-Lin Liu","doi":"10.1002/biot.70076","DOIUrl":"10.1002/biot.70076","url":null,"abstract":"<div>\u0000 \u0000 <p>The liver is recognized as a pivotal tissue contributing to insulin resistance and exerts a crucial influence on the systemic orchestration of glucose metabolism. While biologists have embraced microfluidics for its potential to elucidate hepatic glucose homeostasis and metabolic dynamics, huge challenges remain in the widespread adoption of microfluidic devices, such as complex design and production, and the use of additional pumps for fluid handling. In this study, we report the fabrication of an α-cellulose-modified open microfluidic chip using a 3D printer for the investigation of glucose metabolism in HepG2 cells after insulin and pioglitazone intervention. The Christmas tree-inspired design of the chip integrates a concentration gradient generation zone alongside a 3D cell culture environment. The chip's utility was assessed through the measurement of cellular glucose uptake and ROS expression levels, revealing that the 3D cell model exhibited markedly reduced sensitivity to insulin and pioglitazone compared to the 2D model. Additionally, expression of insulin resistance-related proteins was examined to confirm the improvement of insulin resistance. Collectively, these findings underscore the chip's capacity for stable fluid management and utility in advancing glucose metabolism research. Moreover, this approach provides the new strategy for the development of disease cell models to explore the physiological and pathophysiological intricacies underpinning metabolic disorders.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144582214","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Five-Gene Expression Formula Accurately Detects Hepatocellular Carcinoma Tumors","authors":"Aram Ansary Ogholbake,&nbsp;Qiang Cheng","doi":"10.1002/biot.70073","DOIUrl":"10.1002/biot.70073","url":null,"abstract":"<div>\u0000 \u0000 <p>Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Several diagnostic methods, such as imaging modalities and Serum Alpha-Fetoprotein (AFP) testing, have been used for HCC detection; however, their effectiveness is limited to later stages of the disease. In contrast, transcriptomic analysis of biopsy samples has shown promise for early detection. Although machine learning techniques have been applied to transcriptomic data for cancer detection, their clinical adoption remains limited due to challenges such as poor generalizability across different datasets, lack of interpretability, and high computational complexity. To address these limitations, we developed a novel predictive formula for HCC detection using the Kolmogorov–Arnold Network (KAN). This formula is based on the expression levels of five genes: VIPR1, CYP1A2, FCN3, ECM1, and LIFR. Derived from the GSE25097 dataset, the formula offers a simple, interpretable, efficient, and accessible approach for HCC identification. It achieves 99% accuracy on the GSE25097 test set and demonstrates robust performance on six additional independent datasets, achieving accuracies of above 90% in all cases. These findings highlight the critical role of these five genes as biomarkers for HCC detection, offering a foundation for future research and clinical applications to improve HCC diagnostic approaches.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144582213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of Sensor Based on Circularly Permuted Fluorescent Protein in Molecular Biology, Metabolism, and Cellular Imaging","authors":"Zijian Wang,&nbsp;Guangqing Du,&nbsp;Huan Fang,&nbsp;Bingnan Liu,&nbsp;Dawei Zhang","doi":"10.1002/biot.70071","DOIUrl":"10.1002/biot.70071","url":null,"abstract":"<div>\u0000 \u0000 <p>Circular permutation is a natural phenomenon involving amino acid sequence rearrangement during protein evolution. This approach relies on the native protein sequence and reorganizes the connectivity of secondary structural elements by rearranging the amino acid sequence, without altering the overall protein structure. Circularly permuted fluorescent proteins (cpFPs) have received widespread attention in recent years for their applications in the field of sensors. Rearranging the structure of fluorescent proteins in a circular permutation, the relative positions of their termini can be effectively altered, thereby adjusting their sensitivity to environmental signals such as pH, ion concentration, voltage, or molecular interactions. This property has been widely used to monitor intracellular processes dynamically, particularly in studying neural activity, metabolic reactions, and signaling pathways. This review summarizes the formation mechanism, analysis, and optimization of structure and dynamics, as well as their functional applications in biomedicine and synthetic biology. Furthermore, the future development of sensors based on cpFP is envisioned to provide insights into the design of novel sensors.