The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network

ABSTRACTUnderstanding how complex organ systems are assembled from simple embryonic tissues is a major challenge. Across the animal kingdom a great diversity of visual organs are initiated by a ‘master control gene’ called Pax6, which is both necessary and sufficient for eye development. Yet precisely how Pax6 achieves this deeply homologous function is poorly understood. Using the chick as a model organism, we show that vertebrate Pax6 interacts with a pair of morphogen-coding genes, Tgfb2 and Fst, to form a putative Turing network, which we have computationally modelled. Computer simulations suggest that this gene network is sufficient to spontaneously polarise the developing retina, establishing the first organisational axis of the eye and prefiguring its further development. Our findings reveal how retinal self-organisation may be initiated independently of the highly ordered tissue interactions that help to assemble the eye in vivo. These results help to explain how stem cell aggregates spontaneously self-organise into functional eye-cups in vitro. We anticipate these findings will help to underpin retinal organoid technology, which holds much promise as a platform for disease modelling, drug development and regenerative therapies.

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ThePax6master control gene initiates spontaneous retinal development via a self-organising Turing network

10.1101/583807 ◽

2019 ◽

Author(s):

Timothy Grocott ◽

Estefania Lozano-Velasco ◽

Gi Fay Mok ◽

Andrea E Münsterberg

Keyword(s):

Control Gene ◽

Embryonic Tissues ◽

Tissue Interactions ◽

Organ Systems ◽

Necessary And Sufficient ◽

Regenerative Therapies ◽

Further Development ◽

Master Control

AbstractUnderstanding how complex organ systems are assembled from simple embryonic tissues is a major challenge. Across the animal kingdom a great diversity of visual organs are initiated by a ‘master control gene’ calledPax6, which is both necessary and sufficient for eye development1–6. Yet precisely howPax6achieves this deeply homologous function is poorly understood. Here we show that vertebratePax6interacts with a pair of morphogen-coding genes,Tgfb2andFst, to form a putative Turing network7, which we have computationally modelled. Computer simulations suggest that this gene network is sufficient to spontaneously polarise the developing retina, establishing the eye’s first organisational axis and prefiguring its further development. Our findings reveal how retinal self-organisation may be initiated independent of the highly ordered tissue interactions that help to assemble the eyein vivo. These results help to explain how stem cell aggregates spontaneously self-organise into functional eye-cupsin vitro8. We anticipate these findings will help to underpin retinal organoid technology, which holds much promise as a platform for disease modelling, drug development and regenerative therapies.

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Influence of Dopamine on Fluorescent Advanced Glycation End Products Formation Using Drosophila melanogaster

Biomolecules ◽

10.3390/biom11030453 ◽

2021 ◽

Vol 11 (3) ◽

pp. 453

Author(s):

Ana Filošević Vujnović ◽

Katarina Jović ◽

Emanuel Pištan ◽

Rozi Andretić Waldowski

Keyword(s):

Drosophila Melanogaster ◽

Advanced Glycation End Products ◽

Model Organism ◽

Covalent Modification ◽

Advanced Glycation ◽

Biomarkers Of Aging ◽

Glycation End Products ◽

End Products

Non-enzymatic glycation and covalent modification of proteins leads to Advanced Glycation End products (AGEs). AGEs are biomarkers of aging and neurodegenerative disease, and can be induced by impaired neuronal signaling. The objective of this study was to investigate if manipulation of dopamine (DA) in vitro using the model protein, bovine serum albumin (BSA), and in vivo using the model organism Drosophila melanogaster, influences fluorescent AGEs (fAGEs) formation as an indicator of dopamine-induced oxidation events. DA inhibited fAGEs-BSA synthesis in vitro, suggesting an anti-oxidative effect, which was not observed when flies were fed DA. Feeding flies cocaine and methamphetamine led to increased fAGEs formation. Mutants lacking the dopaminergic transporter or the D1-type showed further elevation of fAGEs accumulation, indicating that the long-term perturbation in DA function leads to higher production of fAGEs. To confirm that DA has oxidative properties in vivo, we fed flies antioxidant quercetin (QUE) together with methamphetamine. QUE significantly decreased methamphetamine-induced fAGEs formation suggesting that the perturbation of DA function in vivo leads to increased oxidation. These findings present arguments for the use of fAGEs as a biomarker of DA-associated neurodegenerative changes and for assessment of antioxidant interventions such as QUE treatment.

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The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis

eLife ◽

10.7554/elife.00947 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 226

Author(s):

Liang Ge ◽

David Melville ◽

Min Zhang ◽

Randy Schekman

Keyword(s):

Autophagosome Formation ◽

Functional Studies ◽

A Cell ◽

Intermediate Compartment ◽

Membrane Isolation ◽

Necessary And Sufficient ◽

Organelle Membrane ◽

Catabolic Process

Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes.

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Preclinical Pharmacokinetics of C118P, a Novel Prodrug of Microtubules Inhibitor and Its Metabolite C118 in Mice, Rats, and Dogs

Molecules ◽

10.3390/molecules23112883 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2883 ◽

Cited By ~ 3

Author(s):

Cang Zhang ◽

Xiaolan Zhang ◽

Guangji Wang ◽

Ying Peng ◽

Xueyuan Zhang ◽

...

