Journal of bioscience and bioengineering最新文献_第9页

Correlation analysis of urinary D/L-amino acid ratios and renal injury by using 5/6 nephrectomy model rats 5/6肾切除模型大鼠尿D/ l -氨基酸比值与肾损伤的相关性分析。

IF 2.9 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-07-10 DOI: 10.1016/j.jbiosc.2025.06.006

Kosuke Fukuda , Yutaka Umakoshi , Kenichi Watanabe , Masahiro Yahata , Izuru Miyawaki , Eiichiro Fukusaki

Renal injury, such as chronic kidney disease (CKD), is a global public health problem. Recent studies have reported that several D/L-amino acid ratios are affected during renal injury; however, most reports have focused on renal dysfunction, and few have focused on the association between D/L-amino acid changes and histopathological alterations in the kidney. Therefore, we aimed to investigate the correlation between D/L-amino acid ratios and the histopathological findings of renal injury in a 5/6 nephrectomy rat model. In this study, the D/L-amino acid ratios were evaluated using urine samples collected 1, 2, 3, and 4 weeks after surgery. The D/L-Ser and D/L-Ala ratios were 3.0-fold and 9.3-fold higher, respectively, and the other D/L-amino acid ratios, such as D/L-Arg, D/L-Asn, D/L-His, D/L-Ile, D/L-Lys, D/L-Phe, and D/L-Thr ratios, also showed higher values than those in the sham group. The D/L-amino acid ratios in the urine were consistently maintained throughout the evaluation period compared to those in the sham group. However, only the D/L-Gln ratio was lower than that in the sham group. Correlation analysis between D/L-amino acid ratios and histopathological alterations in the kidney revealed a good correlation between D/L-Ala, D/L-Glu, and D/L-Gln one week after surgery and tubular dilatation. No other findings showed a strong correlation with the changes in the D/L-amino acid ratios of the aforementioned amino acids. By evaluating urinary D/L-amino acid ratios, we could potentially detect tubular dilatation in the kidney noninvasively and obtain insights into the physiological condition of the kidney.

肾脏损伤，如慢性肾脏疾病（CKD），是一个全球性的公共卫生问题。最近的研究报道了几种D/ l -氨基酸比率在肾损伤期间受到影响；然而，大多数报道都集中在肾功能障碍上，很少有报道关注D/ l -氨基酸变化与肾脏组织病理学改变之间的关系。因此，我们旨在探讨D/ l -氨基酸比例与5/6肾切除术大鼠模型肾损伤的组织病理学结果的相关性。在这项研究中，D/ l -氨基酸比率通过术后1、2、3和4周收集的尿液样本进行评估。D/L-Ser和D/L-Ala比值分别提高了3.0倍和9.3倍，其他D/ l -氨基酸比值，如D/L-Arg、D/L-Asn、D/L-His、D/L-Ile、D/L-Lys、D/L-Phe和D/L-Thr比值也比假手术组高。与假手术组相比，在整个评估期间尿中D/ l -氨基酸比率始终保持不变。但仅D/L-Gln比假手术组低。D/ l -氨基酸比值与肾组织病理学改变的相关性分析显示，术后1周D/L-Ala、D/L-Glu、D/L-Gln与肾小管扩张有良好的相关性。没有其他研究结果显示与上述氨基酸的D/ l -氨基酸比率的变化有很强的相关性。通过评估尿D/ l -氨基酸比率，我们可以无创地检测肾小管扩张，并了解肾脏的生理状况。

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引用次数: 0

Design of a thermostable bilirubin oxidase from Myrothecium verrucaria 一种耐热性胆红素氧化酶的设计。

IF 2.3 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-06-11 DOI: 10.1016/j.jbiosc.2025.05.006

Haruka Kado Horiguchi , Shohei Yamada , Hironori Semba , Hirokazu Tsuboi , Takayuki Bogaki , Akio Koda , Kazuhiko Ishikawa , Yutaro Mori , Chiaki Ogino , Masahiro Takagi , Yoshio Tsujino

