Mechanism of selective induction of apoptosis of HCT116 tumor cells in circulating blood by riboflavin photochemistry.

The removal or inactivation of circulating tumor cells (CTCs) can prevent distant metastasis by hematogenous route, but there is still a lack of mature and effective technical means. In the previous research, our team has initially established a method of riboflavin photosensitized treatment (RPT) for continuous treatment of peripheral blood in vitro for the inactivation of CTCs. The core of this technology is that it can selectively induce apoptosis of CTCs (HCT116 cells) without damaging immunocyte (mainly Peripheral Blood Mononuclear Cells，PBMCs) under specific parameters. To clarify the specific mechanism, firstly, the enrichment of riboflavin in HCT116 cells and PBMCs was observed under fluorescence microscope. Secondly, the apoptotic signaling pathways in HCT116 cells and PBMCs in response to RPT treatment were analyzed by transcriptomics. Finally, the mitochondrial damage in HCT116 cells and PBMCs before and after RPT treatment was observed under electron microscope. The results showed that under the same treatment conditions, HCT116 cells were significantly enriched in riboflavin compared with PBMCs. Besides, RPT treatment reduced the expression of long non﹣coding RNA (lncRNA) NEAT1, an effector gene of HCT116 cells, which further down-regulated the expression of target gene PAX2 and promoted the expression of Bax, leading to mitochondrial outer membrane permeabilization (MOMP), and consequently increased the release of pro-apoptotic factors such as cytochrome c(Cyt C), high-temperature requirement protein A2(HTRA2), apoptosis-inducing factor (AIF), endonuclease G(ENDOG), finally leading to apoptosis of HCT116 cells. In contrast, lncRNA NEAT1 remained unchanged in PBMCs before and after RPT treatment, and was unable to stimulate the PBMCs apoptotic signaling pathway. The results of the study indicated that under the specific treatment conditions, RPT technology could selectively induce apoptosis of HCT116 cells by activating the mitochondrial apoptosis pathway, which would further provide a theoretical and technical support for the effective inactivation of CTCs by RPT technology, thereby reducing the risk of recurrence of malignant tumors and improving the cure rate of malignant tumors.

Role of methylation-related genes CRYAB and SLC39A11 in the occurrence and development of lung adenocarcinoma.

Background: Previous studies have shown that human crystallin alpha B (CRYAB) is highly expressed in human cancers and associated with poor survival in cancer patients. Here we investigated whether SLC39A11 and CRYAB genes were related to the proliferation and development of lung adenocarcinoma (LUAD) to explore their potential as therapeutic targets and prognostic markers for LUAD. Methods: CRYAB and SLC39A11 genes were identified from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database. The human lung cancer cell lines A549 and H1975 were cultured, transfected, and subjected to RNA extraction. After genomic DNA removal, the RNA was reverse-transcribed. Differences between 2 groups were compared using t-test. Results: Knockdown of SLC39A11 inhibited the proliferation of LUAD cells in A549 and H1975. Knockdown of CRYAB promoted the increase of LUAD cell clones, while knockdown of SLC39A11 suppressed LUAD cell clones. In both A549 and H1975 cell lines, knockout of CRYAB inhibited the apoptosis of LUAD cells, whereas knockout of SLC39A11 promoted the apoptosis of LUAD cells. In the H1975 cell line, knockout of CRYAB also lowered the proportion of cells in interphase and increased the proportion of mitotic cells, while knockout of SLC39A11 also slowed down the division cycle of tumor cells. Knockdown of CRYAB promoted the migration of LUAD cells in both the A549 cell line and H1975 cell line. In the H1975 cell line, knockout of SLC39A11 also reduced the invasive ability of LUAD cells. Conclusions: CRYAB and SLC39A11 could be used as prognostic indicators and therapeutic targets for LUAD.

Identification of molecular subtypes in lung adenocarcinoma based on DNA methylation and gene expression profiling-a bioinformatic analysis.

