One-Shot Single-Cell Proteome and Metabolome Analysis Strategy for the Same Single Cell.

Characterizing the profiles of proteome and metabolome at the single-cell level is of great significance in single-cell multiomic studies. Herein, we proposed a novel strategy called one-shot single-cell proteome and metabolome analysis (scPMA) to acquire the proteome and metabolome information in a single-cell individual in one injection of LC-MS/MS analysis. Based on the scPMA strategy, a total workflow was developed to achieve the single-cell capture, nanoliter-scale sample pretreatment, one-shot LC injection and separation of the enzyme-digested peptides and metabolites, and dual-zone MS/MS detection for proteome and metabolome profiling. Benefiting from the scPMA strategy, we realized dual-omic analysis of single tumor cells, including A549, HeLa, and HepG2 cells with 816, 578, and 293 protein groups and 72, 91, and 148 metabolites quantified on average. A single-cell perspective experiment for investigating the doxorubicin-induced antitumor effects in both the proteome and metabolome aspects was also performed.

Triptolide inhibits proliferation and invasion of colorectal cancer cells by blocking Nrf2 expression.

Triptolide (TPL), the main active ingredient of Tripterygium wilfordii, has anti-inflammatory, immunomodulatory, and antitumor actions. It can also inhibit cell proliferation and metastasis while promoting apoptosis of several tumors, such as colorectal cancer (CRC). However, the mechanism of TPL against CRC is not clear. This study was designed to investigate the effects and molecular mechanisms of TPL on the proliferation and invasion ability of CRC cells. A human CRC cell line (HT29 cell line) cultured in vitro was treated with different concentrations of TPL (0, 25, 50, and 100 nmol/L). The proliferation of cells was detected by MTT, the invasion ability of cells by Transwell, and the apoptosis level by flow cytometry. The protein expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), matrix metalloproteinase (MMP)-2, and MMP-9 were detected by western blotting. After transfection with sh-Nrf2, HT29 cells were divided into NC group, NC + TPL group and sh-Nrf2 + TPL group, and the above assays were repeated for each group. TPL significantly inhibited the proliferation and invasion ability of HT29 cells and promoted apoptosis (p < .05). Notably, its inhibitory or promotional effects were concentration-dependent, which were enhanced with increasing drug concentration (p < .05). After silencing Nrf2 expression, the proliferation, and invasion ability of HT29 cells were further significantly inhibited while cells apoptosis was further promoted (p < .05). Besides, the decreased Nrf2 expression reduced the protein expression levels of MMP-2 and MMP-9 (p < .05). TPL can effectively inhibit the proliferation and invasion while promoting apoptosis of HT29 cells. And its mechanism of action may be related to the inhibition of Nrf2 signaling expression.

Efficacy of rhNGF-loaded amniotic membrane transplantation for rabbit corneal epithelial and nerve regeneration.

AIM: To evaluate the efficacy of recombinant human nerve growth factor-loaded amniotic membrane (rhNGF-AM) on corneal epithelial and nerve regeneration in rabbit model. METHODS: Freshly prepared human amniotic membrane (AM) were immersed into PBS buffer containing 100 or 500 µg/mL rhNGF for 15, 30, and 60min at 4°C. The in vitro release kinetics of rhNGF was measured with ELISA. For in vivo evaluation, the AM were immersed with 500 µg/mL rhNGF for 30min. Fifty-seven rabbits were selected to establish corneal epithelial defect model. In addition to the 19 rabbits in control group, 38 rabbits received AM transplantation with or without rhNGF after the removal of central epithelium. Corneal epithelial defect area, sub-epithelial nerve fiber density, corneal sensitivity, rhNGF contents in resident AM and corneas were measured after the surgery. RESULTS: rhNGF was sustained release from the AM within 14d in vitro, with the positive correlation with initial immersion concentration. The immersion of AM in 500 µg/mL rhNGF for 30min achieved the most stable release within 14d. After transplantation in rabbit cornea, a high concentration of rhNGF in resident rhNGF-AM and cornea was maintained within 8d. Corneal epithelial healing, nerve fiber regeneration and the recovery of corneal sensitivity were significantly accelerated after the rhNGF-AM transplantation when compared to simple AM transplantation (all P<0.05). CONCLUSION: Simple immersion of AM achieves the sustained release of rhNGF, and promotes corneal epithelial wound healing and nerve regeneration, as well as the recovery of corneal sensitivity in rabbit.

Upregulation of Long Non-Coding RNA ENST00000429227.1 Is Correlated with Poor Prognosis in Human Hepatocellular Carcinoma.

