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Biotechnology Notes

Pub Date : 2025-01-01

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Biotechnology Notes

Pub Date : 2025-01-01

引用次数: 0

Integrative analysis of candidate MicroRNAs and gene targets for OSA management using in silico and in-vitro approach 应用计算机和体外方法综合分析OSA管理的候选microrna和基因靶点

Biotechnology Notes

Pub Date : 2025-01-01 DOI: 10.1016/j.biotno.2025.01.003

Gaganjyot Kaur Bakshi , Sartaj Khurana , Shambhavee Srivastav , Rohit Kumar , Mukesh Chourasia , Sudeep Bose

MicroRNAs (miRNAs) have been implicated in the pathogenesis of human diseases including sleep disorders. The aim of this study is to address the involvement of miRNAs (miR-21 and miR-29) in the pathophysiology of obstructive sleep apnea (OSA). In this study we have done integrated analysis of miRNAs with their potential gene targets as a strategy for management of OSA.

Methods

miRNA expression levels were quantified in healthy control group and obese vs. Non-obese OSA subjects by Quantitative real-time PCR. In-silico analysis of interplay of miRNAs with potential gene targets was done using Schrödinger Release 2023-1.

Results

The real time expression analysis revealed a differential expression pattern in miRNAs indicating down-regulation of miR-21 in obese OSA while miR-29 showed upregulation as compared to non-obese OSA and healthy subjects with p values of ≤0.01 and <0.0001respectively. A trend was observed where target genes TGFBR2, NAMPT, and NPPB were significantly increased with p-value of ≤0.0001 and TGFBR3 and INSIG2 showed decreasing trend with p-value of ≤0.0001 between obese and non-obese OSA respectively. MD simulation analysis provided valuable information regarding the stability, flexibility, compactness and solvent exposure of the complexes over time.

Conclusion

miR-21 and miR-29 possesses differential expressions in obese OSA subject and exihbits strong molecular interactions with potential target genes, such as TGFBR2, NPPB, NAMPT and INSIG2. Identifying the miRNAs, genes and pathways associated with OSA can help to expand our understanding of the risk factors for the disease as well as provide new avenues for potential treatment.

MicroRNAs （miRNAs）与包括睡眠障碍在内的人类疾病的发病机制有关。本研究的目的是解决mirna （miR-21和miR-29）在阻塞性睡眠呼吸暂停（OSA）病理生理中的参与。在这项研究中，我们对mirna及其潜在基因靶点进行了综合分析，作为OSA管理的策略。方法采用实时荧光定量PCR法测定健康对照组和肥胖与非肥胖OSA患者的smirna表达水平。使用Schrödinger Release 2023-1对mirna与潜在基因靶点的相互作用进行了计算机分析。结果实时表达分析显示，与非肥胖性OSA和健康受试者相比，肥胖性OSA患者miR-21表达下调，miR-29表达上调，p值分别为≤0.01和<；0.0001。肥胖与非肥胖OSA靶基因TGFBR2、NAMPT、NPPB均有显著升高的趋势，p值≤0.0001；TGFBR3、INSIG2均有降低的趋势，p值≤0.0001。MD模拟分析提供了有关配合物随时间的稳定性、柔韧性、致密性和溶剂暴露的有价值的信息。结论mir -21、miR-29在肥胖OSA患者中存在差异表达，与TGFBR2、NPPB、NAMPT、INSIG2等潜在靶基因存在较强的分子相互作用。识别与阻塞性睡眠呼吸暂停相关的mirna、基因和途径有助于扩大我们对该疾病危险因素的理解，并为潜在的治疗提供新的途径。

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引用次数: 0

Agroinfiltration-mediated transient assay for rapid evaluation of constructs in pigeonpea 农业浸润介导的快速评价鸽豌豆构建体的瞬时试验