Background: Molecular typing based on deoxyribonucleic acid (DNA) methylation and gene expression can extend understandings of the molecular mechanisms involved in lung adenocarcinoma (LUAD) and enhance current diagnostic, treatment, and prognosis prediction approaches. Methods: Gene expression and DNA methylation data sets of LUAD were obtained from The Cancer Genome Atlas (TCGA), and the differential gene and methylation expression levels were analyzed. Results: We successfully divided the LUAD samples into 2 clinically relevant subtypes with significantly different survival times and tumor stages according to the transcriptome and methylation data. We found significant differences in the survival status, age, gender, tumor stage, node stage, and clinical stage between the 2 subtypes. The hub genes identified in the subnetworks, including NCAPG, CCNB1, DLGAP5, HLA-DQA1, HLA-DPA1, HLA-DPB1, SFTP, SCGBA1A, and SFTPD, were correlated with the cell cycle and immune system. The Gene Ontology annotation of the hub genes showed that the biological processes included organelle fission mitotic nuclear division, and sister chromatid segregation. The cellular components included chromosomal region, spindle, and kinetochore. The molecular functions included tubulin-binding, microtubule-binding, and DNA replication origin binding. The Kyoto Encyclopedia of Genes and Genomes signaling pathways related to the hub genes mainly included the cell cycle, human T-cell leukemia virus (type 1) infection, inflammatory bowel disease, and the intestinal immune network for immunoglobulin A production. The clinical stage difference was also confirmed in the validation group using the GSE32863 data set. Conclusions: Our findings extend understandings of the pathogenesis of LUAD and can be used to improve current diagnosis, treatment, and prognosis prediction strategies.

[A Novel Lyophilized PRP-Loaded Gel Based on Chitosan and PEG with Hemostatic and Antibacterial Properties].

OBJECTIVE: A dynamic gel loaded with lyophilized platelet-rich plasma-chitosan/difunctionalized polyethylene glycol (LPRP-CP) was prepared to investigate its hemostatic antibacterial and promoting wound healing of scald wounds through in vitro and in vivo experiments. METHODS: In this study, normal gauze/blank tablet (Ctrl), LPRP-CP, Chitosan HUCHUANG Powder(Chito P)and ChitoGauze XP PRO group (Chito G group) were set. The hemostatic effect and promoting healing effect of the four groups of materials were evaluated by establishing rabbit ear artery hemorrhage model and superficial Ⅱ° scalded model of skin on the back. The hemostatic time and bleeding amount were calculated and the gross and histological results of scald healing were observed. The antibacterial effect of the four groups of materials was evaluated by antibacterial test in vitro. RESULTS: In the rabbit ear arterial hemorrhage model, the hemostasis of all materials was successful. The hemostatic time of Ctrl, Chito P, LPRP-CP and Chito G groups was 213.33±38.30, 118.33±24.01, 115.00±8.37 and 111.67±11.69 s, respectively. The blood loss was 1233.83±992.27, 346.67±176.00, 193.33±121.47 and 147.50±80.66 mg, respectively. Compared with Ctrl, the hemostasis time of LPRP-CP, Chito P and Chito G group was significantly shorter (P<0.001), and the amount of blood loss of LPRP-CP and Chito G group was decreased (P<0.05). Compared with LPRP-CP, there were no significant differences in hemostatic time and blood loss between Chito P and Chito G group (P>0.05). In the model of superficial Ⅱ° scalded on the back of rabbit, the wound healing rate of LPRP-CP was faster than that of the other three groups at the same time, and the healing effect was perfect. In the antibacterial test in vitro, only LPRP-CP had better anti-S. aureus effect, and all groups had no anti-E. coli effect. CONCLUSION: LPRP-CP is an excellent hemostatic material for superficial wounds, and has certain antibacterial and wound healing effects, which has a wide academic value and research prospects.