BACKGROUND Long non-coding RNAs (lncRNAs) have been shown to play an important regulatory role in many tumors. This study was designed to investigate the expression of lncRNA ENST00000429227.1 in hepatocellular carcinoma (HCC) and to determine whether the expression of lncRNA ENST00000429227.1 affects the prognosis of HCC. MATERIAL AND METHODS lncRNA ENST00000429227.1 showing differences in expression between M1 and M2 was screened by microarray expression measurements. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of lncRNA ENST00000429227.1 in 161 HCC patients. The chi-square test was used to evaluate the relationship between the expression of ENST00000429227.1 and clinicopathological parameters. A survival curve was drawn and analyzed by Kaplan-Meier method. Cox regression was used for univariate and multivariate analysis to determine whether lncRNA ENST00000429227.1 is an independent factor of the occurrence and prognosis of HCC. RESULTS A total of 3703 differentially expressed lncRNAs were obtained, of which 1777 were upregulated and 1926 were downregulated, with multiple change >1.5. The expression of lncRNA ENST00000429227.1 was upregulated in M2 cells. The expression of lncRNA ENST00000429227.1 in HCC tissues was higher than that in adjacent normal tissues (p<0.05), which was correlated with pathological parameters such as surgical margin (p=0.042), AFP (p=0.022) and Barcelona Clinic Liver Cancer (BCLC) stage (p=0.008). Survival analysis showed that high expression of lncRNA ENST00000429227.1 was associated with a decrease in overall survival (OS) rate of HCC patients. Cox regression analysis showed that high expression of ENST00000429227.1 may be an independent risk factor affecting the prognosis of HCC patients. CONCLUSIONS The results suggest that upregulation of ENST00000429227.1 is associated with poor prognosis of HCC patients, and may be a new biomarker for the diagnosis of HCC.

Downregulation of Macrophage-Derived T-UCR uc.306 Associates with Poor Prognosis in Hepatocellular Carcinoma.

BACKGROUND/AIMS: Increasing evidence suggests that T-UCRs are involved in the development of cancer. In this study, we evaluated the role of a macrophage-derived T-UCR, uc.306, in the prognosis of hepatitis B (HBV)-related hepatocellular carcinoma (HCC). METHODS: The uc.306 was obtained by screening microassay data obtained during the polarization of U937 cells from the M2 to M1 phenotype. Uc.306 and macrophage molecule markers were detected by qPCR. Immunohistochemical (IHC) assays were used to examine the M1/M2 status of 90 paired HCC tissues. Kaplan-Meier tests and multivariable Cox regression models were used to analyze predictive confidences, survival, and risk factors. RESULTS: In total, 2,977 differentially expressed T-UCRs were obtained, of which 257 showed fold changes >1.5. The uc.306 was upregulated in M1 cells and was predicted to be involved in the Wnt pathway. The IHC results showed that M1 macrophages (CD68+) were present in the para-tumor tissues, while the M2 phenotype (CD163+) was mainly in the HCC tissues. Uc.306 had a lower expression in the HCC tissues than in that of the para-tumor tissues in 30 paired HCC training sets (P < 0.0001), and 252 paired HCC testing sets (P < 0.0001). Low expression of uc.306 was significantly associated with a shorter overall survival (P < 0.05). CONCLUSIONS: The uc.306 may be a promising biomarkerfor HBV-related HCC, providing a novel marker for the prognosis of HCC.

Single molecule study of initial structural features on the amyloidosis process.

We employed an α-hemolysin (α-HL) nanopore as a single-molecule tool to investigate the effects of initial structure on the amyloidosis process. The differences in the initial structure of two ß-amyloid (Aß) peptides (Aß25-35 and Aß35-25) could be distinguished in real-time due to their characteristic blockades. More importantly, the distinct aggregate dynamics for these two kinds of Aß fragments can be readily analyzed by monitoring the blockade frequency over time.

BRCA2 and nucleophosmin coregulate centrosome amplification and form a complex with the Rho effector kinase ROCK2.

BRCA2 germline mutations account for the majority of heredity breast and ovarian cancer. Besides its role in DNA damage repair, BRCA2 also plays an important role in cytokinesis, transcription regulation, and cancer cell proliferation. Recently, we reported that BRCA2 localizes to centrosomes as well as nuclei and the dysfunction of BRCA2 in a centrosome causes abnormalities in cell division. Here, we identified a nucleolar phosphoprotein, nucleophosmin (NPM), as a novel BRCA2-associated protein. We also detected the binding of BRCA2 to ROCK2, an effector of Rho small GTPase. Because it is known that ROCK2 binds to NPM at centrosomes, these 3 proteins may form a complex. NPM-binding region was within amino acids 639-1,000 of BRCA2. Exogenous expression of this BRCA2 region resulted in aberrant centrosome amplification and a high frequency of multinucleated cells. Our results suggested that a complex consisting of BRCA2, NPM, and ROCK2 maintains the numerical integrity of centrosomes and accurate cell division and that dysfunction of this regulation might be involved in the tumorigenesis of breast cancer.

[Effects of Lycium barbarum polysaccharide on tumor microenvironment T-lymphocyte subsets and dendritic cells in H22-bearing mice].

OBJECTIVE: To study the effects of Lycium barbarum polysaccharide (LBP) on tumor microenvironment T-lymphocyte subsets and dendritic cells in H22-bearing mice and the mechanisms for intervention of tumor immune escape by LBP. METHODS: H22-bearing mice were given LBP orally for two weeks. T-lymphocyte subsets and the phenotypes of dendritic cells in tumor-infiltrating lymphocytes (TIL) were detected by flow cytometry (FCM). RESULTS: LBP could significantly increase the numbers of CD4(+) and CD8(+) T cells in TIL as compared with those in model control group (P<0.05). In model control group, the number of dendritic cells in tumor microenvironment decreased markedly, while in LBP-treated group, the increased number of dendritic cells and B7-1 expression were observed, but there were no significant differences between these two groups. CONCLUSION: LBP has anti-tumor effect probably by increasing the numbers of CD4(+) and CD8(+) T cells in TIL to relieve the immunosuppression and enhance the anti-tumor function of the immune system. But whether LBP can recover the phenocyte and function of dendritic cells in H22-bearing mice should be further studied.