Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

Objective Newly identified human rhinovirus C(HRV-C) and human bocavirus(HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses. Methods A platform for culturing human airway epithelia in a three-dimensional(3 D) pattern using Matrigel as scaffold was developed. The features of 3 D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3 D cells at designated time points were quantitated by real-time polymerase chain reaction(PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA. Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3 D-cultured human airway epithelial(HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3 D culture system. Conclusion Our data provide a preliminary indication that the 3 D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

SET8 Inhibition Potentiates Radiotherapy by Suppressing DNA Damage Repair in Carcinomas

Objective SET8 is a member of the SET domain-containing family and the only known lysine methyltransferase(KMT)that monomethylates lysine 20 of histone H4(H4 K20 me1).SET8 has been implicated in many essential cellular processes,including cell cycle regulation,DNA replication,DNA damage response,and carcinogenesis.There is no conclusive evidence,however,regarding the effect of SET8 on radiotherapy.In the current study we determined the efficacy of SET8 inhibition on radiotherapy of tumors and the underlying mechanism.Methods First,we explored the radiotherapy benefit of the SET8 expression signature by analyzing clinical data.Then,we measured a series of biological endpoints,including the xenograft tumor growth in mice and apoptosis,frequency of micronuclei,and foci of 53 BP1 andγ-H2 AX in cells to detect the SET8 effects on radiosensitivity.RNA sequencing and subsequent experiments were exploited to verify the mechanism underlying the SET8 effects on radiotherapy.Results Low expression of SET8 predicted a better benefit to radiotherapy in lung adenocarcinoma(LUAD)and invasive breast carcinoma(BRCA)patients.Furthermore,genetic deletion of SET8 significantly enhanced radiation treatment efficacy in a murine tumor model,and A549 and MCF7 cells;SET8 overexpression decreased the radiosensitivity.SET8 inhibition induced more apoptosis,the frequency of micronuclei,and blocked the kinetics process of DNA damage repair as 53 BP1 andγ-H2 AX foci remained in cells.Moreover,RNF8 was positively correlated with the SET8 impact on DNA damage repair.Conclusion Our results demonstrated that SET8 inhibition enhanced radiosensitivity by suppressing DNA damage repair,thus suggesting that SET8 potentiated radiotherapy of carcinomas.As new inhibitors of SET8 are synthesized and tested in preclinical and clinical settings,combining SET8 inhibitors with radiation warrants consideration for precise radiotherapy.

Meso-scale Discovery Assay Detects the Changes of Plasma Cytokine Levels in Mice after Low or High LET Ionizing Irradiation

Objective To obtain precise data on the changes in the levels of 29 cytokines in mice after high or low linear energy transfer(LET)irradiation and to develop an accurate model of radiation exposure based on the cytokine levels after irradiation.Methods Plasma samples harvested from mice at different time points after carbon-ion or X-ray irradiation were analyzed using meso-scale discovery(MSD),a high-throughput and sensitive electrochemiluminescence measurement technique.Dose estimation equations were set up using multiple linear regression analysis.Results The relative levels of IL-6 at 1 h,IL-5 and IL-6 at 24 h,and IL-5,IL-6 and IL-15 at 7 d after irradiation with two intensities increased dose-dependently.The minimum measured levels of IL-5,IL-6 and IL-15 were up to 4.0076 pg/mL,16.4538 pg/mL and 0.4150 pg/mL,respectively.In addition,dose estimation models were established and verified.Conclusions The MSD assay can provide more accurate data regarding the changes in the levels of the cytokines IL-5,IL-6 and IL-15.These cytokines could meet the essential criteria for radiosensitive biomarkers and can be used as radiation indicators.Our prediction models can conveniently and accurately estimate the exposure dose in irradiated organism.

Irradiation Response of Adipose-derived Stem Cells under Three-dimensional Culture Condition

Objective Adipose tissue distributes widely in human body. The irradiation response of the adipose cells in vivo remains to be investigated. In this study we investigated irradiation response of adipose-derived stem cells(ASCs) under three-dimensional culture condition. Methods ASCs were isolated and cultured in low attachment dishes to form three-dimensional(3D) spheres in vitro. The neuronal differentiation potential and stem-liked characteristics was monitored by using immunofluoresence staining and flow cytometry in monolayer and 3D culture. To investigate the irradiation sensitivity of 3D sphere culture, the fraction of colony survival and micronucleus were detected in monolayer and 3D culture. Soft agar assays were performed for measuring malignant transformation for the irradiated monolayer and 3D culture. Results The 3D cultured ASCs had higher differentiation potential and an higher stem-like cell percentage. The 3D cultures were more radioresistant after either high linear energy transfer(LET) carbon ion beam or low LET X-ray irradiation compared with the monolayer cell. The ASCs' potential of cellular transformation was lower after irradiation by soft agar assay. Conclusion These findings suggest that adipose tissue cell are relatively genomic stable and resistant to genotoxic stress.