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Mito-kaede photoactivation and chase experiment for mitophagy: optimizing flux measurement via fluid exchange system. 有丝分裂的光激活和追逐实验:通过流体交换系统优化通量测量。
IF 2.7 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-09-11 DOI: 10.1080/07366205.2024.2372955
Hiroyuki Morinaga,Yoh Sugawara,Yoshinori Kitagawa,Jingyuan Chen,Nobuo Yasuda,Hiroki Ogata,Yoshihiro Yamaguchi,Masao Kaneki,Joseph A Jeevendra Martyn,Shingo Yasuhara
Modulating autophagy and mitophagy, vital cellular quality control systems, offer therapeutic potential for critical illnesses. However, limited drug screening options hinder progress. We present a novel assay using the photoswitchable fluorescent reporter, mito-Kaede, to quantify mitophagy flux. Mito-Kaede's superior UV-induced photoconversion and brightness post-conversion make it ideal for prolonged mitochondrial dynamics tracking. Its specificity in responding to mitophagy, confirmed by parkin-knockout cells, adds value. When coupled with a custom fluid exchange system, enabling efficient medium changes, precise mitophagy observations become feasible. This mitophagy assay, alongside our methodological insights, can decipher mitophagy's role in pathology and supports drug screening efforts.
自噬和有丝分裂是重要的细胞质量控制系统,调节这两个系统可为危重疾病提供治疗潜力。然而,有限的药物筛选方案阻碍了研究的进展。我们介绍了一种利用光开关荧光报告物 mito-Kaede 来量化有丝分裂通量的新型检测方法。Mito-Kaede 具有优异的紫外线诱导光电转换能力和转换后的亮度,因此非常适合用于长时间的线粒体动态跟踪。经 Parkin 基因敲除细胞证实,它对有丝分裂反应的特异性增加了其价值。如果与定制的液体交换系统配合使用,就能有效地更换培养基,从而实现对有丝分裂的精确观察。这种有丝分裂检测方法与我们的方法论见解相结合,可以破译有丝分裂在病理学中的作用,并为药物筛选工作提供支持。
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引用次数: 0
Using Longitudinal Variance Components Models to Assess Hyper-connectivity in Severe Traumatic Brain Injury Patients. 利用纵向变异成分模型评估严重脑损伤患者的超连接性。
IF 2.2 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-07-01 DOI: 10.32598/bcn.2022.3796.1
Keyvan Olazadeh, Nasrin Borumandnia, Mahin Habibi, Hamid Alavi Majd

Introduction: Traumatic brain injury (TBI) is one of the leading causes of death globally and one of the most important diseases indicated by the World Health Organization (WHO). Several studies have concluded that brain damage can dramatically increase functional connectivity (FC) in the brain. The effects of this hyper-connectivity are not yet fully understood and are being studied by neuroscientists. Accordingly, this study identifies areas of the brain where, after brain injury, an acute increase in FC in such areas is observed.

Methods: The data used in this study were downloaded from the accessible open functional magnetic resonance imaging (fMRI) site. The data included fMRI of 14 patients with severe TBI and 12 healthy individuals. The longitudinal model of variance components investigated the difference between FC in the baseline effect and the longitudinal trend between the TBI and control groups.

Results: After fitting the longitudinal model of variance components, no difference was observed between the FC of the two groups due to the baseline effect. However, in the longitudinal trend of FC, there was a statistically significant difference between the three pairs of cerebellum left, cerebellum right, superior frontal gyrus left, superior frontal gyrus right, thalamus left, and thalamus right in the TBI group compared to the control group.

Conclusion: The results showed that FC was sharply increased in 3 pairs of areas in people with TBI. This hyper-connectivity can affect individuals' cognitive functions, including motor and sensory functions. The exact extent of this effect is unclear and requires further investigation by neuroscientists.

