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Emerging models of human and non-human primate placental development - Centre for Trophoblast Research 17th annual meeting 2024. 人类和非人灵长类胎盘发育的新兴模型 - 滋养细胞研究中心第 17 届年会 2024。
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-11-28 DOI: 10.1242/bio.061774
Irving L M H Aye

The 17th annual meeting of the Centre for Trophoblast Research (CTR) took place at the University of Cambridge, UK, on 1-2 July 2024. This year's meeting provided an opportunity to reflect on the significant advancements made recently in modelling the human placenta in vitro. The meeting featured 12 invited speakers and attracted 260 participants from 25 countries. Many of the speakers were leading figures who have developed methods to derive human trophoblast stem cells or organoids from first trimester and term placentas, and from pluripotent stem cells. Accompanying the invited presentations were flash talks selected from the abstract submissions and poster presentations. The meeting concluded with a stimulating panel discussion to evaluate the current human trophoblast models. This Meeting Review aims to capture the spirit of the event and highlight the key themes and take-home messages that emerged.

滋养层研究中心(CTR)第17届年会于2024年7月1-2日在英国剑桥大学举行。今年的会议提供了一个机会,以反思最近在人类胎盘体外建模方面取得的重大进展。会议邀请了 12 位发言人,吸引了来自 25 个国家的 260 名与会者。许多发言人都是开发出从妊娠头三个月和足月胎盘以及多能干细胞中提取人类滋养层干细胞或器官组织的方法的领军人物。在特邀报告的同时,还举行了从提交的摘要和海报中挑选出来的快闪讲座。会议最后进行了小组讨论,对目前的人类滋养细胞模型进行了评估。本会议回顾旨在总结本次会议的精神,并强调会议的关键主题和收获信息。
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引用次数: 0
Disrupting the interaction between AMBRA1 and DLC1 prevents apoptosis while enhancing autophagy and mitophagy. 破坏 AMBRA1 和 DLC1 之间的相互作用可防止细胞凋亡,同时增强自噬和有丝分裂。
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-11-26 DOI: 10.1242/bio.060380
Kate Hawkins, Meg Watt, Sébastien Gillotin, Maya Hanspal, Martin Helley, Jill Richardson, Nicola Corbett, Janet Brownlees

AMBRA1 has critical roles in autophagy, mitophagy, cell cycle regulation, neurogenesis and apoptosis. Dysregulation of these processes are hallmarks of various neurodegenerative diseases and therefore AMBRA1 represents a potential therapeutic target. The flexibility of its intrinsically disordered regions allows AMBRA1 to undergo conformational changes and thus to perform its function as an adaptor protein for various different complexes. Understanding the relevance of these multiple protein-protein interactions will allow us to gain information about which to target pharmacologically. To compare potential AMBRA1 activation strategies, we have designed and validated several previously described mutant constructs in addition to characterising their effects on proliferation, apoptosis, autophagy and mitophagy in SHSY5Y cells. AMBRA1TAT, which is a mutant form of AMBRA1 that cannot interact with DLC1 at the microtubules, produced the most promising results. Overexpression of this mutant protected cells against apoptosis and induced autophagy/mitophagy in SHSY5Y cells in addition to enhancing the switch from quiescence to proliferation in mouse neural stem cells. Future studies should focus on designing compounds that inhibit the protein-protein interaction between AMBRA1/DLC1 and thus have potential to be used as a drug strategy for neurodegeneration.

