Bone marrow mesenchymal stem cells combined with estrogen synergistically promote endometrial regeneration and reverse EMT via Wnt/ß-catenin signaling pathway.

BACKGROUND: Intrauterine adhesion (IUA) is a clinical disease characterized by the uterine cavity occlusion caused by the damage of the endometrial basal layer. Bone marrow mesenchymal stem cells (BMSCs) transplantation have the potential to promote endometrial regeneration mainly through paracrine ability. Estrogen is an indispensable and important factor in the repair of endometrial damage, which has been reported as a promising and adjunctive therapeutic application for stem cell transplantation therapy. This study aims to investigate the synergistic effect of BMSCs and estrogen on improving the endometrial regeneration and restoring the endometrium morphology in a dual damage model of IUA in rabbits and the underlying molecular mechanisms. METHODS: BMSCs were isolated and identified by adipogenic and osteogenic differentiation and flow cytometry assays. The rabbit IUA animal model was established by a dual damage method of mechanical curettage and lipopolysaccharide infection. Additionally, we investigated the therapeutic impact of both BMSCs and estrogen either separately or in combination in a rabbit model. The retention of PKH26-labeled BMSCs was observed by vivo fluorescence imaging.The number of endometrial glands and the degree of fibrosis were observed by H&E and Masson staining respectively. Western blotting, Immunohistochemistry and immunofluorescence staining were performed to detect biomarkers related to endometrial epithelium, endometrial fibrosis and EMT. Finally, the protein expression of core molecules of Wnt/ß-catenin pathway was detected by Western blotting. RESULTS: PKH26-labeled fluorescence results revealed that BMSCs appeared and located in the endometrial glands and extracellular matrix area when orthotopic transplanted into the uterine cavity. Histological assays showed that remarkably increasing the number of endometrial glands and decreasing the area of endometrial fibrosis in the BMSCs combined with estrogen treatment group. Moreover, downregulated expression of fibrosis markers (fibronectin, CollagenI, a-SMA) and interstitial markers (ZEB1, Vimentin, N-cadherin), as well as upregulated E-cadherin expression were found in the combined group. Further study of in vivo staining revealed that fluorescence intensity of CK7 was stronger in the combined group than that of direct BMSCs intrauterine transplantation, while vimentin showed the opposite results. Moreover, the protein levels of ß-catenin, Axin2, C-myc, CycinE of Wnt/ß-catenin signaling pathway increased in the BMSCs combined with estrogen group than in the other treatment groups. CONCLUSION: BMSCs combined with estrogen can promote the differentiation of stem cells into endometrial epithelial cells to facilitate the regeneration of damaged endometrium. The potential mechanism of the synergistic effect may inhibit the occurrence of EMT by activating the Wnt/ß-catenin signaling pathway.

Organoid Transplantation Can Improve Reproductive Prognosis by Promoting Endometrial Repair in Mice.

OBJECTIVE: Intrauterine adhesion (IUA) is one of the major causes of refractory secondary infertility, especially in regions and countries with high abortion rates. In this study, we used the mouse IUA model to evaluate the feasibility of the organoids, a 3D cell structure derived from endometrial tissue, as grafts for the treatment of post-traumatic endometrial regeneration disorders. METHODS: The isolated and cultured endometrial organoid was transplanted into the model IUA uterus by the hydrogel scaffold method. RESULTS: The cultured endometrial organoids were transplanted into the basal layer of the damaged endometrium for 28 days. They were completely implanted and grew normally. They not only reconstructed the structural integrity of the endometrial epithelium but also realized the functional repair of the endometrium through differentiation cultures and secretory functions. CONCLUSION: For severe IUA, this method may be better than stem cell transplantation. These findings provide useful insights into the use of endometrial organoid regeneration in the treatment of injury repair.

Therapeutic Exon Skipping Through a CRISPR-Guided Cytidine Deaminase Rescues Dystrophic Cardiomyopathy in Vivo.