Bilirubin oxidase (BOD), identified as a multicopper oxidase produced by Myrothecium verrucaria, plays a critical role in the oxidation of bilirubin to biliverdin, which is pivotal in various biochemical processes. To construct a highly thermostable BOD, we have used three protein engineering methods: (i) stabilization of the main chain (proline substitution), (ii) design of salt bridges, and (iii) improvement of hydrophobic interactions. Significant enhancement of thermostability was achieved through stabilization of the main chain (L476P, A496P), introduction of a salt bridge (Q495R), and improvement of hydrophobic interactions (A264V). Furthermore, the combination of these point mutations, which contributed to structural stabilization, resulted in a novel thermostable mutant. Utilizing the cumulative effect of point mutations based on the three strategies, we were able to obtain a thermostable enzyme that exhibited approximately 3.9-fold higher residual activity than wild-type BOD (WT) even after incubation at 60 °C for 1 h and nearly 10 °C higher optimum temperature than that of WT. Importantly, these mutations did not affect its 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation activity. This approach provides a valuable strategy for improving the thermostability of multivalent copper oxidases and offers promising prospects for industrial applications.

胆红素氧化酶（Bilirubin oxidase， BOD）是由疣状分枝杆菌（Myrothecium verrucaria）产生的一种多铜氧化酶，在胆红素氧化为胆绿素的过程中起关键作用，在多种生化过程中起关键作用。为了构建高度热稳定的BOD，我们使用了三种蛋白质工程方法：(i)主链稳定（脯氨酸取代），（ii）设计盐桥，（iii）改善疏水相互作用。通过稳定主链（L476P, A496P），引入盐桥（Q495R）和改善疏水相互作用（A264V），显著提高了热稳定性。此外，这些点突变的组合有助于结构稳定，导致了一种新的耐热突变体。利用基于这三种策略的点突变的累积效应，我们能够获得一种耐热酶，即使在60°C孵育1小时后，其残余活性也比野生型BOD （WT）高出约3.9倍，比WT的最适温度高出近10°C。重要的是，这些突变不影响其2,2'-氮化-双（3-乙基苯并噻唑啉-6-硫酸）氧化活性。该方法为提高多价铜氧化酶的热稳定性提供了一种有价值的策略，具有良好的工业应用前景。

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引用次数: 0

Response to the letter to the editor on “Evaluation methods for decellularized tissues: A focus on human amniotic membrane” 回复“脱细胞组织的评价方法：以人羊膜为重点”致编辑的信。

IF 2.3 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-06-10 DOI: 10.1016/j.jbiosc.2025.05.007

Miriam Guadalupe Salgado García , Néstor Fabián Díaz , Guadalupe García López , Ikuri Álvarez Maya , Claudia Hernández Jimenez , Yvonne Roman Maldonado , David José Mendoza Aguayo , Néstor Emmanuel Díaz Martínez

引用次数: 0

Establishment of a nitrate-reductase promoter-driven inducible expression system for metabolic engineering in the marine diatom Fistulifera solaris 硝酸还原酶启动子驱动海洋硅藻代谢工程诱导表达系统的建立。

IF 2.3 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-06-07 DOI: 10.1016/j.jbiosc.2025.05.009

Tomoki Yamanaka , Takashi Yabuuchi , Maeda Yoshiaki , Tomoko Yoshino , Kosuke Kataoka , Tsuyoshi Tanaka

Microalgae have emerged as promising hosts for producing valuable compounds, including lipids and recombinant proteins. The oleaginous diatom Fistulifera solaris has shown potential for industrial applications. However, the constitutive expression of proteins often causes cellular toxicity, necessitating a controlled gene expression system to enhance productivity. In this study, we characterized the endogenous promoter of the nitrate reductase (NR) gene as an inducible expression system. The promoter was activated by nitrate and suppressed by ammonium. We also established a nitrate-spike method for controlled promoter activation by adding NaNO₃ at specific time points, which achieved strong induction without compromising the cellular lipid content. This study provides the first characterization of the NR promoter in an oleaginous diatom and demonstrates its use for controlled compound production. The inducible expression system developed in this study represents fundamental technology for enhancing the productivity of F. solaris.