AMBRA1 在自噬、有丝分裂、细胞周期调控、神经发生和细胞凋亡中发挥着关键作用。这些过程的失调是各种神经退行性疾病的特征,因此 AMBRA1 是一个潜在的治疗靶点。AMBRA1 固有无序区的灵活性使其能够发生构象变化,从而发挥其作为各种不同复合物的适配蛋白的功能。了解这些多重蛋白-蛋白相互作用的相关性将使我们能够获得有关哪些蛋白可以作为药理靶点的信息。为了比较潜在的 AMBRA1 激活策略,我们设计并验证了几种以前描述过的突变构建物,并鉴定了它们对 SHSY5Y 细胞的增殖、凋亡、自噬和有丝分裂的影响。AMBRA1TAT是AMBRA1的一种突变形式,它不能与微管上的DLC1相互作用。过量表达这种突变体可保护细胞免受凋亡,并诱导 SHSY5Y 细胞的自噬/半自噬,此外还能增强小鼠 NSCs 从静止到增殖的转换。未来的研究应侧重于设计能抑制AMBRA1/DLC1之间蛋白-蛋白相互作用的化合物,从而有可能将其用作治疗神经退行性变的药物策略。
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引用次数: 0
Knee position affects medial gastrocnemius and soleus activation during dynamic plantarflexion: no evidence for an inter-muscle compensation in healthy young adults. 在动态跖屈时,膝关节位置影响内侧腓骨肌和比目鱼肌的激活:在健康年轻人中没有肌间代偿的证据。
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-12-30 DOI: 10.1242/bio.061810
Bálint Kovács, Dániel Csala, Song Yang, József Tihanyi, Yaodong Gu, Tibor Hortobágyi

Knee joint position influences ankle torque, but it is unclear whether the soleus compensates to counteract the reductions in gastrocnemius output during knee-flexed versus knee-extended plantarflexions. Therefore, the purpose of this study was to determine the effects of knee joint position and plantarflexion contraction velocity on ankle plantarflexion torque and electromyography activity of the medial gastrocnemius and soleus in healthy young adults. Healthy male participants (n=30) performed concentric plantar flexions in a custom-built dynamometer from 15° dorsiflexion to 30° plantarflexion at gradually increasing velocities during each contraction at 30, 60, 120, 180, and 210° s-1 in a supine position with the knee fully extended and while kneeling with the knee fixed in 90° flexion. Two 16-channel linear electromyographic (EMG) arrays were placed over the medial gastrocnemius and soleus muscles. Plantarflexion torque during flexed-knee versus extended-knee plantarflexions was 31% lower (P=0.002) averaged across the five contraction velocities. The overall EMG activity of the medial gastrocnemius was 35% lower (P=0.002) during knee-flexed versus knee-extended plantarflexions. In the first half of plantarflexions at slower contractions, soleus EMG activity was 15% and 28% higher (both P=0.002) in knee-flexed versus knee-extended plantarflexion, respectively. We conclude that knee position affects medial gastrocnemius and soleus activation during dynamic plantarflexion, with plantarflexion torque being smaller in the knee-flexed versus knee-extended position. However, we found no evidence that changes in soleus activation would compensate for the decrease in medial gastrocnemius activation.

膝关节位置影响踝关节扭矩,但目前尚不清楚比目鱼肌是否在膝关节屈曲和膝关节伸展跖屈时补偿腓肠肌输出的减少。因此,本研究的目的是确定健康年轻人膝关节位置和跖屈收缩速度对踝关节跖屈扭矩和腓骨内侧肌和比目鱼肌肌电活动的影响。健康男性参与者(n=30)在定制的测力计中从15°背屈到30°跖屈,每次收缩时速度逐渐增加,分别为30、60、120、180和210°s-1,仰卧位,膝盖完全伸展,膝盖固定为90°屈,同时跪下。在腓肠肌内侧和比目鱼肌上放置两个16通道线性肌电图(EMG)阵列。在五个收缩速度中,膝关节屈曲时的跖屈扭矩比膝关节伸直时的跖屈扭矩低31% (P=0.002)。膝屈与膝伸跖屈时,腓肠肌内侧的整体肌电活动降低35% (P=0.002)。在较慢收缩的跖屈的前半部分,膝关节屈曲和膝关节伸展的跖屈分别比比目鱼肌肌电活动高15%和28% (P均=0.002)。我们得出结论,在动态跖屈时,膝关节位置影响腓骨内侧肌和比目鱼肌的激活,膝关节屈曲时的跖屈扭矩比膝关节伸直时小。然而,我们没有发现证据表明比目鱼肌激活的变化可以弥补内侧腓肠肌激活的减少。
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引用次数: 0
Effect of cigarette smoke on the proliferation, viability, gene expression, and cellular functions of adipose-derived mesenchymal stem cells from smoking and non-smoking donors. 吸烟对吸烟和不吸烟供体脂肪源性间充质干细胞增殖、活力、基因表达和细胞功能的影响
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-12-03 DOI: 10.1242/bio.061665
Bareqa Salah, Diana Shahin, Momen Sarhan, Joud Al-Karmi, Ban Al-Kurdi, Renata Al-Atoom, Mohammad A Ismail, Nouran Hammad, Hanan Jafar, Abdalla Awidi, Nidaa A Ababneh