Biotechnology Notes

Pub Date : 2025-01-01 DOI: 10.1016/j.biotno.2025.02.005

Kalenahalli Yogendra, Harika Gadeela, Koppula Nithya Sree, Wricha Tyagi

The process of generating stable transformants is time-consuming, labor-intensive, and genotype-dependent. In contrast, transient gene expression techniques, such as agroinfiltration, offer a rapid assessment of gene function and expression. Agroinfiltration, widely employed for studying gene function, has been extensively applied in leaf tissues of Nicotiana benthamiana and various other plant species. Despite its broad utility in various plants, to our knowledge, no prior investigation has been reported in pigeonpea. In this study, we developed an agroinfiltration method for transiently expressing a green fluorescent protein (mGFP5) reporter gene in four pigeonpea genotypes using syringe infiltration at the seedling stage under greenhouse conditions. The expression of the reporter gene mGFP5 was assessed at 72-, 96-, and 120 h post-infiltration (hpi). Additionally, we assessed the effect of morphogenic genes, specifically growth-regulating factor 4 (GRF4) and GRF-interacting factor 1 (GIF1), from both rice and pigeonpea on the expression of mGFP5 in four pigeonpea genotypes. Our findings demonstrate that OsGRF4-GIF1 led to enhanced mGFP5 expression compared to CcGRF4-GIF1 in four diverse pigeonpea genotypes. Fluorescence could be detected till 120 hpi. Furthermore, PCR, RT-PCR, and fluorescence quantification confirmed the presence and expression of mGFP5 at 72 hpi. Our results highlight the efficacy of agroinfiltration in quickly evaluating candidate genes in four genetically diverse pigeonpea genotypes, thereby reducing the time required for the initial assessment of constructs suitable for diverse molecular biology analyses.

产生稳定的转化体的过程是耗时的，劳动密集型的，并且依赖于基因型。相比之下，瞬时基因表达技术，如农业渗透，提供了基因功能和表达的快速评估。农业渗透技术被广泛应用于研究基因功能，已广泛应用于烟叶和其他植物的叶组织中。尽管它在各种植物中广泛应用，但据我们所知，尚无关于鸽子豆的研究报道。在这项研究中，我们建立了一种农业渗透方法，在温室条件下，在苗期使用注射器渗透，在四种基因型的鸽子豌豆中瞬时表达绿色荧光蛋白（mGFP5）报告基因。在浸润后72、96和120 h （hpi）评估报告基因mGFP5的表达。此外，我们评估了水稻和鸽豌豆中形态发生基因，特别是生长调节因子4 （GRF4）和grf相互作用因子1 （GIF1）对四种鸽豌豆基因型中mGFP5表达的影响。我们的研究结果表明，与CcGRF4-GIF1相比，OsGRF4-GIF1在四种不同的鸽豆基因型中导致mGFP5的表达增强。直到120 hpi都能检测到荧光。此外，PCR、RT-PCR和荧光定量证实了72hpi时mGFP5的存在和表达。我们的研究结果强调了农业渗透在四种遗传多样性的鸽豌豆基因型中快速评估候选基因的有效性，从而减少了初始评估适合不同分子生物学分析的构建体所需的时间。

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Straightforward MALDI-TOF MS based screening approach for selection of recombinant protein-expressing E. coli 直接基于MALDI-TOF MS筛选表达重组蛋白大肠杆菌的方法

Biotechnology Notes

Pub Date : 2025-01-01 DOI: 10.1016/j.biotno.2025.02.004

I.N. Kravtsov , A.I. Solovyev , E.A. Potemkina , A.V. Kartashova , M.A. Dmitrieva , K.V. Danilova , I.L. Tutykhina , N.B. Polyakov , V.D. Desinov , D.A. Egorova , A.L. Gintsburg

Recombinant protein production is a milestone of modern biotechnology, drug development and scientific research. When obtaining recombinant protein producers, differences in expression levels among clones necessitate screening. Traditional widely used methods include protein electrophoresis and western blot hybridization. This protocol provides high-throughput advantages by eliminating time-consuming steps inherent to traditional methods, such as cell lysis, protein extraction, purification, antibody-based detection, and gel-based analysis. MALDI-TOF MS represents a simple, rapid and cost-effective method for bacterial species identification through protein fingerprint signature in clinical diagnostics, but not practically integrated into biotechnological workflow. This study proposes a fast and easy method for screening E. coli clones producing recombinant proteins with MALDI-TOF MS. The proposed method demonstrated efficiency in screening of E. coli producing several recombinant proteins with different properties: sfGFP; bacterial DNA binding proteins IHFα, IHFβ, HU; bacteriophage protein GP46 and camelid VHH antibody fragments.

重组蛋白的生产是现代生物技术、药物开发和科学研究的一个里程碑。在获得重组蛋白生产者时，需要筛选克隆之间表达水平的差异。传统的广泛使用的方法包括蛋白质电泳和western blot杂交。该方案通过消除传统方法固有的耗时步骤提供高通量优势，例如细胞裂解，蛋白质提取，纯化，基于抗体的检测和基于凝胶的分析。MALDI-TOF质谱在临床诊断中是一种简单、快速、具有成本效益的蛋白质指纹图谱细菌种类鉴定方法，但尚未真正融入生物技术工作流程。本研究提出了一种快速简便的利用MALDI-TOF ms筛选产生重组蛋白的大肠杆菌克隆的方法，该方法在筛选产生不同性质重组蛋白的大肠杆菌中表现出高效率：sfGFP；细菌DNA结合蛋白IHFα、IHFβ、HU；噬菌体蛋白GP46和骆驼类VHH抗体片段。

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Biotechnology Notes

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Biotechnology Notes

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Biotechnology Notes

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