Development of Human Lung Induction Models for Air Pollutants' Toxicity Assessment.

There is an urgent need for reliable and effective models to study air pollution health effects on human lungs. Here, we report the utilization of human pluripotent stem cell (hPSC) induction models for human lung progenitor cells (hLPs) and alveolar type 2 epithelial cell-like cells (ATLs) for the toxicity assessment of benzo(a)pyrene, nano-carbon black, and nano-SiO2, as common air pollutants. We induced hPSCs to generate ATLs, which recapitulated key features of human lung type 2 alveolar epithelial cells, and tested the induction models for cellular uptake of nanoparticles and toxicity evaluations. Our findings reveal internalization of nano-carbon black, dose-dependent uptake of nano-SiO2, and interference with surfactant secretion in ATLs exposed to benzo(a)pyrene/nano-SiO2. Thus, hLP and ATL induction models could facilitate the evaluation of environmental pollutants potentially affecting the lungs. In conclusion, this is one of the first studies that managed to adopt hPSC pulmonary induction models in toxicology studies.

TBBPA, TBBPS, and TCBPA disrupt hESC hepatic differentiation and promote the proliferation of differentiated cells partly via up-regulation of the FGF10 signaling pathway.

Halogenated flame retardants (HFRs), including Tetrabromobisphenol A (TBBPA), Tetrabromobisphenol S (TBBPS), and Tetrachlorobisphenol A (TCBPA), are widely applied in the manufacturing industry to improve fire safety and can be detected in pregnant women's serum at nanomolar levels. Thus, it is necessary to pay attention to the three HFR potential development toxicity, which has not been conclusively addressed yet. The liver is the main organ that detoxifies our body; TBBPA exposure may lead to increased liver weight in rodents. Therefore, in this study, we assessed the developmental hepatic toxicity of the three HFRs with a human embryonic stem cell hepatic differentiation-based system and transcriptomics analyses. We mostly evaluated lineage fate alterations and demonstrated the three HFRs may have common disruptive effects on hepatic differentiation, with TCBPA being significantly more potent. More specifically, the three HFRs up-regulated genes related to cell cycle and FGF10 signaling, at late stages of the hepatic differentiation. This indicates the three chemicals promoted hepatoblast proliferation likely via up-regulating the FGF10 cascade. At the same time, we also presented a powerful way to combine in vitro differentiation and in silico transcriptomic analyses, to efficiently evaluate hazardous materials' adverse effects on lineage fate decisions during early development.

Evaluation of the effects of low nanomolar bisphenol A-like compounds' levels on early human embryonic development and lipid metabolism with human embryonic stem cell in vitro differentiation models.

The widely used chemical bisphenol A (BPA) has been associated with several health effects. In recent years, many derivatives were developed to replace BPA although without thorough toxicological evaluation. Here, we employed a human embryoid body (EB)-based in vitro global differentiation and hepatic specification models, followed by RNA-seq analyses, to comprehensively study the potential developmental toxicity of six BPA replacements (BPS, BPF, BPZ, BPB, BPE, and BPAF), as compared to BPA. We found that those bisphenols may disrupt lineage commitment and lipid metabolism during early embryonic development. These effects mostly manifested via the dysregulation of HOX and APO family genes. Moreover, among the seven bisphenols analyzed, BPE seemed to have the mildest effects.

F-53B and PFOS treatments skew human embryonic stem cell in vitro cardiac differentiation towards epicardial cells by partly disrupting the WNT signaling pathway.