导言:创伤性脑损伤(TBI)是导致全球死亡的主要原因之一,也是世界卫生组织(WHO)指出的最重要的疾病之一。一些研究认为,脑损伤会显著增加大脑的功能连接性(FC)。这种超连接性的影响尚未完全明了,神经科学家正在对其进行研究。因此,本研究确定了大脑中哪些区域在脑损伤后功能连通性会急剧增加:本研究使用的数据是从可访问的开放式功能磁共振成像(fMRI)网站下载的。数据包括 14 名严重创伤性脑损伤患者和 12 名健康人的 fMRI。方差成分纵向模型研究了创伤性脑损伤组和对照组之间基线效应和纵向趋势中FC的差异:结果:在拟合了方差分量纵向模型后,没有观察到基线效应导致的两组 FC 之间的差异。然而,在 FC 的纵向趋势中,创伤性脑损伤组的小脑左侧、小脑右侧、额上回左侧、额上回右侧、丘脑左侧和丘脑右侧三对之间的差异与对照组相比具有统计学意义:结论:研究结果表明,创伤性脑损伤患者的三对区域的FC急剧增加。结论:研究结果表明,创伤性脑损伤患者 3 对区域的 FC 均急剧增加,这种超连接性会影响患者的认知功能,包括运动和感觉功能。这种影响的确切程度尚不清楚,需要神经科学家进一步研究。
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引用次数: 0
Phylobook: a tool for display, clade annotation and extraction of sequences from molecular phylogenies. Phylobook:从分子系统进化论中显示、注释和提取序列的工具。
IF 2.7 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-03 DOI: 10.2144/btn-2023-0056
Jeffrey C Furlong, Peter D Darley, Wenjie Deng, James I Mullins, Roger E Bumgarner
As the volume of sequence data from variable pathogens increases, means of analyzing, annotating and extracting specific taxa for study becomes more difficult. To meet these challenges for datasets with hundreds to thousands of taxa, 'Phylobook' was developed. Starting with a sequence alignment file, Phylobook generates and displays phylogenetic trees adjacent to highlighter plots showing the position of mutations, and allows the user to identify lineages and recombinants, annotate and export selected subsets of sequences for downstream analysis. Accurate lineage assignment, which is difficult to automate, is aided using annotations created by different clustering methods. Phylobook provides web-based display combined with automated clustering and manual editing to allow for expert assessment and correction of lineage assignments and extraction for downstream analysis.
随着来自不同病原体的序列数据量的增加,分析、注释和提取特定类群进行研究的手段变得更加困难。为了应对这些挑战,"Phylobook "应运而生。从序列比对文件开始,Phylobook 生成并显示系统发生树,旁边的荧光笔图显示突变的位置,并允许用户识别世系和重组体,注释和导出选定的序列子集进行下游分析。利用不同聚类方法创建的注释,可以帮助进行难以自动化的准确系谱分配。Phylobook 提供基于网络的显示,结合自动聚类和手动编辑,允许专家评估和校正谱系分配,并提取用于下游分析。
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引用次数: 0
Comparing robotic and manual injection methods in zebrafish embryos for high-throughput RNA silencing using CRISPR-RfxCas13d. 比较使用 CRISPR-RfxCas13d 在斑马鱼胚胎中进行高通量 RNA 沉默的机器人和人工注射方法。
IF 2.5 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-02-29 DOI: 10.2144/btn-2023-0062
Joaquin Abugattas-Nuñez Del Prado, Yi Ding, Jan de Sonneville, Kees-Jan van der Kolk, Miguel A Moreno-Mateos, Edward Málaga-Trillo, Herman P Spaink

In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail (TBXTA) gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for TBXTA. Cas9 protein exhibited a higher percentage of severe phenotypes compared with mRNA, while CasRx protein and mRNA showed similar efficiency. Both robotic and manual injections demonstrated comparable phenotype percentages and mortality rates. The findings highlight the potential of RNA-targeting CRISPR effectors for precise gene knockdown and endorse automated microinjection at a speed of 1.0 s per embryo as a high-throughput alternative to manual methods.

在这项研究中,作者比较了CRISPR-RfxCas13d(CasRx)系统在斑马鱼胚胎中进行mRNA敲除和Cas9介导的DNA靶向的自动机器人和人工注射方法的效率。他们以无尾(TBXTA)基因为靶标,评估了诱导的胚胎表型,并进行了原理验证。Cas9 和 CasRx 系统都导致了 TBXTA 的功能缺失表型。与 mRNA 相比,Cas9 蛋白表现出更高比例的严重表型,而 CasRx 蛋白和 mRNA 表现出相似的效率。机器人注射和人工注射的表型比例和死亡率相当。研究结果凸显了 RNA 靶向 CRISPR 效应器在精确基因敲除方面的潜力,并支持以每个胚胎 1.0 秒的速度进行自动显微注射,作为人工方法的高通量替代方法。
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引用次数: 0
Comparison and optimization of protocols and whole-genome capture conditions for ancient DNA samples. 比较和优化古代 DNA 样本的方案和全基因组捕获条件。
IF 2.5 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-03-26 DOI: 10.2144/btn-2023-0107
Reyhan Yaka, Maja Krzewińska, Vendela Kempe Lagerholm, Anna Linderholm, Füsun Özer, Mehmet Somel, Anders Götherström