微藻已成为生产有价值化合物（包括脂质和重组蛋白）的有希望的宿主。富含油脂的硅藻具有潜在的工业应用价值。然而，蛋白质的组成表达经常引起细胞毒性，需要一个受控的基因表达系统来提高生产力。在本研究中，我们将硝酸还原酶（NR）基因的内源性启动子描述为一个诱导表达系统。促进剂被硝酸盐激活，被铵抑制。我们还建立了一种硝酸盐尖峰法，通过在特定时间点添加NaNO3来控制启动子的激活，该方法在不影响细胞脂质含量的情况下实现了强诱导。本研究首次对产油硅藻中的NR促进剂进行了表征，并证明了其在控制化合物生产中的应用。本研究建立的诱导表达体系是提高凤头莲产量的基础技术。

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引用次数: 0

Recovery of hyaluronic acid using thermosensitive polymer-salt aqueous biphasic system with polymer recycling 热敏聚合物-盐双水相体系回收透明质酸的研究。

IF 2.3 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-06-06 DOI: 10.1016/j.jbiosc.2025.04.007

Nagaraju Marimuthu , Yin Hui Chow , Phei Er Kee , John Chi-Wei Lan , Zee Wei Lai , Li Wan Yoon , Yew Joon Tam

Microbial fermentation emerges as a viable alternative to animal tissue-based extraction for industrial hyaluronic acid (HA) production. However, microbial HA requires rigorous separation and purification processes to meet high purity standards for cosmeceutical use. Aqueous biphasic system (ABS) presents a promising strategy for HA recovery due to its cost-effectiveness, high yield, purity and eco-friendly nature. Thermosensitive polymer, specifically ethylene oxide-propylene oxide (EOPO)-salt ABS, was proposed in the present study to recover microbial HA from Streptococcus zooepidemicus fermentation broth. The effects of molecular weight (MW) of EOPO, types of salt, phase composition, and the amount of crude extract on the efficiency of ABS for HA recovery were studied. Furthermore, the recovery efficiency of ABS formed using recycled EOPO was investigated. HA exhibited a preference for the salt-rich bottom phase of ABS because of its hydrophilic surface features. Highest partition coefficient of 11.41 ± 0.00 and yield of 93.42 % ± 0.00 % were achieved in ABS containing 14 % (w/w) EOPO₂₅₀₀, 12 % (w/w) sodium citrate at pH 8.2 and 15 % (w/w) crude extract. More than 70 % of EOPO₂₅₀₀ was recovered through the thermo-separation process, and the 1st recycling of EOPO₂₅₀₀ maintained a recovery yield of HA above 80 % in ABS. Moreover, Fourier transform infrared spectroscopy validated that HA retained its structure after the recovery process. In conclusion, this study demonstrates the effectiveness of EOPO-salt ABS for microbial HA recovery, achieving high recovery yield. The recycling of EOPO enhances the economic viability and environmental sustainability of the system. These findings pave the way for greener, scalable HA production practices while maintaining product integrity.