Cigarette smoking negatively impacts mesenchymal stem cell functionality, including proliferation, viability, and differentiation potential. Adipose-derived mesenchymal stem cells (ADMSCs) are increasingly used for therapeutic purposes, but the specific effects of smoking in vivo on these cells are poorly understood. This study investigates the effects of cigarette smoke on the proliferation, viability, gene expression, and cellular functions of ADMSCs from smoking and non-smoking donors. In this study, ADMSCs were isolated from healthy smokers and non-smokers, and cell proliferation was assessed using the MTT assay, viability with apoptosis assays, mitochondrial membrane potential (MMP), and gene expression related to oxidative stress and cellular functions. Cell cycle analysis was also conducted. Our findings reveal a significant decrease in the proliferation of ADMSCs from smokers. Apoptosis assays showed reduced viable cells in smokers without a significant change in MMP, suggesting alternative pathways contributing to decreased viability. Gene expression analysis indicated the upregulation of genes associated with oxidative stress response and cellular defense mechanisms and the downregulation of genes related to inflammatory signaling, detoxification, and cellular metabolism. Cell cycle analysis indicates cycle arrest or delay in smokers, possibly due to stress and potential DNA damage. Smoking negatively affects ADMSCs' proliferation, viability, and function through oxidative stress and gene expression alterations. These findings highlight the importance of considering smoking status in ADMSC therapies and the need for further research to mitigate the effect of smoking on stem cells.

吸烟对间充质干细胞的功能有负面影响,包括增殖、活力和分化潜力。脂肪源性间充质干细胞(ADMSCs)越来越多地用于治疗目的,但体内吸烟对这些细胞的具体影响尚不清楚。本研究探讨了吸烟和不吸烟供体对ADMSCs增殖、活力、基因表达和细胞功能的影响。在这项研究中,从健康吸烟者和非吸烟者中分离出ADMSCs,并使用MTT法、凋亡活性法、线粒体膜电位(MMP)和与氧化应激和细胞功能相关的基因表达来评估细胞增殖。同时进行细胞周期分析。我们的研究结果显示吸烟者ADMSCs的增殖显著减少。细胞凋亡实验显示,吸烟者的活细胞减少,但MMP没有显著变化,这表明有其他途径导致了活细胞减少。基因表达分析显示,与氧化应激反应和细胞防御机制相关的基因上调,与炎症信号、解毒和细胞代谢相关的基因下调。细胞周期分析表明吸烟者的周期阻滞或延迟,可能是由于压力和潜在的DNA损伤。吸烟通过氧化应激和基因表达改变对ADMSCs的增殖、活力和功能产生负面影响。这些发现强调了在ADMSC治疗中考虑吸烟状况的重要性,以及进一步研究减轻吸烟对干细胞影响的必要性。
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引用次数: 0
Patient-matched tumours, plasma, and cell lines reveal tumour microenvironment- and cell culture-specific microRNAs. 患者匹配的肿瘤、血浆和细胞系显示肿瘤微环境和细胞培养特异性microrna。
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-12-23 DOI: 10.1242/bio.060483
Latasha Ludwig, Emma N Vanderboon, Heather Treleaven, R Darren Wood, Courtney R Schott, Geoffrey A Wood

