Biotechnology Journal最新文献

{"title":"Optimizing Conditions of Polyethylene Glycol Precipitation for Exosomes Isolation From MSCs Culture Media for Regenerative Treatment","authors":"Junjun Yu,&nbsp;Daqiang Huang,&nbsp;Hanwen Liu,&nbsp;Haibo Cai","doi":"10.1002/biot.202400374","DOIUrl":"10.1002/biot.202400374","url":null,"abstract":"<div>\u0000 \u0000 <p>Mesenchymal stem cell (MSC)-derived exosomes, as a cell-free alternative to MSCs, offer enhanced safety and significant potential in regenerative medicine. However, isolating these exosomes poses a challenge, complicating their broader application. Commonly used methods like ultracentrifugation (UC) and tangential flow filtration are often impractical due to the requirement for costly instruments and ultrafiltration membranes. Additionally, the high cost of commercial kits limits their use in processing large sample volumes. Polyethylene glycol (PEG) precipitation offers a more convenient and cost-effective alternative, but there is a critical need for optimized and standardized isolation protocols using PEG precipitation across different cell types and fluids to ensure consistent quality and yield. In this work, we optimized the PEG precipitation method for exosomes isolation and compared its effectiveness to two commonly used methods: UC and commercial exosome isolation kits (ExoQuick). The recovery rate of the optimized PEG method (about 61.74%) was comparable to that of the commercial ExoQuick kit (about 62.19%), which was significantly higher than UC (about 45.80%). Exosome cargo analysis validated no significant differences in miRNA and protein profiles associated with the proliferation and migration of exosomes isolated by UC and PEG precipitation, which was confirmed by gap closure and CCK8 assays. These findings suggest that PEG-based exosomes isolation could be a highly efficient and high-quality method and may facilitate the development of exosome-based therapies for regenerative medicine.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256069","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":"Comparison of Lung Tissue-Derived Extracellular Vesicles Using Multiple Dissociation Methods for Profiling Protein Biomarkers","authors":"Yue Han,&nbsp;Meng Ye,&nbsp;Sheng Ye,&nbsp;Bowen Liu","doi":"10.1002/biot.202400329","DOIUrl":"10.1002/biot.202400329","url":null,"abstract":"<p>Extracellular vesicles (EVs) operate as chemical messengers that facilitate intercellular communication. Emerging evidence has demonstrated that lung tissue-derived EVs play pivotal roles in pulmonary physiological processes and have potential as biomarkers and therapeutics for lung diseases. Multiple methods have been proposed for the isolation of lung tissue-derived EVs. However, the effects of different tissue pre-treatments on lung EV isolation and subsequent disease biomarker discovery have not yet been comprehensively investigated. In this study, we compared the physical characteristics, recovery yields, and protein compositions of EVs isolated from lung tissues using three methods based on different tissue dissociation principles. Methodologically, the beneficial roles of blood perfusion and gentle meshing were emphasized based on their impact on EV yield and purity. These results demonstrate that different methods enrich distinct subpopulations of EVs that exhibit significant differences in their protein cargo and surface properties. These disparities directly affect the diagnostic detection of marker proteins related to lung diseases, including lung tumors, asthma, and pulmonary fibrosis. Collectively, these findings highlight the variations in EV characteristics resulting from the applied approaches and offer compelling suggestions for guiding researchers in selecting a suitable isolation method based on downstream functional studies and clinical applications.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400329","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256066","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":"Redesigning glycolaldehyde synthase for the synthesis of biorefinery feedstock D-xylulose from C1 compounds","authors":"Yue Yan,&nbsp;Haodong Zhao,&nbsp;Dingyu Liu,&nbsp;Jie Zhang,&nbsp;Yuwan Liu,&nbsp;Huifeng Jiang","doi":"10.1002/biot.202400360","DOIUrl":"10.1002/biot.202400360","url":null,"abstract":"<p>Global climate deterioration intensifies the demand for exploiting efficient CO₂ utilization approaches. Converting CO₂ to biorefinery feedstock affords an alternative strategy for third-generation biorefineries. However, upcycling CO₂ into complex chiral carbohydrates remains a major challenge. Previous attempts at sugar synthesis from CO₂ either produce mixtures with poor stereoselectivity or require ATP as a cofactor. Here, by redesigning glycolaldehyde synthase, the authors constructed a synthetic pathway for biorefinery feedstock D-xylulose from CO₂ that does not require ATP as a cofactor. The artificial D-xylulose pathway only requires a three-step enzyme cascade reaction to achieve the stereoselective synthesis of D-xylulose at a concentration of 1.2 g L⁻¹. Our research opens up an alternative route toward future production of chemicals and fuels from CO₂.