微生物发酵作为一种可行的替代动物组织为基础的提取工业透明质酸（HA）生产。然而，微生物透明质酸需要严格的分离和纯化过程，以满足药妆使用的高纯度标准。双相水相体系（ABS）具有成本效益高、收率高、纯度高、环境友好等优点，是一种很有前途的HA回收方法。本研究提出了一种热敏聚合物，即环氧乙烷-环氧丙烷(EOPO)-盐ABS，用于回收动物流行病链球菌发酵液中的微生物HA。研究了EOPO分子量、盐种类、物相组成、粗提物用量等因素对ABS回收HA效率的影响。此外，还研究了回收EOPO形成ABS的回收效率。由于ABS的亲水表面特征，HA表现出对富盐底相的偏好。在含有14% (w/w) EOPO2500、12% （w/w）柠檬酸钠和15% （w/w）粗提物的ABS中，分配系数最高，为11.41±0.00，产率为93.42%±0.00 %。通过热分离工艺回收了70%以上的EOPO2500，第一次回收的EOPO2500在ABS中保持了80%以上的HA回收率，并且傅里叶变换红外光谱验证了回收过程后HA保持了原有的结构。综上所述，本研究验证了eopo盐ABS对微生物HA回收的有效性，实现了较高的回收率。EOPO的回收利用提高了系统的经济可行性和环境可持续性。这些发现为更环保、可扩展的高可用性生产实践铺平了道路，同时保持了产品的完整性。

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引用次数: 0

Discovery and affinity maturation of antibody fragments from an unfavorably enriched phage display selection by deep sequencing and machine learning 通过深度测序和机器学习从不利富集的噬菌体展示选择中发现抗体片段并进行亲和成熟。

IF 2.3 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-06-04 DOI: 10.1016/j.jbiosc.2025.05.004

Sakiya Kawada , Yoichi Kurumida , Tomoyuki Ito , Thuy Duong Nguyen , Hafumi Nishi , Hikaru Nakazawa , Yutaka Saito , Tomoshi Kameda , Koji Tsuda , Mitsuo Umetsu

Phage display selection has been used for directed evolution of antibody fragments. However, variants with binding affinity cannot be always identified due to undesirable enrichment of target-unrelated variants in the biopanning process. Here, our goal was to obtain functional variants by deep sequencing and machine learning from a phage display library where functional variants were not appropriately enriched. Deep sequencing of the previously biopanned pools revealed that amplification bias might have prevented the enrichment of target-binding phages. We performed a sequence similarity search based on the deep sequencing analysis so that the influence of bias was decreased, leading to discovery of a variant with binding affinity, which could not be discovered by a conventional screening method alone. We applied machine learning to the deep sequencing data; the machine learning proposed effective mutations for increasing affinity, allowing us to identify a variant with improved affinity (EC₅₀ = 3.46 μM). In summary, we present the possibility of obtaining functional variants even from unfavorably enriched phage libraries by using deep sequencing and machine learning.

噬菌体展示选择已被用于抗体片段的定向进化。然而，由于在生物筛选过程中不希望富集与靶标无关的变异，具有结合亲和力的变异并不总是被识别出来。在这里，我们的目标是通过深度测序和机器学习从功能变体没有适当丰富的噬菌体展示库中获得功能变体。对先前生物筛选池的深度测序显示，扩增偏差可能阻止了靶结合噬菌体的富集。我们在深度测序分析的基础上进行了序列相似性搜索，减少了偏倚的影响，从而发现了一个具有结合亲和力的变异，这是传统筛选方法无法发现的。我们将机器学习应用于深度测序数据；机器学习提出了增加亲和力的有效突变，使我们能够识别出具有提高亲和力的变体（EC50 = 3.46 μM）。总之，我们提出了通过使用深度测序和机器学习从不利富集的噬菌体文库中获得功能变体的可能性。

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引用次数: 0

Preparation of minicircle DNA vectors for gene knock-in to mammalian cells 哺乳动物细胞基因敲入用小环DNA载体的制备。

IF 2.3 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-05-29 DOI: 10.1016/j.jbiosc.2025.05.005

Yoshinori Kawabe, Makoto Hamaoka, Akio Kuno, Yusaku Yoshimura, Masamichi Kamihira