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144582212","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid Differential Diagnosis of Influenza A and B Viruses With RT-RPA Centrifugal Microfluidic Chip","authors":"Xiaorui Feng,&nbsp;Shuyuan Han,&nbsp;Yong Bian,&nbsp;Chengkai Wang,&nbsp;Ran Chen,&nbsp;Fan Tang,&nbsp;Leilei Zhang,&nbsp;Su Yang,&nbsp;Han Jiang,&nbsp;Ran Tao,&nbsp;Chenze Lu","doi":"10.1002/biot.70072","DOIUrl":"10.1002/biot.70072","url":null,"abstract":"<div>\u0000 \u0000 <p>Influenza A H1N1 and Influenza B are two of the most prevalent airborne viruses, capable of triggering seasonal epidemics that affect millions of individuals globally. Rapid on-site identification of these two viruses is crucial to accurate clinical diagnosis and prompt control of public health risks. This study presents a novel one-step reverse transcription recombinase polymerase amplification (RT-RPA) method integrated with a centrifugal microfluidic chip for rapid differential diagnosis of Influenza A H1N1 and Influenza B viruses. Addressing limitations of conventional techniques, the platform merges reverse transcription and amplification into a single step, reducing aerosol contamination risk and enabling on-site detection. Specific primers and probes targeting H1N1-HA, H1N1-NA, IVB-HA, and IVB-NA genes were designed, achieving detection limits of 10² copies/mL for H1N1-HA and 10¹ copies/mL for other targets within 25 min. The system demonstrated 100% specificity against common respiratory viruses and no cross-reactivity. Validation with 26 aerosol samples collected from public areas using bioaerosol sampler identified two H1N1-positive cases, showing 96.15%–100% consistency with off-chip qRT-RPA. The chip required only 6.25 µL of sample, exhibited high reproducibility (CV &lt;5%). This microfluidic-RT-RPA system offers a practical solution for point-of-care influenza subtyping, enhancing outbreak control and clinical decision-making in resource-limited settings.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144573413","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effect of Apoptosis and Autophagy on Recombinant Protein Expression in Chinese Hamster Ovary Cells","authors":"Hui-Jie Zhang,&nbsp;Qi Zhao,&nbsp;Jumai Abiti,&nbsp;Yan-Ping Gao,&nbsp;Ming-Ming Han,&nbsp;Hai-Tong Wang,&nbsp;Xi Zhang,&nbsp;Jia-Ning Wang,&nbsp;Jia-Liang Guo,&nbsp;Xiao-Yin Wang,&nbsp;Zi-Chun Hua,&nbsp;Tian-Yun Wang,&nbsp;Yan-Long Jia","doi":"10.1002/biot.70069","DOIUrl":"10.1002/biot.70069","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <p>Chinese hamster ovary (CHO) cells serve as a cornerstone platform for producing diverse therapeutic recombinant proteins, including monoclonal antibodies, vaccines, and hormones. Apoptosis and autophagy emerge as critical biological factors that directly impair cell proliferation and limit recombinant protein production. These cellular processes diminish CHO cell viability and reduce culture density, ultimately compromising protein yield and product quality. While apoptosis and autophagy exhibit distinct molecular mechanisms, they demonstrate functional interdependence in cellular regulation. This interrelationship highlights the importance of coordinated pathway modulation as an effective approach to improve both cell growth performance and recombinant protein synthesis. This review examines the complex interplay between apoptosis and autophagy pathways, their collective impact on recombinant protein expression, and contemporary strategies for developing stable anti-apoptotic/anti-autophagy cell lines. Through systematic analysis of these critical elements, we present optimized engineering approaches to enhance CHO cell culture systems and biopharmaceutical manufacturing processes, with the goal of facilitating more efficient therapeutic protein production.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> SUMMARY</h3>\u0000 \u0000 <div>\u0000 <ul>\u0000 \u0000 <li>\u0000 <p>Crosstalk mechanisms between apoptosis and autophagy in CHO cell cultures.</p>\u0000 </li>\u0000 \u0000 <li>\u0000 <p>Recent advances in real-time monitoring of apoptosis and autophagy.</p>\u0000 </li>\u0000 \u0000 <li>\u0000 <p>Strategic mitigation of apoptosis/autophagy to improve recombinant protein yields.