MicroRNAs (miRNAs) are small non-coding RNA molecules that are present in all cell types and bodily fluids and are commonly dysregulated in cancer. miRNAs in cancer have been studied by measuring levels in cell lines, tumour tissues, and in circulation; however, no study has specifically investigated miRNA expression in patient-matched samples across all three sample types. Canine osteosarcoma is a well-established spontaneously occurring model of human osteosarcoma for which matched samples are available. We analysed a panel of miRNAs by real-time quantitative PCR and compared across patients and sample types. While some miRNAs are highly expressed in all three sample types, tumour tissue and cell lines had the most in common. There were several miRNAs that were highly expressed in plasma and tumour tissue but not in cell lines and likely represent miRNAs produced in the tumour microenvironment. Two highly expressed miRNAs were exclusive to plasma and are known to be expressed in circulating cells. This study highlights the importance of considering sample type when studying miRNAs in cancer and demonstrates the power of using patient-matched samples.

MicroRNAs (miRNAs)是存在于所有细胞类型和体液中的小的非编码RNA分子,在癌症中通常是失调的。通过测量细胞系、肿瘤组织和血液循环中的mirna水平,研究了癌症中的mirna;然而,没有研究专门调查了所有三种样本类型的患者匹配样本中的miRNA表达。犬骨肉瘤是一种完善的自发发生的人类骨肉瘤模型,其匹配的样本是可用的。我们通过实时定量PCR分析了一组mirna,并在患者和样本类型之间进行了比较。虽然一些mirna在所有三种样品类型中都高度表达,但肿瘤组织和细胞系的共同点最多。有几种mirna在血浆和肿瘤组织中高表达,但在细胞系中不表达,可能代表肿瘤微环境中产生的mirna。两种高表达的mirna仅存在于血浆中,并且已知在循环细胞中表达。这项研究强调了在研究癌症中的mirna时考虑样本类型的重要性,并展示了使用患者匹配样本的力量。
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引用次数: 0
Early-life challenge enhances cortisol regulation in zebrafish larvae. 生命早期的挑战会增强斑马鱼幼体的皮质醇调节能力。
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-11-28 DOI: 10.1242/bio.061684
Luis A Castillo-Ramírez, Ulrich Herget, Soojin Ryu, Rodrigo J De Marco

The hypothalamic-pituitary-adrenal (HPA) axis in mammals and the hypothalamic-pituitary-interrenal (HPI) axis in fish are open systems that adapt to the environment during development. Little is known about how this adaptation begins and regulates early stress responses. We used larval zebrafish to examine the impact of prolonged forced swimming at 5 days post-fertilization (dpf), termed early-life challenge (ELC), on cortisol responses, neuropeptide expression in the nucleus preopticus (NPO), and gene transcript levels. At 6 dpf, ELC-exposed larvae showed normal baseline cortisol but reduced reactivity to an initial stressor. Conversely, they showed increased reactivity to a second stressor within the 30-min refractory period, when cortisol responses are typically suppressed. ELC larvae had fewer corticotropin-releasing hormone (crh), arginine vasopressin (avp), and oxytocin (oxt)-positive cells in the NPO, with reduced crh and avp co-expression. Gene expression analysis revealed upregulation of genes related to cortisol metabolism (hsd11b2, cyp11c1), steroidogenesis (star), and stress modulation (crh, avp, oxt). These results suggest that early environmental challenge initiates adaptive plasticity in the HPI axis, tuning cortisol regulation to balance responsiveness and protection during repeated stress. Future studies should explore the broader physiological effects of prolonged forced swimming and its long-term impact on cortisol regulation and stress-related circuits.