Minicircle DNA vectors (MCs) are gene carriers with favorable characteristics for gene therapy and the production of recombinant cells using animal cells as hosts. Typically, MCs are prepared by inserting target gene fragments as an expression unit into a standard plasmid vector, followed by the removal of bacterial sequences. In this study, we report a method for MC preparation utilizing the Cre-loxP recombination system. By inserting the target gene fragments between two loxP sites introduced into the plasmid vector, the plasmid backbone can be removed through the action of Cre recombinase. We explored optimal reaction conditions for MC generation using Cre. Furthermore, site-specific knock-ins into the CHO cell genome were performed using the remaining loxP sequence in the generated MCs via the Cre-loxP reaction. When MCs were used as gene donors, significant improvements in gene integration efficiency and enhanced gene expression in CHO cells were observed compared with conventional plasmids. The MC preparation and gene knock-in techniques developed in this study are highly useful for CHO cell engineering.

微环DNA载体是一种基因载体，具有良好的基因治疗和以动物细胞为宿主生产重组细胞的特性。通常，mc是通过将目标基因片段作为表达单元插入标准质粒载体中，然后去除细菌序列来制备的。在本研究中，我们报告了一种利用Cre-loxP复合体系制备MC的方法。将目的基因片段插入到导入质粒载体的两个loxP位点之间，可通过Cre重组酶的作用去除质粒骨架。我们探索了用Cre生成MC的最佳反应条件。此外，利用Cre-loxP反应生成的MCs中剩余的loxP序列，对CHO细胞基因组进行了位点特异性敲入。当MCs作为基因供体时，与常规质粒相比，CHO细胞中的基因整合效率显著提高，基因表达增强。本研究开发的MC制备和基因敲入技术在CHO细胞工程中具有重要的应用价值。

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引用次数: 0

Construction of contractile human iPSC-derived skeletal muscle tissues on 96-well scale microdevices 在96孔级微设备上构建可收缩的人类ipsc衍生骨骼肌组织。

IF 2.3 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-05-29 DOI: 10.1016/j.jbiosc.2025.05.003

Seitaro Nakamura , Yuhei Kamei , Ayumu Matsushima , Kazuki Yamamoto , Hirokazu Akiyama , Muhammad Irfanur Rashid , Yohei Okada , Tomoya Uchimura , Hidetoshi Sakurai , Hiroyuki Honda , Kazunori Shimizu

Tissue-engineered three-dimensional (3D) skeletal muscles can be potentially used in contractile force-based phenotypic screening to elucidate the mechanisms of skeletal muscle dysfunction and develop preventive and therapeutic strategies. Human induced pluripotent stem cells (hiPSCs) expressing tetracycline-inducible myogenic differentiation 1 (MYOD1) are a promising cell source for construction of tissue-engineered skeletal muscles. Although we successfully constructed contractile tissues using these hiPSCs in a previous study, further improvements are required because of their weak contractile force and inefficient screening capabilities. In this study, we aimed to construct iPSC-derived muscle tissues with high contractile force using a 96-well scale microdevice that we had previously developed. To increase the contractile force, we optimized the time of supplementation of the transforming growth factor-β (TGF-β) inhibitor, SB431542 (SB), to identify culture conditions that enhance contractile force. The maximum contractile force with addition of SB was approximately five times greater than that without SB (58.45 ± 20.14 μN with SB compared to 11.64 ± 4.86 μN without SB). Various analyses, including immunostaining, transmission electron microscopy, gene expression analysis, and proteomics, revealed enhanced myotube differentiation and muscle tissue maturation in the presence of SB. Experiments using inhibitors indicated that TGFβ1, not myostatin, is partially involved in these effects. Furthermore, we confirmed that tissues constructed from iPSCs derived from patients with Duchenne muscular dystrophy also showed improved contractility following addition of SB. Therefore, iPSC-derived muscle tissues cultured with SB on the 96-well microdevice provide a promising platform for screening compounds that can ameliorate disease pathology.

