gene reporters
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Small Methods ◽  
2020 ◽  
Vol 4 (12) ◽  
pp. 2000491
Author(s):  
Arshad Ahmed Padhiar ◽  
Abdullah Faqeer ◽  
Shimin Sun ◽  
Md Rana Hamid ◽  
Jinqi Liao ◽  
...  

ACS Nano ◽  
2019 ◽  
Vol 13 (7) ◽  
pp. 8114-8123 ◽  
Author(s):  
Felix Sigmund ◽  
Susanne Pettinger ◽  
Massimo Kube ◽  
Fabian Schneider ◽  
Martina Schifferer ◽  
...  

2019 ◽  
Author(s):  
Felix Sigmund ◽  
Susanne Pettinger ◽  
Massimo Kube ◽  
Fabian Schneider ◽  
Martina Schifferer ◽  
...  

Multi-colored gene reporters such as fluorescent proteins are indispensable for biomedical research, but equivalent tools for electron microscopy (EM), a gold standard for deciphering mechanistic details of cellular processes1,2and uncovering the network architecture of cell-circuits3,4, are still sparse and not easily multiplexable. Semi-genetic EM reporters are based on the precipitation of exogenous chemicals5–9which may limit spatial precision and tissue penetration and can affect ultrastructure due to fixation and permeabilization. The latter technical constraints also affect EM immunolabeling techniques10–13which may furthermore be complicated by limited epitope accessibility. The fully genetic iron storage protein ferritin generates contrast via its electron-dense iron core14–16, but its small size complicates differentiation of individual ferritin particles from cellular structures. To enable multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM), we here introduce the encapsulin system ofQuasibacillus thermotolerans(Qt) as a fully genetic iron-biomineralizing nanocompartment. We reveal by cryo-electron reconstructions that the Qt monomers (QtEnc) self-assemble to nanospheres with T=4 icosahedral symmetry and an ~44 nm diameter harboring two putative pore regions at the fivefold and threefold axes. We furthermore show that the native cargo (QtlMEF) auto-targets to the inner surface of QtEnc and exhibits ferroxidase activity leading to efficient iron sequestration inside mammalian cells. We then demonstrate that QtEnc can be robustly differentiated from the non-intermixing encapsulin ofMyxococcus xanthus17(Mx, ~32 nm) via a deep-learning model, thus enabling automated multiplexed EM gene reporter imaging in mammalian cells.


2006 ◽  
Vol 25 (2) ◽  
pp. 242-249 ◽  
Author(s):  
Freddy J. Troost ◽  
Robert-Jan M. Brummer ◽  
Guido R. M. M. Haenen ◽  
Aalt Bast ◽  
Rachel I. van Haaften ◽  
...  

Iron-induced oxidative stress in the small intestine may alter gene expression in the intestinal mucosa. The present study aimed to determine which genes are mediated by an iron-induced oxidative challenge in the human small intestine. Eight healthy volunteers [22 yr(SD2)] were tested on two separate occasions in a randomized crossover design. After duodenal tissue sampling by gastroduodenoscopy, a perfusion catheter was inserted orogastrically to perfuse a 40-cm segment of the proximal small intestine with saline and, subsequently, with either 80 or 400 mg of iron as ferrous gluconate. After the intestinal perfusion, a second duodenal tissue sample was obtained. Thiobarbituric acid-reactive substances, an indicator of lipid peroxidation, in intestinal fluid samples increased significantly and dose dependently at 30 min after the start of perfusion with 80 or 400 mg of iron, respectively ( P < 0.001). During the perfusion with 400 mg of iron, the increase in thiobarbituric acid-reactive substances was accompanied by a significant, momentary rise in trolox equivalent antioxidant capacity, an indicator of total antioxidant capacity ( P < 0.05). The expression of 89 gene reporters was significantly altered by both iron interventions. Functional mapping showed that both iron dosages mediated six distinct processes. Three of those processes involved G-protein receptor coupled pathways. The other processes were associated with cell cycle, complement activation, and calcium channels. Iron administration in the small intestine induced dose-dependent lipid peroxidation and a momentary antioxidant response in the lumen, mediated the expression of at least 89 individual gene reporters, and affected at least six biological processes.


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