Keyword(s):

Clinical Development ◽

High Dose ◽

Protein Inhibitor ◽

Promising Candidate ◽

Preclinical Pharmacokinetics ◽

Tumor Bearing ◽

Microtubule Protein ◽

Further Development

C118P, a phosphate prodrug of C118, which is a novel microtubule protein inhibitor, is currently under Phase I clinical development in China for treating ovarian cancer and lung cancer. The preclinical pharmacokinetics of prodrug C118P and its metabolite C118 were extensively characterized in vivo in mice, rats, and dogs and in vitro to support the further development of C118P. The preclinical tissue distribution and excretion were investigated in rats. Plasma protein binding in mice, rat, and human, and hepatic microsomal metabolic stability in mice, rat, dog, monkey, and human, were also evaluated. The (AUC0-inf) and C30s of C118P at 50 mg/kg in rats and 6 mg/kg in dogs, and the C2min of C118 at 6 mg/kg in dogs increased less than the dosage increase, suggested nonlinear pharmacokinetic occurred at high dose. As a prodrug, C118P can be quickly hydrolyzed into C118 after an intravenous administration. The unbound C118 in plasma is slightly higher than C118P. C118P can hardly penetrate the tissue, while C118 can distribute widely into tissues. In tumor-bearing nude mice, the concentration of C118 is high in lung, ovary, and tumor, with an extended half-life in tumor. C118P is a promising candidate prodrug for further clinical development.

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In Vitro and in Vivo Imaging of Nitroxyl with Copper Fluorescent Probe in Living Cells and Zebrafish

Molecules ◽

10.3390/molecules23102551 ◽

2018 ◽

Vol 23 (10) ◽

pp. 2551 ◽

Cited By ~ 3

Author(s):

Sathyadevi Palanisamy ◽

Yu-Liang Wang ◽

Yu-Jen Chen ◽

Chiao-Yun Chen ◽

Fu-Te Tsai ◽

...

Keyword(s):

Critical Role ◽

Model Organism ◽

Living Cells ◽

Biological Species ◽

Zebrafish Model ◽

Physiological Processes ◽

Collective Data ◽

Angeli's Salt

Nitroxyl (HNO) plays a critical role in many physiological processes which includes vasorelaxation in heart failure, neuroregulation, and myocardial contractility. Powerful imaging tools are required to obtain information for understanding the mechanisms involved in these in vivo processes. In order to develop a rapid and high sensitive probe for HNO detection in living cells and the zebrafish model organism, 2-((2-(benzothiazole-2yl)benzylidene) amino)benzoic acid (AbTCA) as a ligand, and its corresponding copper(II) complex Cu(II)-AbTCA were synthesized. The reaction results of Cu(II)-AbTCA with Angeli’s salt showed that Cu(II)-AbTCA could detect HNO quantitatively in a range of 40–360 µM with a detection limit of 9.05 µM. Furthermore, Cu(II)-AbTCA is more selective towards HNO over other biological species including thiols, reactive nitrogen, and reactive oxygen species. Importantly, Cu(II)-AbTCA was successfully applied to detect HNO in living cells and zebrafish. The collective data reveals that Cu(II)-AbTCA could be used as a potential probe for HNO detection in living systems.

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Functional optical coherence tomography of retinal photoreceptors

Experimental Biology and Medicine ◽

10.1177/1535370218816517 ◽

2018 ◽

Vol 243 (17-18) ◽

pp. 1256-1264 ◽

Cited By ~ 9

Author(s):

Xincheng Yao ◽

Taeyoon Son ◽

Tae-Hoon Kim ◽

Yiming Lu

Keyword(s):

Vision Loss ◽

Objective Assessment ◽

Age Related Macular Degeneration ◽

Retinal Stimulation ◽

Age Related ◽

Retinal Photoreceptors ◽

Severe Vision Loss ◽

Further Development

Age-related macular degeneration (AMD) is the leading cause of severe vision loss and legal blindness. It is known that retinal photoreceptors are the primary target of AMD. Therefore, a reliable method for objective assessment of photoreceptor function is needed for early detection and reliable treatment evaluation of AMD and other eye diseases such as retinitis pigmentosa that are known to cause photoreceptor dysfunctions. Stimulus-evoked intrinsic optical signal (IOS) changes promise a unique opportunity for objective assessment of physiological function of retinal photoreceptor and inner neurons. Instead of a comprehensive review, this mini-review is to provide a brief summary of our recent in vitro and in vivo optical coherence tomography (OCT) studies of stimulus-evoked IOS changes in animal retinas. By providing excellent axial resolution to differentiate individual retinal layers, depth-resolved OCT revealed rapid IOS response at the photoreceptor outer segment. The fast photoreceptor-IOS occurred almost right away (∼ 2 ms) after the onset of retinal stimulation, differentiating itself from slow IOS changes correlated with inner neural and hemodynamic changes. Further development of the functional IOS instruments and retinal stimulation protocols may provide a feasible solution to pursue clinical application of functional IOS imaging for objective assessment of human photoreceptors. Impact statement Retinal photoreceptors are the primary target of age-related macular degeneration (AMD) which is the leading cause of severe vision loss and legal blindness. An objective method for functional assessment of photoreceptor physiology can benefit early detection and better treatment evaluation of AMD and other eye diseases that are known to cause photoreceptor dysfunctions. This article summarizes in vitro study of IOS mechanisms and in vivo demonstration of IOS imaging of intact animals. Further development of the functional IOS imaging may provide a revolutionary solution to achieve objective assessment of human photoreceptors.

