piggyBac-mediated genomic integration of linear dsDNA-based library for deep mutational scanning in mammalian cells.

Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here, we developed two novel strategies for DMS library construction in mammalian cells, namely 'piggyBac-in vitro ligation' and 'piggyBac-in vitro ligation-PCR'.

Construction and Applications of Mammalian Cell-Based DNA-Encoded Peptide/Protein Libraries.

DNA-encoded peptide/protein libraries are the starting point for protein evolutionary modification and functional peptide/antibody selection. Different display technologies, protein directed evolution, and deep mutational scanning (DMS) experiments employ DNA-encoded libraries to provide sequence variations for downstream affinity- or function-based selections. Mammalian cells promise the inherent post-translational modification and near-to-natural conformation of exogenously expressed mammalian proteins and thus are the best platform for studying transmembrane proteins or human disease-related proteins.

Construction of Peptide Library in Mammalian Cells by dsDNA-Based Strategy.

While different display technologies, represented by phage display, have been widely used in drug discovery, they still can hardly achieve function-based peptide screening, which in most cases is performed in mammalian cells. And most attempts to screen functional peptides with mammalian platforms utilized plasmids to store coding information. Our previous work established double-stranded DNAs (dsDNAs) as innovative biological parts to implement AND-gate genetic circuits in mammalian cells.

MZF1 mediates oncogene-induced senescence by promoting the transcription of p16.

Oncogene induced senescence is a tumor suppressing defense mechanism, in which the cell cycle-dependent protein kinase (CDK) inhibitor p16 (encoded by the CDKN2A gene) plays a key role. We previously reported that a transcriptional co-activator chromodomain helicase DNA binding protein 7 (CHD7) mediates oncogenic ras-induced senescence by inducing transcription of the p16 gene. In the current study, we identified myeloid zinc finger 1 (MZF1) as the transcriptional factor that recruits CHD7 to the p16 promoter, where it mediates oncogenic ras-induced p16 transcription and senescence through CHD7, in primary human cells from multiple origins.

Construction of Synthetic Nanobody Library in Mammalian Cells by dsDNA-Based Strategies*.

A nanobody is an antibody fragment consisting of a single monomeric variable antigen-binding domain. Mammalian cells are ideal platforms for identifying nanobodies targeting hard-to-display transmembrane proteins and nanobodies that function as modulators of cellular phenotypes. However, the introduction of a high-diversity nanobody library into mammalian cells is challenging.

Development of a CRISPR-Cas9 Based Luciferase Turn-On System as Nonhomologous End Joining Pathway Reporter.

There is a need of a non-homologous end joining (NHEJ) pathway reporter system that facilitates screening and discovery of NHEJ chemical inhibitors. In this study, we developed a CRISPR-Cas9 based luciferase turn-on system as a NHEJ pathway reporter. By substituting nucleotide 205C with ATC, we introduced a reading-frame shift and a pre-stop codon into the luciferase coding region and thereby generated a bioluminescent signal mute HEK293T reporter cell line.

Ferroptosis and Its Potential Role in Human Diseases.

Ferroptosis is a novel regulated cell death pattern discovered when studying the mechanism of erastin-killing RAS mutant tumor cells in 2012. It is an iron-dependent programmed cell death pathway mainly caused by an increased redox imbalance but with distinct biological and morphology characteristics when compared to other known cell death patterns. Ferroptosis is associated with various diseases including acute kidney injury, cancer, and cardiovascular, neurodegenerative, and hepatic diseases.

CD137 promotes bone metastasis of breast cancer by enhancing the migration and osteoclast differentiation of monocytes/macrophages.

Bone is one of the most common metastatic sites of breast cancer. CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor superfamily, is mainly expressed in activated leukocytes. Previous study demonstrates the effect of CD137-CD137L bidirectional signaling pathway on RANKL-mediated osteoclastogenesis.

Linear double-stranded DNAs as innovative biological parts to implement genetic circuits in mammalian cells.

Synthetic biology employs engineering principles to redesign biological systems for biomedical or industrial purposes. Innovation and application of original biological parts for genetic circuit construction will significantly facilitate and expedite the development of synthetic biology. Here, we built two- or three-input linear double-stranded DNA (ldsDNA)-based Boolean AND gate genetic circuits in mammalian cells.

PFKFB4 Promotes Breast Cancer Metastasis via Induction of Hyaluronan Production in a p38-Dependent Manner.

Background/aims: The bi-functional enzyme 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase-4 (PFKFB4) is highly expressed in many types of cancer and its requirement for tumor survival has been demonstrated in glioma, lung, and prostate cancers. However, whether PFKFB4 plays a role in the tumor metastasis remains uncertain. This study explores the role of PFKFB4 in tumor metastasis and its underlying mechanisms in breast cancer cells.

miR-30 disrupts senescence and promotes cancer by targeting both p16 and DNA damage pathways.

miR-30 is a microRNA frequently overexpressed in human cancers. However, the biological consequence of miR-30 overexpression in cancer has been unclear. In a genetic screen, miR-30 was found to abrogate oncogenic-induced senescence, a key tumor-suppressing mechanism that involves DNA damage responses, activation of p53 and induction of p16.