组织工程三维（3D）骨骼肌可以潜在地用于基于收缩力的表型筛选，以阐明骨骼肌功能障碍的机制，并制定预防和治疗策略。表达四环素诱导的肌源分化1 （MYOD1）的人诱导多能干细胞（hiPSCs）是构建组织工程骨骼肌的一种有前途的细胞来源。虽然我们在之前的研究中成功地使用这些hiPSCs构建了可收缩组织，但由于它们的收缩力弱，筛选能力低，需要进一步改进。在这项研究中，我们的目标是使用我们之前开发的96孔规模的微型设备构建具有高收缩力的ipsc衍生肌肉组织。为了提高收缩力，我们优化了转化生长因子-β (TGF-β)抑制剂SB431542 （SB）的添加时间，以确定增强收缩力的培养条件。添加SB的最大收缩力（58.45±20.14 μN）约为未添加SB的5倍（11.64±4.86 μN）。包括免疫染色、透射电镜、基因表达分析和蛋白质组学在内的各种分析显示，在SB的存在下，肌管分化和肌肉组织成熟增强。使用抑制剂的实验表明，TGFβ1，而不是肌肉生长抑制素，部分参与了这些作用。此外，我们证实了杜氏肌营养不良症患者的ipsc构建的组织在添加SB后也显示出改善的收缩性。因此，在96孔微设备上用SB培养的ipsc来源的肌肉组织为筛选可以改善疾病病理的化合物提供了一个有希望的平台。

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引用次数: 0

Novel promoter of Pseudozyma antarctica exhibits stable glucose-mediated activation and copper-mediated suppression of flanking genes 新的南极假酶启动子表现出稳定的葡萄糖介导的激活和铜介导的对侧翼基因的抑制。

IF 2.3 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-05-29 DOI: 10.1016/j.jbiosc.2025.05.002

Tohru Yarimizu , Atsuhiro Miura , Xiao-hong Cao , Takumi Tanaka , Tomotake Morita , Hitoshi Shimoi , Hirokazu Ueda , Hiroko Kitamoto

Pseudozyma antarctica is a leaf-colonizing basidiomycetous yeast that produces a plastic-degrading enzyme (PaE) using xylose as an inducer and carbon source. Notably, glucose, a readily available carbon source, has not been utilized for PaE production. Herein, we identified two promoters of P. antarctica for stable induction of PaE gene transcription at high levels in commonly used yeast media with glucose, along with assessing the effect of copper supplementation. Through microarray analysis, the top five genes with high expression in glucose cultures were identified and their promoter activities were evaluated by reporter assay. The first and second genes were located 903 nucleotides apart on the P. antarctica chromosome and shared reciprocal inverted sequences as promoters, namely PaFRE1 and PaCTR1, respectively, owing to their weak similarity to FRE and CTR in Saccharomyces cerevisiae genome, which regulate the copper uptake. When P. antarctica was grown in three common media using glucose as a carbon source, both PaFRE1 and PaCTR1 induced high and stable expression and produced more heterologous artificial luciferase and endogenous PaE than that by the highly expressed actin promoter. Additionally, the activities of PaFRE1 and PaCTR1 promoters were suppressed by copper addition. Altogether, this study shows that these P. antarctica promoters can be used to produce various proteins such as PaE.

南极假酶是一种以木糖为诱导剂和碳源产生塑料降解酶（PaE）的叶定殖担子菌酵母。值得注意的是，葡萄糖是一种容易获得的碳源，尚未用于PaE的生产。这里,我们确定了两个p .南极洲的倡导者在高水平稳定的PaE诱导基因转录与葡萄糖在媒体常用的酵母,以及评估补充铜的影响。通过微阵列分析，鉴定出葡萄糖培养中高表达的前5个基因，并通过报告子实验评估其启动子活性。第一和第二基因与酿酒酵母基因组中调控铜摄取的FRE和CTR具有较弱的相似性，因此它们作为启动子具有互反序列，分别为PaFRE1和PaCTR1，在南极p.a ntera染色体上相距903个核苷酸。p .南极时生长在三种常见的媒体利用葡萄糖作为碳源,PaFRE1和PaCTR1诱导高和稳定表达和产生更多不同的人工荧光素酶和内源性PaE肌动蛋白的高表达启动子。此外，添加铜抑制了PaFRE1和PaCTR1启动子的活性。总之,这项研究表明,这些p .南极启动子可用于生产各种蛋白质如PaE。

