generation-of-3d-tumor-spheroids-for-drug-evaluation-studies

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Southeast University

Jiangsu Avatarget Biotechnology Co., Ltd.

Subunit-subunit interactions of the classical and alternate chaperone-usher (CU) systems have been shown to proceed through a donor strand exchange (DSE) mechanism. However, it is not known whether DSE is required for intersubunit interactions in the archaic CU system. We have previously shown that the Myxococcus xanthus Mcu system, a member of the archaic CU family that functions in spore coat formation, is likely to use the principle of donor strand complementation to medicate chaperone-subunit interactions analogous to the classical CU pathway. Here we describe the results of studies on Mcu subunit-subunit interactions. We constructed a series of N-terminal-deleted, single amino acid-mutated and donor strand-complemented Mcu subunits, and characterized their abilities to participate in subunit-subunit interactions. It appears that certain residues in both the N and C termini of McuA, a subunit of the Mcu system, play a critical role in intersubunit interactions and these interactions may involve the general principle of DSE of the classical and alternate CU systems. In addition, the specificity of the M. xanthus CU system for Mcu subunits over other spore coat proteins is demonstrated. 

The archaic chaperone-usher pathways may depend on donor strand exchange for intersubunit interactions.

Gene clusters coding for the chaperone/usher (CU) pathway are widely distributed in many important environmental and pathogenic microbes; however, information about the regulatory machineries controlling CU gene expression during multicellular morphogenesis is missing. The Myxococcus xanthus Mcu system, encoded by the mcuABCD gene cluster, represents a prototype of the archaic CU family that functions in spore coat formation. Using genome-wide transposon mutagenesis, we identified MXAN2872 to be a potential regulator of the mcuABC operon and demonstrated the necessity of MXAN2872 for mcuABC expression and fruiting body morphogenesis in early development. In silico, biochemical, and genetic analyses suggest that MXAN2872 encodes a Baeyer-Villiger monooxygenase (BVMO) of flavoproteins, and the potential cofactor-binding site as well as the BVMO fingerprint sequence is important for the regulatory role of the MXAN2872 protein. The expression profile of mcuA in strains with an MXAN2872 deletion and point mutation agrees well with the timing of cell aggregation of these mutants. Furthermore, McuA could not be detected either in a fruA-null mutant, where starvation-induced aggregation was completely blocked, or in the glycerol-induced spores, where sporulation was uncoupled from cell aggregation. In sum, the present work uncovers a positive role for MXAN2872, a metabolic enzyme-encoding gene, in controlling M. xanthus development. MXAN2872 functions by affecting the onset of cell aggregation, thereby leading to a secondary effect on the timing of mcuABC expression of this model organism.

Identification of a putative flavin adenine dinucleotide-binding monooxygenase as a regulator for Myxococcus xanthus development.

A Myxococcus xanthus gene, MXAN3487, was identified by transposon mutagenesis to be required for the expression of mcuABC, an operon coding for part of the chaperone-usher (CU) system in this bacterium. The MXAN3487 protein displays sequence and structural homology to adenosine 5'-phosphosulphate (APS) kinase family members and contains putative motifs for ATP and APS binding. Although the MXAN3487 locus is not linked to other sulphate assimilation genes, its protein product may have APS kinase activity in vivo and the importance of the ATP-binding site for activity was demonstrated. Expression of MXAN3487 was not affected by sulphate availability, suggesting that MXAN3487 may not function in a reductive sulphate assimilation pathway. Deletion of MXAN3487 significantly delayed fruiting body formation and the production of McuA, a spore coat protein secreted by the M. xanthus Mcu CU system. Based on these observations and data from our previous studies, we propose that MXAN3487 may phosphorylate molecules structurally related to APS, generating metabolites necessary for M. xanthus development, and that MXAN3487 exerts a positive effect on the mcuABC operon whose expression is morphogenesis dependent.

A gene encoding a potential adenosine 5'-phosphosulphate kinase is necessary for timely development of Myxococcus xanthus.

Matrix nanotopography plays an essential role in regulating cell behaviors including cell proliferation, differentiation, and migration. While studies on isolated single cell migration along the nanostructural orientation have been reported for various cell types, there remains a lack of understanding of how nanotopography regulates the behavior of collectively migrating cells during processes such as epithelial wound healing. We demonstrated that collective migration of epithelial cells was promoted on nanogratings perpendicular to, but not on those parallel to, the wound-healing axis. We further discovered that nanograting-modulated epithelial migration was dominated by the adhesion turnover process, which was Rho-associated protein kinase activity-dependent, and the lamellipodia protrusion at the cell leading edge, which was Rac1-GTPase activity-dependent. This work provides explanations to the distinct migration behavior of epithelial cells on nanogratings, and indicates that the effect of nanotopographic modulations on cell migration is cell-type dependent and involves complex mechanisms.

Nanotopography-Modulated Epithelial Cell Collective Migration.

Organ-on-a-chip (OoC) is a new and promising technology, which aims to improve the efficiency of drug development and realize personalized medicine by simulating  environment . Physiologically based pharmacokinetic (PBPK) modeling is believed to have the advantage of better reflecting the absorption, distribution, metabolism and excretion process of drugs  than traditional compartmental or non-compartmental pharmacokinetic models. The combination of PBPK modeling and organ-on-a-chip is believed to provide a strong new tool for new drug development and have the potential to replace animal testing. This article provides the recent development of organ-on-a-chip technology and PBPK modeling including model construction, parameter estimation and validation strategies. Application of PBPK modeling on Organ-on-a-Chip (OoC) has been emphasized, and considerable progress has been made. PBPK modeling on OoC would become an essential part of new drug development, personalized medicine and other fields.

Article	Year
The archaic chaperone-usher pathways may depend on donor strand exchange for intersubunit interactions. Microbiology (Reading, England)\| PubMed ID: 25035068	2014
Identification of a putative flavin adenine dinucleotide-binding monooxygenase as a regulator for Myxococcus xanthus development. Journal of bacteriology\| PubMed ID: 25605309	2015
A gene encoding a potential adenosine 5'-phosphosulphate kinase is necessary for timely development of Myxococcus xanthus. Microbiology (Reading, England)\| PubMed ID: 26860640	2016
Nanotopography-Modulated Epithelial Cell Collective Migration. Journal of biomedical nanotechnology\| PubMed ID: 34167622	2021
PBPK Modeling on Organs-on-Chips: An Overview of Recent Advancements. Frontiers in bioengineering and biotechnology\| PubMed ID: 35497341	2022

Shihui Xu

Biography

JoVE Journal Publications

Other Publications