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256064","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":"Overexpression of SLC2A1, ALDOC, and PFKFB4 in the glycolysis pathway drives strong drug resistance in 3D HeLa tumor cell spheroids","authors":"Tong Wang,&nbsp;Xueting Wang,&nbsp;Xuli Zheng,&nbsp;Zhongfang Guo,&nbsp;Ali Mohsin,&nbsp;Yingping Zhuang,&nbsp;Guan Wang","doi":"10.1002/biot.202400163","DOIUrl":"10.1002/biot.202400163","url":null,"abstract":"<p>The 3D multicellular tumor spheroid (MTS) model exhibits enhanced fidelity in replicating the tumor microenvironment and demonstrates exceptional resistance to clinical drugs compared to the 2D monolayer model. In this study, we used multiomics (transcriptome, proteomics, and metabolomics) tools to explore the molecular mechanisms and metabolic differences of the two culture models. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways revealed that the differentially expressed genes between the two culture models were mainly enriched in cellular components and biological processes associated with extracellular matrix, extracellular structural organization, and mitochondrial function. An integrated analysis of three omics data revealed 11 possible drug resistance targets. Among these targets, seven genes, <i>AKR1B1</i>, <i>ALDOC</i>, <i>GFPT2</i>, <i>GYS1</i>, <i>LAMB2</i>, <i>PFKFB4</i>, and <i>SLC2A1</i>, exhibited significant upregulation. Conversely, four genes, <i>COA7</i>, <i>DLD</i>, <i>IFNGR1</i>, and <i>QRSL1</i>, were significantly downregulated. Clinical prognostic analysis using the TCGA survival database indicated that high-expression groups of <i>SLC2A1</i>, <i>ALDOC</i>, and <i>PFKFB4</i> exhibited a significant negative correlation with patient survival. We further validated their involvement in chemotherapy drug resistance, indicating their potential significance in improving prognosis and chemotherapy outcomes. These results provide valuable insights into potential therapeutic targets that can potentially enhance treatment efficacy and patient outcomes.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256067","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":"A novel laccase for alkaline medium temperature environments in the textile industry","authors":"Kaifeng Xu,&nbsp;Ying Huo,&nbsp;Shiming Tang,&nbsp;Shuangyan Han,&nbsp;Ying Lin,&nbsp;Suiping Zheng","doi":"10.1002/biot.202400383","DOIUrl":"10.1002/biot.202400383","url":null,"abstract":"<p>Laccases are extensively used in the textile industry due to their ability to decolorize dyes, modify fabric surfaces, and bleach textiles. Identifying a laccase with both high thermal stability and alkali tolerance suitable for textile applications presents a significant challenge. A novel alkaline laccase, LacCT, was discovered from <i>Caldalkalibacillus thermarum</i> and successfully expressed it in <i>Escherichia coli</i>. LacCT displayed optimal activity at 65°C and maintained high stability across a pH range of 6.0–10.0, with an optimal pH of 7.5. Through rational design, the thermal stability of the best variant, G190P/Q254Y/G336M/D510F (LacCT-11), was significantly enhanced, resulting in a half-life of 63.2 min at 60°C – 1.8 times longer than that of the wild type. This research introduces a promising new laccase with considerable potential for decolorizing textile wastewater and improving the ramie degumming process.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256062","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":"Synergistic effects of biological stimuli and flexion induce microcavities promote hypertrophy and inhibit chondrogenesis during in vitro culture of human mesenchymal stem cell aggregates","authors":"Bo Zhang,&nbsp;Jim Berilla,&nbsp;Sungwoo Cho,&nbsp;Rodrigo A. Somoza,&nbsp;Jean F. Welter,&nbsp;Peter E. Alexander,&nbsp;Harihara Baskaran","doi":"10.1002/biot.202400060","DOIUrl":"10.1002/biot.202400060","url":null,"abstract":"<p>Interzone/cavitation are key steps in early stage joint formation that have not been successfully developed in vitro. Further, current models of endochondral ossification, an important step in early bone formation, lack key morphology morphological structures such as microcavities found during development in vivo. This is possibly due to the lack of appropriate strategies for incorporating chemical and mechanical stimuli that are thought to be involved in joint development. We designed a bioreactor system and investigated the synergic effect of chemical stimuli (chondrogenesis-inducing [CIM] and hypertrophy-inducing medium [HIM]) and mechanical stimuli (flexion) on the growth of human mesenchymal stem cells (hMSCs) based linear aggregates under different conditions over 4 weeks of perfusion culture. Computational studies were used to evaluate tissue stress qualitatively. After harvesting, both Safranin-O and hematoxylin &amp; eosin (H&amp;E) staining histology demonstrated microcavity structures and void structures in the region of higher stresses for tissue aggregates cultured only in HIM under flexion. In comparison to either HIM treatment or flexion only, increased glycosaminoglycan (GAG) content in the extracellular matrix (ECM) at this region indicates the morphological change resembles the early stage of joint cavitation; while decreased type