Epigenetic Regulation of Elf5 Is Associated with Epithelial-Mesenchymal Transition in Urothelial Cancer

E74-like factor 5 (Elf5) has been associated with tumor suppression in breast cancer. However, its role in urothelial cancer (UC) is completely unknown. Immunohistochemistry (IHC) and methylation specific PCR (MSP) were done to detect Elf5 expression level and its promoter methylation. Results revealed that low expression of Elf5 on protein and mRNA levels were associated with tumor progression, early relapse and poor survival. In vitro, down-regulation of Elf5 can increase epithelial-mesenchymal transition (EMT). Aberrant Elf5 methylation was identified as major mechanism for Elf5 gene silence. Accordingly, restoration of Elf5 by infection or demethylating treatment effectively reversed EMT processes. In conclusion, we identified Elf5 as a novel biomarker of UC on several biological levels and established a causative link between Elf5 and EMT in UC.     

Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum

Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 10³ CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5–25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.     

Diterpenoids from the Whole Plants of Ajuga nipponensis and Their Inhibition of RANKL-Induced Osteoclastogenesis

Two new diterpenoids, ajudecunoid A ( 1 ) and ajudecunoid B ( 14 ), along with thirteen known diterpenoids, were isolated from the whole plants of Ajuga nipponensis Makino. Their structures were elucidated by the extensive spectroscopic analysis (UV, IR, MS, and NMR). The absolute configurations of ajudecunoid A ( 1 ) and ajudecunoid B ( 14 ) were defined through analysis of X-ray crystallography. Fifteen compounds were evaluated for inhibition of the formation of osteoclasts in bone marrow-derived macrophages (BMM) cells. Two neo-clerodane diterpenoids ajuganipponin B ( 5 ) and (12S)-6α,19-diacetoxy-18-chloro-4α-hydroxy-12-tigloyloxy-neo-clerod-13-en-15,16-olide ( 12 ) showed significant inhibition of osteoclastogenesis with IC₅₀ values of 0.88 and 0.79 μM, respectively. Here we firstly reported diterpenoids with anti-osteoclastogenesis activity from A. nipponensis.     

The Role of Angiopoietins as Potential Therapeutic Targets in Renal Cell Carcinoma

Angiopoietin 2 (Ang2) is a secreted glycoprotein upregulated at sites of angiogenesis and has been implicated in cancer neovascularization. Recent studies have suggested efficacy of combined Ang and vascular endothelial growth factor receptor (VEGFR) inhibition for patients with metastatic renal cell carcinoma (mRCC). We measured Ang2 expression in human tissue and plasma, and tested the effect of dual Ang1/2 (trebananib; AMG386) or Ang2 alone (L1-7) inhibition with VEGFR inhibition on murine RCC growth and blood flow. Ang2 levels were higher in human tumors than normal tissues with RCC ranking highest for Ang2 expression across all tumor types tested. Plasma Ang2 was significantly higher in patients with mRCC compared to controls or patients with stage I disease. Plasma Ang2 decreased with sunitinib treatment and increased at time of disease progression. In the RCC mouse, dual Ang1/2 and Ang2 inhibition improved the activity of sunitinib. Combined Ang1/2 and VEGFR inhibition prevented the resumption of blood flow associated with sunitinib resistance. Thus, Ang2 inhibition, independent of Ang1 inhibition, improves the activity of sunitinib and plasma Ang2 increases in the setting of progression on sunitinib possibly contributing to resistance. Further, arterial spin-labeled perfusion magnetic resonance imaging might be a non-invasive marker of the antiangiogenic activity of Ang inhibitors.     

Initiation of focal adhesion assembly by talin and kindlin: A dynamic view

Focal adhesions (FAs) are integrin‐containing protein complexes regulated by a network of hundreds of protein–protein interactions. They are formed in a spatiotemporal manner upon the activation of integrin transmembrane receptors, which is crucial to trigger cell adhesion and many other cellular processes including cell migration, spreading and proliferation. Despite decades of studies, a detailed molecular level understanding on how FAs are organized and function is lacking due to their highly complex and dynamic nature. However, advances have been made on studying key integrin activators, talin and kindlin, and their associated proteins, which are major components of nascent FAs critical for initiating the assembly of mature FAs. This review will discuss the structural and functional findings of talin and kindlin and their immediate interaction network, which will shed light upon the architecture of nascent FAs and how they act as seeds for FA assembly to dynamically regulate diverse adhesion‐dependent physiological and pathological responses.     

Pharmacokinetic and pharmacodynamic studies of CD19 CAR T cell in human leukaemic xenograft models with dual-modality imaging

In recent years, chimeric antigen receptor T (CAR T)-cell therapy has shown great potential in treating haematologic disease, but no breakthrough has been achieved in solid tumours. In order to clarify the antitumour mechanism of CAR T cell in solid tumours, the pharmacokinetic (PK) and pharmacodynamic (PD) investigations of CD19 CAR T cell were performed in human leukaemic xenograft mouse models. For PK investigation, we radiolabelled CD19 CAR T cell with ⁸⁹Zr and used PET imaging in the CD19-positive and the CD19-negative K562-luc animal models. For PD evaluation, optical imaging, tumour volume measurement and DNA copy-number detection were performed. Unfortunately, the qPCR results of the DNA copy number in the blood were below the detection limit. The tumour-specific uptake was higher in the CD19-positive model than in the CD19-negative model, and this was consistent with the PD results. The preliminary PK and PD studies of CD19 CAR T cell in solid tumours are instructive. Considering the less efficiency of CAR T-cell therapy of solid tumours with the limited number of CAR T cells entering the interior of solid tumours, this study is suggestive for the subsequent CAR T-cell design and evaluation of solid tumour therapy.