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CRISPR-induced deletion with SaCas9 restores dystrophin expression in dystrophic models in vitro and in vivo

10.1101/378331 ◽

2018 ◽

Author(s):

Benjamin L. Duchêne ◽

Khadija Cherif ◽

Jean-Paul Iyombe-Engembe ◽

Antoine Guyon ◽

Joel Rousseau ◽

...

Keyword(s):

Hereditary Disease ◽

Reading Frame ◽

Dystrophin Expression ◽

Dmd Gene ◽

Dystrophin Protein ◽

Further Development ◽

Muscle Biopsies ◽

Complete Deletion

AbstractDuchenne Muscular Dystrophy (DMD), a severe hereditary disease, affecting 1 boy out of 3500, mainly results from the deletion of one or more exons leading to a reading frame shift of the DMD gene that abrogates dystrophin protein synthesis. We used the Cas9 of Staphylococcus aureus (SaCas9) to edit the human DMD gene. Pairs of sgRNAs were meticulously chosen to induce a genomic deletion to not only restore the reading frame but also produced a dystrophin protein with normally phased spectrin-like repeats. The formation of a dystrophin protein with spectrin-like repeats normally phased is not usually obtained by skipping or by deletion of complete exons. This can however be obtained in rare instances where the exon/intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion are at similar positions in the spectrin-like repeat. We used pairs of sgRNAs, targeting exons 47 and 58 and a normal reading frame was restored in 67 to 86% of the resulting hybrid exons in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. The restoration of the DMD reading frame and restoration of the dystrophin expression was also obtained in vivo in the heart of the del52hDMD/mđx. Our results provide a proof-of-principle that SaCas9 could be used to edit the human DMD gene and could be considered for the further development of a therapy for DMD.

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Repurposing celecoxib analogues as leads for antibiotics

Future Medicinal Chemistry ◽

10.4155/fmc-2021-0030 ◽

2021 ◽

Author(s):

Baowei Yang ◽

Yicheng Mei ◽

Qianhui Li ◽

Mengyuan Zhang ◽

Huiling Tang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Enzyme Inhibition ◽

Mild Improvement ◽

New Antibiotics ◽

Cytotoxicity Assessment ◽

Improvement In Survival ◽

In Vivo Cytotoxicity ◽

Further Development

There is an urgent need for new antibiotics and alternative strategies to combat bacterial pathogens. Molecular docking, antibacterial evaluation in vitro and in vivo, cytotoxicity assessment and enzyme inhibition analyses were performed. Compound 12 exhibited antimicrobial activity against Staphylococcus aureus (MIC: 4 μg/ml), various clinically isolated strains of MRSA (MIC: 4–16 μg/ml) and Acinetobacter baumannii (MIC: 4 μg/ml) when combined with subinhibitory concentrations of colistin B. Compound 12 (20 mg/kg) yielded mild improvement in survival of methicillin-resistant Staphylococcus aureus (MRSA)-infected mice. Additionally, enzyme inhibition tests showed that compound 12 exhibited inhibitory effects against S. aureus dihydrofolate reductase (105.1 μg/ml) and DNA gyrase (122.8 μg/ml). Compound 12 is a promising antibacterial candidate for further development.

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An amphipathic helix enables septins to sense micrometer-scale membrane curvature

The Journal of Cell Biology ◽

10.1083/jcb.201807211 ◽

2019 ◽

Vol 218 (4) ◽

pp. 1128-1137 ◽

Cited By ~ 26

Author(s):

Kevin S. Cannon ◽

Benjamin L. Woods ◽

John M. Crutchley ◽

Amy S. Gladfelter

Keyword(s):

Membrane Curvature ◽

Amphipathic Helix ◽

Curvature Sensing ◽

C Terminus ◽

Number Of Binding Sites ◽

Curved Membranes ◽

Necessary And Sufficient ◽

Micrometer Scale

Cell shape is well described by membrane curvature. Septins are filament-forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single-septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.

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Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro

Blood ◽

10.1182/blood.v94.6.1926 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1926-1932 ◽

Cited By ~ 44

Author(s):

Tomoaki Fujisaki ◽

Marc G. Berger ◽

Stefan Rose-John ◽

Connie J. Eaves

Keyword(s):

Growth Factors ◽

Fold Increase ◽

Semisolid Medium ◽

Response Characteristics ◽

Adult Human ◽

Liquid Suspension ◽

Normal Duration ◽

Necessary And Sufficient

Abstract Recently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.

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