Ancient DNA (aDNA) obtained from human remains is typically fragmented and present in relatively low amounts. Here we investigate a set of optimal methods for producing aDNA data by comparing silica-based DNA extraction and aDNA library preparation protocols. We also test the efficiency of whole-genome enrichment (WGC) on ancient human samples by modifying a number of parameter combinations. We find that the Dabney extraction protocol performs significantly better than alternatives. We further observed a positive trend with the BEST library protocol indicating lower clonality. Notably, our results suggest that WGC is effective at retrieving endogenous DNA, particularly from poorly-preserved human samples, by increasing human endogenous proportions by 5x. Thus, aDNA studies will be most likely to benefit from our results.

从人类遗骸中获取的古 DNA(aDNA)通常是片段化的,且含量相对较低。在这里,我们通过比较硅基 DNA 提取和 aDNA 文库制备方案,研究了一套生成 aDNA 数据的最佳方法。我们还通过修改一些参数组合来测试古人类样本的全基因组富集(WGC)效率。我们发现,Dabney 提取方案的性能明显优于其他方案。我们进一步观察到,BEST 文库方案的阳性趋势表明克隆性较低。值得注意的是,我们的研究结果表明,WGC 能有效地提取内源性 DNA,尤其是从保存较差的人类样本中提取,能将人类内源性比例提高 5 倍。因此,aDNA 研究最有可能从我们的结果中获益。
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引用次数: 0
A free and user-friendly software protocol for the quantification of microfauna swimming behavior. 用于量化微型动物游泳行为的免费且用户友好的软件协议。
IF 2.5 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-02-29 DOI: 10.2144/btn-2024-0001
Colleen M McMaken, Kristin E Gribble

Characterizing swimming behavior can provide a holistic assessment of the health, physiology and ecology of microfaunal species when done in conjunction with measuring other biological parameters. However, tracking and quantifying microfauna swimming behavior using existing automated tools is often difficult due to the animals' small size or transparency, or because of the high cost, expertise, or labor needed for the analysis. To address these issues, we created a cost-effective, user-friendly protocol for behavior analysis that employs the free software packages HitFilm and ToxTrac along with the R package 'trajr' and used the method to quantify the behavior of rotifers. This protocol can be used for other microfaunal species for which investigators may face similar issues in obtaining measurements of swimming behavior.

在测量其他生物参数的同时,游泳行为的特征描述可以对微小动物的健康、生理和生态进行整体评估。然而,由于动物体型小或透明度低,或者由于分析所需的成本、专业知识或劳动力较高,使用现有的自动化工具跟踪和量化微型动物的游泳行为通常比较困难。为了解决这些问题,我们创建了一种成本效益高、用户友好的行为分析方案,该方案采用了免费的软件包 HitFilm 和 ToxTrac 以及 R 软件包 "trajr",并使用该方法量化了轮虫的行为。该方案可用于其他微动物物种,研究人员在获取游泳行为测量结果时可能会遇到类似的问题。
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引用次数: 0
Isolation and characterization of Paenibacillus peoriae JC-3jx from Dendrobium nobile. 从金钗石斛中分离并鉴定 peoriae JC-3jx Paenibacillus。
IF 2.5 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-03-12 DOI: 10.2144/btn-2023-0083
ZhiPing Li, Yuan Lin, FeiFei Song, RuoNan Zheng, QinGeng Huang

Dendrobium is a rich source of high-value natural components. Endophytic fungi are well studied, yet bacteria research is limited. In this study, endophytic bacteria from Dendrobium nobile were isolated using an improved method, showing inhibition of pathogens and growth promotion. JC-3jx, identified as Paenibacillus peoriae, exhibited significant inhibitory activity against tested fungi and bacteria, including Escherichia coli. JC-3jx also promoted corn seed rooting and Dendrobium growth, highlighting its excellent biocontrol and growth-promoting potential.