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引用次数: 0

Glycolytic inhibition by resveratrol facilitates chondrocyte survival under glucose-deprived conditions and improves the viability of 3D-cultured cartilage tissue 白藜芦醇的糖酵解抑制促进了葡萄糖剥夺条件下软骨细胞的存活，提高了3d培养软骨组织的活力。

IF 2.3 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Journal of bioscience and bioengineering

Pub Date : 2025-05-23 DOI: 10.1016/j.jbiosc.2025.05.001

Rui Zhang, Keita Kanki

Decreased cell viability resulting from severe nutrient deprivation is a major obstacle in three-dimensional (3D) tissue construction. Therefore, technical improvements that prevent cell death in the core region of cell aggregates are desired for the development of large, thick tissues. We focused on the anti-glycolytic effects of resveratrol (RSV), a polyphenol known as a caloric restriction mimetic, and investigated its cytoprotective effects under glucose-deprived conditions in two-dimensional (2D) and 3D-cell culture systems using rat chondrocytes. In 2D culture, the low-glucose (LG, 0.5 mg/mL) condition caused time- and dose-dependent cell death in chondrocytes, whereas co-treatment with 50 μM RSV significantly restored cell viability under glucose deprivation. In RSV-treated cells, the expression levels of glycolytic genes (GLUT1, PKM, and LDHA) and glucose uptake were significantly downregulated, and phospho-AMPK levels were upregulated, indicating energy stress. RSV treatment restored the expression of extracellular matrix genes (COL1A1 and COL2A1), which were downregulated under the LG condition, and augmented the pro-chondrogenic effect of TGF-β1 and ascorbic acid. In a 3D-culture model, spheroids constructed with RSV-pretreated chondrocytes had a more viable core region than dimethyl-sulfoxide-treated control spheroids. TGF-β-induced cartilage maturation led these spheroids to develop larger and more viable tissues than control spheroids. These results suggested that glycolytic inhibition by RSV decreased chondrocyte glucose usage, thereby preventing cell death caused by glucose deprivation. Our findings provide useful information for improving cell viability under hyponutrition conditions and can be applied to 3D tissue construction.

严重的营养剥夺导致的细胞活力下降是三维（3D）组织构建的主要障碍。因此，防止细胞聚集核心区域的细胞死亡的技术改进是大而厚的组织发育所需要的。我们专注于白藜芦醇（RSV）的抗糖酵解作用，白藜芦醇是一种多酚，被称为热量限制模拟物，并在使用大鼠软骨细胞的二维（2D）和三维细胞培养系统中研究了它在葡萄糖剥夺条件下的细胞保护作用。在2D培养中，低糖（LG, 0.5 mg/mL）条件导致软骨细胞的时间和剂量依赖性细胞死亡，而与50 μM RSV共处理可显著恢复葡萄糖剥夺下的细胞活力。在rsv处理的细胞中，糖酵解基因（GLUT1、PKM和LDHA）和葡萄糖摄取的表达水平显著下调，磷酸化ampk水平上调，表明能量应激。RSV处理恢复了LG条件下下调的细胞外基质基因COL1A1和COL2A1的表达，增强了TGF-β1和抗坏血酸的促软骨形成作用。在3d培养模型中，用rsv预处理的软骨细胞构建的球体比二甲亚砜处理的对照球体具有更有活力的核心区域。TGF-β诱导的软骨成熟导致这些球体比对照球体发育更大、更有活力的组织。这些结果表明RSV抑制糖酵解降低了软骨细胞葡萄糖的使用，从而防止了葡萄糖剥夺引起的细胞死亡。我们的发现为在低营养条件下提高细胞活力提供了有用的信息，并可应用于3D组织构建。

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引用次数: 0