BACKGROUND: Loss of dystrophin protein causes Duchenne muscular dystrophy (DMD), characterized by progressive degeneration of cardiac and skeletal muscles, and mortality in adolescence or young adulthood. Although cardiac failure has risen as the leading cause of mortality in patients with DMD, effective therapeutic interventions remain underdeveloped, in part, because of the lack of a suitable preclinical model. METHODS: We analyzed a novel murine model of DMD created by introducing a 4-bp deletion into exon 4, one of the exons encoding the actin-binding domain 1 of dystrophin (referred to as DmdE4* mice). Echocardiography, microcomputed tomography, muscle force measurement, and histological analysis were performed to determine cardiac and skeletal muscle defects in these mice. Using this model, we examined the feasibility of using a cytidine base editor to install exon skipping and rescue dystrophic cardiomyopathy in vivo. AAV9-based CRISPR/Cas9-AID (eTAM) together with AAV9-sgRNA was injected into neonatal DmdE4* mice, which were analyzed 2 or 12 months after treatment to evaluate the extent of exon skipping, dystrophin restoration, and phenotypic improvements of cardiac and skeletal muscles. RESULTS: DmdE4* mice recapitulated many aspects of human DMD, including shortened life span (by ≈50%), progressive cardiomyopathy, kyphosis, profound loss of muscle strength, and myocyte degeneration. A single-dose administration of AAV9-eTAM instituted >50% targeted exon skipping in the Dmd transcripts and restored up to 90% dystrophin in the heart. As a result, early ventricular remodeling was prevented and cardiac and skeletal muscle functions were improved, leading to an increased life span of the DmdE4* mice. Despite gradual decline of AAV vector and base editor expression, dystrophin restoration and pathophysiological rescue of muscular dystrophy were long lasted for at least 1 year. CONCLUSIONS: Our study demonstrates the feasibility and efficacy to institute exon skipping through an enhanced TAM (eTAM) for therapeutic application(s).

Analysis of Immune Landscape Reveals Prognostic Significance of Cytotoxic CD4+ T Cells in the Central Region of pMMR CRC.

BACKGROUND: Mismatch repair proficient colorectal cancer (pMMR CRC) lacks effective treatments and has a poor prognosis, which can be attributed to the complexity of tumor microenvironment. The coordinated function of immune cells is vital to anti-tumor immunity. However, the spatial characteristics of immune cells in the pMMR CRC immune microenvironment and their relationship with clinical prognosis are not fully understood. Meanwhile, the immune modulatory effect of neoadjuvant chemotherapy (NCT), which is the first-line treatment of pMMR CRC, needs further investigation. Therefore, this study aims to explore the spatial dynamics of immune cells and its prognostic value in pMMR CRC. METHODS: We analyzed the various immune cells in formalin-fixed, paraffin-embedded tumor tissues which were collected from 77 patients with stage II/III of pMMR CRC, including 39 non-NCT treated and 38 NCT treated patients. We used the optimized multiplex immunohistochemistry (mIHC) to identify and quantify the density, type and location of immune cells in pMMR CRC. Multivariate survival analysis was performed to assess the relationship of immune profiles and clinical prognosis of pMMR CRC patients. RESULTS: The densities of most T cell subsets, B cells and macrophages were higher in the central region of the pMMR CRC than in the invasion margin. Tumor infiltrating lymphocytes (TILs), especially the infiltration of CD4+ GzmB+ T cells in the central region of the tumor was identified to be positively correlated with the prognosis of the patients. Multivariate analysis confirmed that CD4+ GzmB+ T cells population was an independent predictor of disease-free survival (DFS) in non-NCT group. Meanwhile, NCT enhanced the infiltration of CD4+ GzmB+ T cells in the central region of the pMMR CRC, which was also identified as an independent protective factor of overall survival (OS) and DFS in NCT group. CONCLUSION: We demonstrated that the level of CD4+ GzmB+ T cells located in the center of tumor could provide great prognostic value for pMMR CRC patients. And the application of neoadjuvant chemotherapy further improves the infiltration of CD4+ GzmB+ T cells in the central compartment. Further studies into the application of CD4+ GzmB+ T cells in tumor immunotherapy are needed.

Lipid Metabolism in Tumor-Infiltrating T Cells.