哺乳动物的下丘脑-垂体-肾上腺(HPA)轴和鱼类的下丘脑-垂体-肾上腺(HPI)轴是开放系统,在发育过程中适应环境。人们对这种适应如何开始并调节早期应激反应知之甚少。我们利用斑马鱼幼体研究了受精后 5 天(dpf)长时间强迫游泳(称为早期生命挑战(ELC))对皮质醇反应、视前核(NPO)中神经肽表达和基因转录水平的影响。6 dpf时,暴露于ELC的幼虫皮质醇基线正常,但对初始应激源的反应性降低。相反,在皮质醇反应通常受到抑制的 30 分钟难抑期内,它们对第二种应激源的反应性有所提高。ELC幼虫在NPO中的促肾上腺皮质激素释放激素(crh)、精氨酸加压素(avp)和催产素(otocin)阳性细胞较少,crh和avp共表达减少。基因表达分析表明,与皮质醇代谢(hsd11b2、cyp11c1)、类固醇生成(star)和应激调节(crh、avp、otx)有关的基因上调。这些结果表明,早期环境挑战会启动 HPI 轴的适应性可塑性,调整皮质醇调节,从而在反复应激过程中平衡反应性和保护性。未来的研究应探索长时间强迫游泳的广泛生理效应及其对皮质醇调节和应激相关回路的长期影响。
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引用次数: 0
EFEMP1 contributes to light-dependent ocular growth in zebrafish. EFEMP1有助于斑马鱼依赖光的眼球生长。
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-11-28 DOI: 10.1242/bio.061741
Jiaheng Xie, Bang V Bui, Patrick T Goodbourn, Patricia R Jusuf

Myopia (short-sightedness) is the most common ocular disorder. It generally develops after over-exposure to aberrant visual environments, disrupting emmetropization mechanisms that should match eye growth with optical power. A pre-screening of strongly associated myopia-risk genes identified through human genome-wide association studies implicates efemp1 in myopia development, but how this gene impacts ocular growth remains unclear. Here, we modify efemp1 expression specifically in the retina of zebrafish. We found that under normal lighting, efemp1 mutants developed axial myopia, enlarged eyes, reduced spatial vision and altered retinal function. However, under myopia-inducing dark-rearing, compared to control fish, mutants remained emmetropic and showed changes in retinal function. Efemp1 modification changed the expression of efemp1, egr1, tgfb1a, vegfab and rbp3 genes in the eye, and changed the inner retinal distributions of myopia-associated EFEMP1, TIMP2 and MMP2 proteins. Efemp1 modification also impacted dark-rearing-induced responses of vegfab and wnt2b genes and above-mentioned myopia-associated proteins. Together, we provided robust evidence that light-dependent ocular growth is regulated by efemp1.

近视(近视眼)是最常见的眼部疾病。近视一般是由于过度暴露于异常的视觉环境,破坏了本应使眼睛生长与光学能力相匹配的屈光机制而形成的。通过人类全基因组关联研究确定的与近视风险密切相关的基因预筛选发现,efemp1 与近视的形成有关,但该基因如何影响眼球生长仍不清楚。在这里,我们特异性地改变了斑马鱼视网膜中 efemp1 的表达。我们发现,在正常光照条件下,efemp1突变体会出现轴性近视、眼球增大、空间视觉减弱和视网膜功能改变。然而,在诱发近视的黑暗饲养条件下,与对照组相比,突变体仍具有向光性,视网膜功能也发生了变化。Efemp1修饰改变了眼睛中efemp1、egr1、tgfb1a、vegfab和rbp3基因的表达,并改变了与近视相关的EFEMP1、TIMP2和MMP2蛋白在视网膜内部的分布。Efemp1的修饰还影响了黑暗后退诱导的vegfab和wnt2b基因以及上述近视相关蛋白的反应。总之,我们提供了强有力的证据,证明光依赖性眼球生长受efemp1调控。
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引用次数: 0
Age- and oxidative stress-induced centrosome amplification and renal stones in Drosophila Malpighian tubules. 年龄和氧化应激诱导的果蝇马氏小管中心体扩增和肾结石。
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-12-16 DOI: 10.1242/bio.061743
Hyun-Jin Na, Mi-Jeong Sung, Joung-Sun Park