铁皮石斛是高价值天然成分的丰富来源。对内生真菌的研究很多,但对细菌的研究却很有限。在这项研究中,使用改进的方法从金钗石斛中分离出了内生细菌,显示出抑制病原体和促进生长的作用。JC-3jx被鉴定为Paenibacillus peoriae,对测试的真菌和细菌(包括大肠杆菌)具有显著的抑制活性。JC-3jx 还能促进玉米种子生根和铁皮石斛的生长,凸显了其卓越的生物防治和生长促进潜力。
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引用次数: 0
Extraterrestrial agriculture: plant cultivation in space. 地外农业:太空植物栽培。
IF 2.7 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-04-11 DOI: 10.2144/btn-2024-0033
Beatrice Bowlby
[Formula: see text] Researchers are using various techniques and technologies to study how plants grow in extraterrestrial conditions with the hopes of sustaining longer missions for exploring deep space as well as being able to one day cultivate crops on other planets.
[公式:见正文] 研究人员正在利用各种技术和工艺来研究植物如何在地外条件下生长,希望能够维持更长时间的深空探索任务,以及有朝一日能够在其他星球上种植作物。
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引用次数: 0
Development and evaluation of a single-tube nested PCR with colorimetric assay for Mycobacterium tuberculosis detection. 开发和评估用于检测结核分枝杆菌的单管巢式 PCR 比色法。
IF 2.7 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-04-11 DOI: 10.2144/btn-2023-0080
Marcela Pereira Salazar, Juliana Figueiredo da Costa Lima Suassuna Monteiro, Wlisses Henrique Veloso Carvalho-Silva, George Tadeu Nunes Diniz, Roberto Pereira Werkhauser, Lílian Maria Lapa Montenegro, Haiana Charifker Schindler
Molecular techniques have revolutionized tuberculosis (TB) diagnosis by offering a faster and more sensitive approach, detecting Mycobacterium tuberculosis (Mtb) DNA directly from samples. Single-tube nested PCR (STNPCR) combines two PCR reactions with separate oligonucleotide sets in a single tube. Moreover, colorimetric methods in PCR products have been studied for pathogen detection. Thus, this study aimed to establish a novel system based on colorimetric STNPCR for Mtb detection using microtiter plates with IS6110-amplified fragments. The results showed a general colorimetric STNPCR detection limit of 1 pg/μl. Its general sensitivity and specificity were 76.62 and 60.53%, respectively, with kappa index agreement of 0.166.
分子技术提供了一种更快、更灵敏的方法,可直接从样本中检测结核分枝杆菌(Mtb)DNA,从而彻底改变了结核病(TB)诊断方法。单管嵌套 PCR (STNPCR) 将两个 PCR 反应与不同的寡核苷酸组合在一个试管中。此外,还研究了用于病原体检测的 PCR 产物比色法。因此,本研究旨在建立一种基于比色 STNPCR 的新型系统,使用微孔板和 IS6110 扩增片段检测 Mtb。结果显示,比色 STNPCR 的一般检测限为 1 pg/μl。其一般灵敏度和特异性分别为 76.62% 和 60.53%,卡帕指数一致度为 0.166。
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引用次数: 0
Fully in vitro iterative construction of a 24 kb-long artificial DNA sequence to store digital information. 完全体外迭代构建 24 kb 长的人工 DNA 序列,用于存储数字信息。
IF 2.7 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-04-04 DOI: 10.2144/btn-2023-0109
Julien Leblanc, Olivier Boulle, Emeline Roux, Jacques Nicolas, Dominique Lavenier, Yann Audic
In the absence of a DNA template, the ab initio production of long double-stranded DNA molecules of predefined sequences is particularly challenging. The DNA synthesis step remains a bottleneck for many applications such as functional assessment of ancestral genes, analysis of alternative splicing or DNA-based data storage. In this report we propose a fully in vitro protocol to generate very long double-stranded DNA molecules starting from commercially available short DNA blocks in less than 3 days using Golden Gate assembly. This innovative application allowed us to streamline the process to produce a 24 kb-long DNA molecule storing part of the Declaration of the Rights of Man and of the Citizen of 1789 . The DNA molecule produced can be readily cloned into a suitable host/vector system for amplification and selection.
在没有 DNA 模板的情况下,从头开始生产具有预定序列的长双链 DNA 分子尤其具有挑战性。DNA 合成步骤仍然是许多应用的瓶颈,如祖先基因的功能评估、替代剪接分析或基于 DNA 的数据存储。在本报告中,我们提出了一种完全体外的方案,利用黄金门组装技术,从市售的短 DNA 块开始,在不到 3 天的时间内生成超长的双链 DNA 分子。这一创新应用使我们能够简化流程,生成一个 24 kb 长的 DNA 分子,其中存储了 1789 年《人权和公民权利宣言》的部分内容。生成的 DNA 分子可以很容易地克隆到合适的宿主/载体系统中进行扩增和选择。
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引用次数: 0
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BioTechniques
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