Inflammatory Human Umbilical Cord-Derived Mesenchymal Stem Cells Promote Stem Cell-Like Characteristics of Cancer Cells in an IL-1-Dependent Manner.

To ensure the safety of clinical applications of MSCs, thorough understanding of their impacts on tumor initiation and progression is essential. Here, to further explore the complex dialog between MSCs and tumor cells, umbilical cord-derived mesenchymal stem cells (UC-MSCs) were employed to be cocultured with either breast or ovarian cancer cells. Though having no obvious influence on proliferation or apoptosis, UC-MSCs exerted intense stem cell-like properties promoting effects on both cancer models.

Inactivation of p38 MAPK contributes to stem cell-like properties of non-small cell lung cancer.

Cancer stem cells (CSCs) are recognized as the major source for cancer initiation and recurrence. Yet, the mechanism by which the cancer stem cell properties are acquired and maintained in a cancer cell population is not well understood. In the current study, we observed that the level of active p38 MAPK is downregulated, while the level of the stemness marker SOX2 is upregulated in lung cancer tissues as compared to normal tissues.

Artificial Virus Delivers CRISPR-Cas9 System for Genome Editing of Cells in Mice.

CRISPR-Cas9 has emerged as a versatile genome-editing platform. However, due to the large size of the commonly used CRISPR-Cas9 system, its effective delivery has been a challenge and limits its utility for basic research and therapeutic applications. Herein, a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) was constructed for the delivery of CRISPR-Cas9 system.

Paclitaxel and Tacrolimus Coencapsulated Polymeric Micelles That Enhance the Therapeutic Effect of Drug-Resistant Ovarian Cancer.

The combination of chemotherapy drugs and multidrug-resistant reversing agents for treating multidrug resistance in tumors has attracted increasing attention. However, the poor water solubility of some anticancer drugs restricted their clinical application. In this work, we prepared poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) micelles as a codelivery system to load the chemotherapy drug paclitaxel (PTX) and the multidrug-resistant reversing agent tacrolimus (FK506).

Activating transcription factor 4 promotes angiogenesis of breast cancer through enhanced macrophage recruitment.

Angiogenesis plays an important role in the progression of tumor. Besides being regulated by tumor cells per se, tumor angiogenesis is also influenced by stromal cells in tumor microenvironment (TME), for example, tumor associated macrophages (TAMs). Activating transcription factor 4 (ATF4), a member of the ATF/CREB family, has been reported to be related to tumor angiogenesis.

Biodegradable polymeric micelle-encapsulated doxorubicin suppresses tumor metastasis by killing circulating tumor cells.

Circulating tumor cells (CTCs) play a crucial role in tumor metastasis, but it is rare for any chemotherapy regimen to focus on killing CTCs. Herein, we describe doxorubicin (Dox) micelles that showed anti-metastatic activity by killing CTCs. Dox micelles with a small particle size and high encapsulation efficiency were obtained using a pH-induced self-assembly method.

Gene and MicroRNA Profiling of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

Introduction: The differentiated cell lineages from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have shown their potential in regenerative medicine. However, the functional and transcriptional microRNA (miRNA) expression pattern during endothelial differentiation has yet to be characterized.

Methods: In this study, hESCs and hiPSCs were differentiated into endothelial cells (ECs).

Biodegradable polymeric micelles encapsulated JK184 suppress tumor growth through inhibiting Hedgehog signaling pathway.

JK184 can specially inhibit Gli in the Hedgehog (Hh) pathway, which showed great promise for cancer therapeutics. For developing aqueous formulation and improving anti-tumor activity of JK184, we prepared JK184 encapsulated MPEG-PCL micelles by the solid dispersion method without using surfactants or toxic organic solvents. The cytotoxicity and cellular uptake of JK184 micelles were both increased compared with the free drug.

Polymeric micelles encapsulating fisetin improve the therapeutic effect in colon cancer.

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) was discovered to possess antitumor activity, revealing its potential value in future chemotherapy. However, its poor water solubility makes it difficult for intravenous administration. In this study, the monomethyl poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) copolymer was applied to prepare nanoassemblies of fisetin by a self-assembly procedure.

Molecular imaging for assessment of mesenchymal stem cells mediated breast cancer therapy.

The tumor tropism of mesenchymal stem cells (MSCs) makes them an excellent delivery vehicle used in anticancer therapy. However, the exact mechanisms of MSCs involved in tumor microenvironment are still not well defined. Molecular imaging technologies with the versatility in monitoring the therapeutic effects, as well as basic molecular and cellular processes in real time, offer tangible options to better guide MSCs mediated cancer therapy.