II collagen (Col II), and increased type X collagen (Col X) and vascular endothelial growth factor (VEGF) with a clear boundary in the staining section indicates it resembles the early stage of ossification. Further, cell alignment analysis indicated that cells were mostly oriented toward the direction of flexion in high-stress region only in HIM under flexion, resembling cell morphology in both joint cavitation and hypertrophic cartilage in growth plate. Collectively, our results suggest that flexion and HIM inhibit chondrogenesis and promote hypertrophy and development of microcavities that resemble the early stage of joint cavitation and endochondral ossification. We believe the tissue model described in this work can be used to develop in vitro models of joint tissue for applications such as pathophysiology and drug discovery.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400060","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256063","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":"Physiological Metabolic Analysis of VB12 Accumulation in Ensifer adhaerens Casida A Enhanced by Oxygen Limitation","authors":"Bo Li,&nbsp;Xinyi Chen,&nbsp;Dujuan Zhao,&nbsp;Zebo Liu,&nbsp;Junming Li,&nbsp;Muhammad Safwan Siddique,&nbsp;Jiequn Wu,&nbsp;Yingping Zhuang,&nbsp;Zejian Wang","doi":"10.1002/biot.202400305","DOIUrl":"10.1002/biot.202400305","url":null,"abstract":"<div>\u0000 \u0000 <p>Cobalamin (VB₁₂) is in enormous demand across the fields of medicine, food, and feed additives. However, the oxygen supply plays a critical role in VB₁₂ biosynthesis by <i>Ensifer adhaerens Casida A</i> and has been identified as a bottleneck for economical substrate consumption. This study elucidates the relationship between oxygen limitation and VB₁₂ accumulation with transcriptomic and metabolomic analyses. Under oxygen limitation, <i>E. adhaerens</i> enhances oxygen transport and storage by increasing expression of flavin hemoglobin (<i>Hmp</i>), which was up-regulated 6-fold at 24 h of oxygen restriction compared to the oxygen restriction of 4 h (<i>p</i> &lt; 0.01). Because of the cofactor of <i>Hmp</i> is heme, the demand for heme increases, leading to the upregulation of genes in the heme biosynthesis pathway. Similarly, genes involved in biosynthesis of its precursor, 5-ALA, were upregulated as well. 5-ALA is also a direct precursor of VB₁₂, further leading to the upregulation of genes in the VB₁₂ biosynthesis pathway. This process initiates biosynthesis and accumulation of VB₁₂. As VB₁₂ and heme biosynthesis progresses, genes associated with the biosynthesis and transportation pathways of compounds related to their biosynthesis were likewise upregulated, including genes involved in S-adenosyl methionine (SAM) biosynthesis, and the transport of Fe²⁺ and Co²⁺. Additionally, amino acids and organic acids associated with biosynthesis were also extensively consumed, such as methionine, which is used for synthesizing SAM, decreased by 310% after 24 h of oxygen limitation compared to 20% dissolved oxygen (<i>p</i> &lt; 0.05). At the same time, genes related to growth-associated metabolic pathways, such as pentose phosphate pathway (PPP), were significantly downregulated. Therefore, the potential mechanism by which <i>E. adhaerens</i> accumulates VB₁₂ under oxygen-limited conditions by enhancing <i>Hmp</i> expression, which facilitates the porphyrin metabolic pathway and promotes VB₁₂ biosynthesis. This research provides valuable insights for increasing VB₁₂ production through metabolic engineering and process optimization.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142256068","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":"Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production","authors":"Peter Andorfer,&nbsp;Carolin-Isabel Kahlig,&nbsp;Doris Pakusic,&nbsp;Robert Pachlinger,&nbsp;Christiane John,&nbsp;Irene Schrenk,&nbsp;Peter Eisenhut,&nbsp;Johannes Lengler,&nbsp;Bernd Innthaler,&nbsp;Lucia Micutkova,&nbsp;Barbara Kraus,&nbsp;Corey Brizzee,&nbsp;Jack Crawford,&nbsp;Juan A. Hernandez Bort","doi":"10.1002/biot.202400415","DOIUrl":"10.1002/biot.202400415","url":null,"abstract":"<p>In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400415","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142152667","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":"Issue Information: Biotechnology Journal 9/2024","authors":"","doi":"10.1002/biot.202470091","DOIUrl":"https://doi.org/10.1002/biot.202470091","url":null,"abstract":"","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202470091","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142165482","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":"Partitioning behavior of short DNA fragments in polymer/salt aqueous two-phase systems","authors":"Rafaela Meutelet,&nbsp;Lea J. Bisch,&nbsp;Benedikt C. Buerfent,&nbsp;Markus Müller,&nbsp;Jürgen Hubbuch","doi":"10.1002/biot.202400394","DOIUrl":"10.1002/biot.202400394","url":null,"abstract":"<p>The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"19 9","pages":""},"PeriodicalIF":3.2,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.202400394","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142152668","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}