</p>\u0000 </li>\u0000 </ul>\u0000 </div>\u0000 </section>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144573412","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transcriptomic Insights Into Serum-Free Medium Adaptation and Temperature Reduction in Chinese Hamster Ovary Cell Cultures","authors":"Benjamin F. Synoground,&nbsp;Yogender Gowtham,&nbsp;Timothy Lindquist,&nbsp;Junessa Pressley,&nbsp;Derrick C. Scott,&nbsp;Christopher S. Saski,&nbsp;Sarah W. Harcum","doi":"10.1002/biot.70055","DOIUrl":"10.1002/biot.70055","url":null,"abstract":"<p>Chinese hamster ovary (CHO) cells are widely used in recombinant biopharmaceutical production; yet, yields remain low, leading to high market prices. Improving product yield and quality has heavily relied on empirical characterization with limited insight into internal molecular dynamics. RNA-seq offers a powerful alternative to understand intracellular responses to process changes through gene expression measurement. In this study, three RNA-seq datasets across three CHO cell lines and four industrially relevant treatments were integrated to characterize the global transcriptome changes, construct a weighted gene co-expression network, assess the impact on recombinant anti-interleukin 8 (anti-IL8) immunoglobulin heavy and light chain transcript abundance, and expression of glycosylation genes. Treatments included adaptation to serum-free medium, low temperature, low pH, and low glucose concentration in the medium. The findings suggest upregulation of cholesterol biosynthesis is critical for serum-free medium adaptation, and the rate-limiting enzymes in the sterol regulatory element-binding protein pathway (<i>Insig1</i> and <i>Srebf2</i>) could be targeted to accelerate adaptation. Temperature-induced cell cycle suppression was likely mediated by p53 activation, consistent with previous reports, with the p53-targets, <i>Zmat3</i> and <i>Btg2</i>, identified as key hub genes. Conversely, glucose and pH were observed to have negligible impacts on the transcriptome. This study uniquely identifies novel genes mediating temperature-induced cell cycle arrest, distinct glycosylation-related gene responses impacting product quality, and new stable housekeeping genes for accurate gene expression normalization in CHO cells.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70055","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144551164","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Neuroprotective Potential of Human Platelet Lysate in Parkinson's Disease: Insights Into Oxidative Stress, Mitochondrial Dysfunction, Cell Death, and Reactive Gliosis in Experimental Models","authors":"Samir Kumar Beura,&nbsp;Abhishek Kumar Maurya,&nbsp;Sneha Kumari,&nbsp;Divya Soni,&nbsp;Prajjwal Sharma,&nbsp;Nisha Yadav,&nbsp;Abhishek Ramachandra Panigrahi,&nbsp;Pooja Yadav,&nbsp;Puneet Kumar,&nbsp;Dibbanti Harikrishna Reddy,&nbsp;Debapriya Garabadu,&nbsp;Sunil Kumar Singh","doi":"10.1002/biot.70064","DOIUrl":"10.1002/biot.70064","url":null,"abstract":"<div>\u0000 \u0000 <p>Parkinson's disease (PD) entails complex pathology, with current treatments managing symptoms but failing to halt neurodegeneration. Preclinical evidence suggests human platelet lysate (HPL) from healthy donors as a neuroprotective candidate due to its rich neurotrophic content, though its potential in PD models remains largely underexplored. In this study, we present substantial experimental findings demonstrating the neuroprotective effects of HPL administration in both rotenone (ROT)-induced in vitro as well as in vivo models of PD. Our findings reveal that freshly prepared HPL from healthy humans, obtained through freeze-thaw cell lysis followed by heat treatment and ultrafiltration, exhibits significant neuroprotective effects. This protection, evidenced by the attenuation of ROT-induced cell death in SH-SY5Y cells, was mediated through the reduction of oxidative stress, mitochondrial dysfunction, calcium dysregulation, and apoptosis. Additionally, in PD rats, intranasal HPL administration at various doses counteracted ROT-induced weight loss, improved motor function, balance, and grip strength, and alleviated anxiety, stress, and depression. Additionally, HPL promoted neuronal regeneration, suppressed astrocytic and microglial activation in the substantia nigra and striatum, enhanced antioxidant enzyme expression (glutathione and catalase), and reduced pro-oxidants (malondialdehyde and nitric oxide). These findings underscore HPL's potential as a promising therapeutic strategy for PD, representing a significant advancement in regenerative medicine.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 7","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144551091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}