Renal diseases, including cancer, are rapidly increasing worldwide, driven by rising temperatures and changing diets, especially among younger people. Renal stones, a major risk for chronic renal disease, are increasingly common due to various health issues. Research on the underlying mechanisms, drug discovery, and the effects of aging and stress is limited. We used Drosophila, due to its similarity to the human renal system and ease of use, to identify cancer hallmarks and renal stone formation related to aging and oxidative stress. Our results indicate that centrosome amplification and stone formation increase with age and oxidative stress, and high sucrose feeding also heightens stone formation in the renal system. Our results show a close relationship between these diseases and aging, reactive oxygen species (ROS) stress, and chronic diseases. We suggest that the Drosophila renal model could be a powerful tool to study the relationship between age and age-related diseases and to discovering new agents for nephropathy.

在气温上升和饮食变化的推动下,包括癌症在内的肾脏疾病在全球范围内迅速增加,尤其是在年轻人中。肾结石是慢性肾脏疾病的主要危险因素,由于各种健康问题,肾结石越来越常见。对潜在机制、药物发现以及衰老和压力的影响的研究是有限的。由于果蝇与人类肾脏系统相似且易于使用,我们使用果蝇来识别与衰老和氧化应激相关的癌症标志和肾结石形成。我们的研究结果表明,中心体扩增和结石形成随着年龄和氧化应激的增加而增加,高蔗糖喂养也会增加肾脏系统的结石形成。我们的研究结果表明,这些疾病与衰老、活性氧(ROS)应激和慢性疾病密切相关。我们认为果蝇肾脏模型可以成为研究年龄和年龄相关疾病之间关系以及发现肾病新药物的有力工具。
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引用次数: 0
Elemental stoichiometry and insect chill tolerance: evolved and plastic changes in organismal Na+ and K+ content in Drosophila. 元素化学计量学和昆虫抗寒性:果蝇有机Na+和K+含量的进化和塑性变化。
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-12-30 DOI: 10.1242/bio.060597
Sarah C Chalmer, Seth M Rudman, Mads K Andersen, Paul Schmidt, Heath A MacMillan

Acclimation and evolutionary adaptation can produce phenotypic changes that allow organisms to cope with challenges. Determining the relative contributions and the underlying mechanisms driving phenotypic shifts from acclimation and adaptation is of central importance to understanding animal responses to change. Rates of evolution have traditionally been considered slow relative to ecological processes that shape biodiversity. Many organisms nonetheless show patterns of genetic variation that suggest that adaptation may act sufficiently fast to allow continuous change in phenotypes in response to environmental change (called 'adaptive tracking'). In Drosophila, both plastic and evolved differences in chill tolerance are associated with ionoregulation. Here, we combine an acclimation experiment, field collections along a well-characterized latitudinal cline, and a replicated field experiment to assess the concordance in the direction, magnitude, and potential mechanisms of acclimation and adaptation on chill coma recovery and elemental (Na and K) stoichiometry in both sexes of Drosophila melanogaster. Acclimation strongly shaped chill coma recovery, spatial adaptation produced comparatively modest effects, and temporal adaptation had no significant effect. Leveraging knowledge on the mechanisms underlying variation in chill tolerance traits, we find that relationships between elemental stoichiometry and chill coma recovery in the context of acclimation may differ from those that are associated with spatial adaptive change.

