Epigenetic modifications and miRNAs determine the transition of somatic cells into somatic embryos.

KEY MESSAGE: This review discusses the epigenetic changes during somatic embryo (SE) development, highlights the genes and miRNAs involved in the transition of somatic cells into SEs as a result of epigenetic changes, and draws insights on biotechnological opportunities to study SE development. Somatic embryogenesis from somatic cells occurs in a series of steps. The transition of somatic cells into somatic embryos (SEs) is the most critical step under genetic and epigenetic regulations. Major regulatory genes such as SERK, WUS, BBM, FUS3/FUSA3, AGL15, and PKL, control SE steps and development by turning on and off other regulatory genes. Gene transcription profiles of somatic cells during SE development is the result of epigenetic changes, such as DNA and histone protein modifications, that control and decide the fate of SE formation. Depending on the type of somatic cells and the treatment with plant growth regulators, epigenetic changes take place dynamically. Either hypermethylation or hypomethylation of SE-related genes promotes the transition of somatic cells. For example, the reduced levels of DNA methylation of SERK and WUS promotes SE initiation. Histone modifications also promote SE induction by regulating SE-related genes in somatic cells. In addition, miRNAs contribute to the various stages of SE by regulating the expression of auxin signaling pathway genes (TIR1, AFB2, ARF6, and ARF8), transcription factors (CUC1 and CUC2), and growth-regulating factors (GRFs) involved in SE formation. These epigenetic and miRNA functions are unique and have the potential to regenerate bipolar structures from somatic cells when a pluripotent state is induced. However, an integrated overview of the key regulators involved in SE development and downstream processes is lacking. Therefore, this review discusses epigenetic modifications involved in SE development, SE-related genes and miRNAs associated with epigenetics, and common cis-regulatory elements in the promoters of SE-related genes. Finally, we highlight future biotechnological opportunities to alter epigenetic pathways using the genome editing tool and to study the transition mechanism of somatic cells.

Engineered exosomes for targeted co-delivery of miR-21 inhibitor and chemotherapeutics to reverse drug resistance in colon cancer.

BACKGROUND: 5-Fluorouracil (5-FU) has been commonly prescribed for patients with colorectal cancer (CRC), but resistance to 5-FU is one of the main reasons for failure in CRC. Recently, microRNAs (miRNAs) have been established as a means of reversing the dilemma by regulating signaling pathways involved in initiation and progression of CRC. However, how to safely and effectively deliver miRNA to target cells becomes a main challenge. RESULTS: In this study, Engineered exosomes were exploited to simultaneously deliver an anticancer drug 5-FU and miR-21 inhibitor oligonucleotide (miR-21i) to Her2 expressing cancer cells. Purified engineered exosomes from the donor cells loaded with 5-FU and miR-21i via electroporation to introduce into 5-FU-resistant colorectal cancer cell line HCT-1165FR. Furthermore, systematic administration of 5-FU and miR-21i loaded exosomes in tumor bearing mice indicated a significantly anti-tumor effect. The results showed that the engineered exosome-based 5-FU and miR-21i co-delivery system could efficiently facilitate cellular uptake and significantly down-regulate miR-21 expression in 5-FU resistant HCT-1165FR cell lines. Consequently, the down-regulation of miR-21 induced cell cycle arrest, reduced tumor proliferation, increased apoptosis and rescued PTEN and hMSH2 expressions, regulatory targets of miR-21. Of particular importance was the significant reduction in tumor growth in a mouse model of colon cancer with systematic administration of the targeting miR-21i. More excitedly, the combinational delivery of miR-21i and 5-FU with the engineered exosomes effectively reverse drug resistance and significantly enhanced the cytotoxicity in 5-FU-resistant colon cancer cells, compared with the single treatment with either miR-21i or 5-FU. CONCLUSION: The strategy for co-delivering the functional small RNA and anticancer drug by exosomes foreshadows a potential approach to reverse the drug resistance in CRC and thus to enhance the efficacy of the cancer treatment.

Evaluation of cytotoxicity levels of poly vinyl ether silicone, polyether, and poly vinyl siloxane impression materials: An in vitro study.

AIM: To assess the cytotoxicity level of newly introduced poly vinyl ether silicone (PVES) compared to poly vinyl siloxane (PVS) and polyether (PE) elastomeric impression materials. SETTINGS AND DESIGN: Comparative -Invitro study design. MATERIALS AND METHODS: Mouse cell line NIH/3T3 was grown in Dulbecco's modified Eagle's medium. Samples of three elastomers were dissolved in dimethyl sulfoxide and were tested at various concentrations. Twenty-four well plates with NIH/3T3 cells with different concentrations of elastomeric solutions were incubated at 37°C. 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay was performed on day 1, 3, and 7, with a time interval of 15 min, 30 min, 60 min, and 24th h to estimate the cytotoxicity for all three elastomers. STATISTICAL ANALYSIS USED: Kruskal-Wallis ANOVA test and the period effect within the subjects, repeated-measure ANOVA was done using the Greenhouse-Geisser correction method. RESULTS: The mean cell viability (survival rate) of NIH 3T3 cells at the concentrations tested was measured. A repeated-measure Kruskal-Wallis ANOVA determined the mean survival concentration on day 1, 3, and 7. PVES showed significant decrease in the survival rate on day 1 than PVS and PE, while PVS and PE had significant decrease in the survival rates of cells on day 3 and 7 which were statistically significant (P < 0.001). CONCLUSION: PVES shows early cytotoxic signs as compared to PVS and PE, and cell viability for PVS was the highest among all. When making impression with PVES and PE, it is always better to evaluate the impression and gingival sulcus carefully with magnification to prevent adverse reaction, if any material is left inadvertently for longer period of time.