T cells recognize "foreign" antigens and induce durable humoral and cellular immune responses, which are indispensable for defending pathogens, as well as maintaining the integrity and homeostasis of tissues and organs. T cells are the major immune cell population in the tumor microenvironment which play a critical role in the antitumor immune response and cancer immune surveillance. Defective immune response of tumor-infiltrating T cells is the main cause of cancer immune evasion. The antitumor response of T cells is affected by multiple factors in the tumor microenvironment, including immunosuppressive cells, immune inhibitory cytokines, tumor-derived suppressive signals like PD-L1, immnuogenicity of tumor cells, as well as metabolic factors like hypoxia and nutrient deprivation. Abundant studies in past decades have proved the metabolic regulations of the immune response of T cells and the tumor-infiltrating T cells. In this chapter, we will discuss the regulations of the antitumor response of tumor-infiltrating T cells by lipid metabolism, which is one of the main components of metabolic regulation.

Potentiating CD8+ T cell antitumor activity by inhibiting PCSK9 to promote LDLR-mediated TCR recycling and signaling.

Metabolic regulation has been proven to play a critical role in T cell antitumor immunity. However, cholesterol metabolism as a key component of this regulation remains largely unexplored. Herein, we found that the low-density lipoprotein receptor (LDLR), which has been previously identified as a transporter for cholesterol, plays a pivotal role in regulating CD8+ T cell antitumor activity. Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion, we also found a non-canonical function of LDLR in CD8+ T cells: LDLR interacts with the T-cell receptor (TCR) complex and regulates TCR recycling and signaling, thus facilitating the effector function of cytotoxic T-lymphocytes (CTLs). Furthermore, we found that the tumor microenvironment (TME) downregulates CD8+ T cell LDLR level and TCR signaling via tumor cell-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs. Moreover, genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8+ T cells by alleviating the suppressive effect on CD8+ T cells and consequently inhibit tumor progression. While previously established as a hypercholesterolemia target, this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well.

Genetic Modulation of RNA Splicing with a CRISPR-Guided Cytidine Deaminase.

RNA splicing is a critical mechanism by which to modify transcriptome, and its dysregulation is the underlying cause of many human diseases. It remains challenging, however, to genetically modulate a splicing event in its native context. Here, we demonstrate that a CRISPR-guided cytidine deaminase (i.e., targeted-AID mediated mutagenesis [TAM]) can efficiently modulate various forms of mRNA splicing. By converting invariant guanines to adenines at either 5' or 3' splice sites (SS), TAM induces exon skipping, activation of alternative SS, switching between mutually exclusive exons, or targeted intron retention. Conversely, TAM promotes downstream exon inclusion by mutating cytidines into thymines at the polypyrimidine tract. Applying this approach, we genetically restored the open reading frame and dystrophin function of a mutant DMD gene in patient-derived induced pluripotent stem cells (iPSCs). Thus, the CRISPR-guided cytidine deaminase provides a versatile genetic platform to modulate RNA splicing and to correct mutations associated with aberrant splicing in human diseases.

Composition and antimicrobial activity of the essential oil from the branches of Jacaranda cuspidifolia Mart. growing in Sichuan, China.

This study represents the first report on the chemical composition and antimicrobial activity of the essential oil from the branches of Jacaranda cuspidifolia Mart. Thirty-three compounds were identified by Gas Chromatography-Mass Spectrometry (GC-MS) and the major constituents of the essential oil were Palmitic acid (31.36%), (Z) - 9,17-Octadecadienal (12.06%), Ethyl palmitate (3.81%), Perhydrofarnesyl acetone (2.07%), γ-Maaliene (1.88%), and Cedro (1.42%) and 9,12-Octadecadienoic acid ethyl ester (1.42%). The in vitro antimicrobial activities of the essential oil were evaluated by the disc diffusion method, and the inhibition zones against Escherichia coli, Staphylococcus aureus and Candida albicans were 7.10, 8.20 and 7.25 mm, respectively. The oil showed moderate activities against E. coli, S. aureus and C. albicans with minimum inhibition concentration (MIC) values of 17.3 mg/mL, 12.9 mg/mL and 16.0 mg/mL, respectively.

The Prognostic Value of Nanog Overexpression in Lung Cancer: A Meta-Analysis.