驯化和进化适应可以产生表型变化,使生物体能够应对挑战。确定从驯化和适应中驱动表型转变的相关贡献和潜在机制对于理解动物对变化的反应至关重要。与形成生物多样性的生态过程相比,进化的速度历来被认为是缓慢的。然而,许多生物体表现出的遗传变异模式表明,适应可能足够快,从而允许表型随着环境变化而持续变化(称为“适应性跟踪”)。在果蝇中,可塑性和耐寒性的进化差异都与离子调节有关。在此,我们结合驯化实验、沿特定纬度梯度的野外采集和重复的野外实验,评估了驯化和适应对黑腹果蝇两性冷醒恢复和元素(Na和K)化学计量的方向、幅度和潜在机制的一致性。驯化对冷昏迷恢复的影响较大,空间适应对冷昏迷恢复的影响较小,时间适应对冷昏迷恢复的影响不显著。利用对耐寒性状变异机制的了解,我们发现在驯化背景下,元素化学计量与冷昏迷恢复之间的关系可能不同于与空间适应性变化相关的关系。
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引用次数: 0
Spectral scanning and fluorescence lifetime imaging microscopy (FLIM) enable separation and characterization of C. elegans autofluorescence in the cuticle and gut. 光谱扫描和荧光寿命成像显微镜(FLIM)可以分离和表征秀丽隐杆线虫角质层和肠道中的自身荧光。
IF 1.8 4区 生物学 Q3 BIOLOGY Pub Date : 2024-12-15 Epub Date: 2024-12-30 DOI: 10.1242/bio.060613
Heino J Hulsey-Vincent, Elizabeth A Cameron, Caroline L Dahlberg, Domenico F Galati

Caenorhabditis elegans gut and cuticle produce a disruptive amount of autofluorescence during imaging. Although C. elegans autofluorescence has been characterized, it has not been characterized at high resolution using both spectral and fluorescence lifetime-based approaches. We performed high resolution spectral scans of whole, living animals to characterize autofluorescence of adult C. elegans. By scanning animals at 405 nm, 473 nm, 561 nm, and 647 nm excitations, we produced spectral profiles that confirm the brightest autofluorescence has a clear spectral overlap with the emission of green fluorescent protein (GFP). We then used fluorescence lifetime imaging microscopy (FLIM) to further characterize autofluorescence in the cuticle and the gut. Using FLIM, we were able to isolate and quantify dim GFP signal within the sensory cilia of a single pair of neurons that is often obscured by cuticle autofluorescence. In the gut, we found distinct spectral populations of autofluorescence that could be excited by 405 nm and 473 nm lasers. Further, we found lifetime differences between subregions of this autofluorescence when stimulated at 473 nm. Our results suggest that FLIM can be used to differentiate biochemically unique populations of gut autofluorescence without labeling. Further studies involving C. elegans may benefit from combining high resolution spectral and lifetime imaging to isolate fluorescent protein signal that is mixed with background autofluorescence and to perform useful characterization of subcellular structures in a label-free manner.

秀丽隐杆线虫的肠道和角质层在成像过程中产生破坏性的自身荧光。虽然秀丽隐杆线虫的自身荧光已经被表征,但它还没有在高分辨率下使用光谱和荧光寿命为基础的方法进行表征。我们对整个活体动物进行了高分辨率的光谱扫描,以表征成年秀丽隐杆线虫的自身荧光。通过在405 nm、473 nm、561 nm和647 nm的激发下扫描动物,我们得到了光谱曲线,证实了最亮的自身荧光与绿色荧光蛋白(GFP)的发射有明显的光谱重叠。然后,我们使用荧光寿命成像显微镜(FLIM)进一步表征角质层和肠道中的自身荧光。使用FLIM,我们能够在一对神经元的感觉纤毛中分离和量化微弱的GFP信号,这些信号通常被角质层自身荧光所掩盖。在肠道中,我们发现了不同光谱的自体荧光,可以被405 nm和473 nm的激光激发。此外,我们发现在473nm刺激下,该自身荧光的亚区之间存在寿命差异。我们的研究结果表明,FLIM可以用来区分生物化学上独特的肠道自身荧光群体,而无需标记。对秀丽隐杆线虫的进一步研究可能受益于结合高分辨率光谱和寿命成像来分离与背景自身荧光混合的荧光蛋白信号,并以无标记的方式对亚细胞结构进行有用的表征。
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引用次数: 0
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