BACKGROUND: Recent several studies have showed that the nanog overexpression leads to poor prognosis in some kinds of cancer including hepatocellular carcinoma and gastrointestinal luminal cancer. However, the correlations between prognosis and clinic-pathological features and nanog overexpression in lung cancer are still not well-known. Thus, we performed a meta-analysis to evaluate the role of nanog in lung cancer. METHODS: An electronic retrieval for related studies was conducted in PubMed, Cochrane Library, Web of Science, EMBASE databases, Chinese CNKI, and the Chinese Wan Fang database up to May 2018. The relationships between nanog overexpression and overall survival (OS) and disease-free survival (DFS) as well as clinic-pathological features in lung cancer were investigated. Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by STATA12. RESULTS: 11 studies containing 1422 patients were identified in our meta-analysis. The overexpression of nanog showed decreased OS (HR = 1.83, 95% CI = 1.49-2.25, P ≤ 0.001) and DFS (HR = 1.86, 95% CI = 1.2-2.9, P = 0.006). Moreover, overexpression of nanog was significantly related to differentiation (OR = 4.17, 95% CI = 2.17-6.43, P ≤ 0.001), lymph node metastasis (OR = 1.76, 95% CI = 1.06-2.91, P = 0.028) and tumor size (OR = 1.93, 95% CI = 1.17-3.20, P = 0.010), and no correlation with T stage, TNM, stage, and gender. CONCLUSIONS: Our results suggested that nanog overexpression, a hazard factor of differentiation, lymph node metastasis, and tumor size, may predicate decreased OS and DFS for lung cancer.

Flavones and Lignans from the Stems of Wikstroemia scytophylla Diels.

BACKGROUND: The genus Wikstroemia has about 70 species, but only a limited number of species have been studied chemically. Wikstroemia indica has long been used as a traditional crude drug in China. However, there is no report about the bioactivity of Wikstroemia scytophylla. OBJECTIVE: This paper reports the chemical investigation and biological evaluation of the W. scytophylla. MATERIALS AND METHODS: The EtOAc extraction of W. scytophylla was isolated using chromatographic methods, and the compounds were analyzed by spectroscopic methods. The in vitro antitumor activities against five human cancer cell lines were performed according to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: The chemical investigation of the stems of W. scytophylla resulted in the isolation of 12 compounds mainly including one biflavone (1), five flavones (2-6) compounds, and six lignans (7-12), in which compound 8 was a new natural product. Compounds 1 and 7-12 were evaluated for their antitumor activities while these compounds showed weak cytotoxicity with the half maximal inhibitory (IC50) values more than 40 µM. CONCLUSION: All of these compounds were isolated from this plant for the first time, and compounds 2-12 were first reported from genus Wikstroemia, in which compound 8 was a new natural product. Compounds 1 and 7-12 exhibited weak antitumor activities (IC50>40 µM). The chemotaxonomic significance of all the isolations was summarized. SUMMARY: The chemical investigation of the stems of W. scytophylla resulted in the isolation of 12 compoundsThe 12 compounds including six lignans (7-12), in which compound 8 was a new natural productThe isolated compounds 1 and 7-12 were evaluated for their antitumor activitiesThe chemotaxonomic significance of all the isolations was summarized. Abbreviations used: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC50: Half maximal inhibitory; HL-60: Human leukemia cell line; SMMC-7721: Human hepatocellular carcinoma cell line; A549: Human lung tumor cell line; MCF-7: Human breast cancer cell line; SW480: Human colon cancer cell line; MS: Mass spectrometry; NMR: Nuclear Magnetic Resonance.

A new phenolic glycoside from Lindera nacusua.

The present study was designed to investigate the chemical constituents of Lindera nacusua and their antitumor activities. A new phenolic glycoside, namely 1-O-3-hydroxyphenyl-5-methoxyphenol-(6'-O-vanilloyl)-ß-d-glucopyranoside (1), together with five known phenolic glycosides (2-6), two anthraquinones (7, 8) and two γ-butanolides (9, 10), was isolated, and its structure was elucidated by spectroscopic and chemical methods. Compounds 1-10 were screened for their in vitro cytotoxicities against HL-60, SMMC-7721, A549, MCF-3 and SW480 cell lines by the MTS method. Compounds 9 and 10 showed moderate cytotoxicities with IC50 values ranging from 17.40 to 35.21 µM.