F-53B and PFOS are two per- and polyfluoroalkyl substances (PFASs) widely utilized in the metal plating industry as mist suppressants. Recent epidemiological studies have linked PFASs to cardiovascular diseases and alterations in heart geometry. However, we still have limited understanding of the effects of F-53B and PFOS on the developing heart. In this study, we employed a human embryonic stem cell (hESC)-based cardiac differentiation system and whole transcriptomics analyses to evaluate the potential developmental cardiac toxicity of F-53B and PFOS. We utilized F-53B and PFOS concentrations of 0.1-60 µM, covering the levels detected in human blood samples. We demonstrated that both F-53B and PFOS inhibited cardiac differentiation and promoted epicardial specification via upregulation of the WNT signaling pathway. Most importantly, the effects of F-53B were more robust than those of PFOS. This was because F-53B treatment disrupted the expression of more genes and led to lower cardiac differentiation efficiency. These findings imply that F-53B may not be a safe replacement for PFOS.

Bisphenol A and several derivatives exert neural toxicity in human neuron-like cells by decreasing neurite length.

Bisphenol A (BPA) and its derivatives, including bisphenols S (BPS), F (BPF), E (BPE), B (BPB), Z (BPZ), and AF (BPAF), are widely used in consumer products. Moreover, they are typically detected in the environment, food, and humans. Previous studies have linked BPA to several health risks, but it is still unclear whether BPA replacements are safe. In this study, we developed an in vitro model based on human embryonic stem cells (hESCs) to explore the potential neural toxicity of these compounds. We observed that the bisphenols affected the viability of hESCs and hESC-derived neural stem cells (NSCs) at high concentrations, with BPS being the least cytotoxic and BPAF the strongest cytotoxic compound. At human-relevant concentrations, the bisphenols did not significantly interfere with gene expression and protein levels during hESC differentiation into the neural epithelium, as well as during specification of neuron-like cells from NSCs. Nevertheless, monitoring of cell morphology changes indicated that exposure to BPA and its derivatives impaired neurite length in neuron-like cells. Thus, our findings provide insights into the molecular mechanisms of bisphenol-dependent neurotoxicity at low nanomolar levels and support the view that BPA substitutes may not be sufficiently safe for widespread use as industrial chemicals.

A human embryonic stem cell-based in vitro model revealed that ultrafine carbon particles may cause skin inflammation and psoriasis.

Air pollution has been linked to many health issues, including skin conditions, especially in children. Among all the atmospheric pollutants, ultrafine particles have been deemed very dangerous since they can readily penetrate the lungs and skin, and be absorbed into the bloodstream. Here, we employed a human embryonic stem cell (hESC)-based differentiation system towards keratinocytes, to test the effects of ultrafine carbon particles, which mimic ambient ultrafine particles, at environment related concentrations. We found that 10 ng/mL to 10 µg/mL ultrafine carbon particles down-regulated the expression of the pluripotency marker SOX2 in hESCs. Moreover, 1 µg/mL to 10 µg/mL carbon particle treatments disrupted the keratinocyte differentiation, and up-regulated inflammation- and psoriasis-related genes, such as IL-1ß, IL-6, CXCL1, CXCL2, CXCL3, CCL20, CXCL8, and S100A7 and S100A9, respectively. Overall, our results provide a new insight into the potential developmental toxicity of atmospheric ultrafine particles.

New insights into mechanism of bisphenol analogue neurotoxicity: implications of inhibition of O-GlcNAcase activity in PC12 cells.

Bisphenol analogues including bisphenol A and its derivatives are ubiquitous environmental contaminants and have been linked to adverse neurodevelopment effects on animals and humans. Most toxicological research focused on estrogen receptor mediated pathways and did not comprehensively clarify the observed toxicity. O-GlcNAcase (OGA), the highest level in brain, plays a critical role in controlling neuronal functions at multi-levels from molecule to animal behaviors. In this work, we intend to investigate the underlying molecular mechanisms for the neurotoxicity of bisphenol analogues by identifying their cellular targets and the resultant effects. The inhibitory actions of seven bisphenol analogues on the OGA activity at molecular level were investigated by our developed electrochemical biosensor. We found that their potency varied with substituent groups, in which tetrabromo bisphenol A (TBBPA) was the strongest. The seven bisphenol analogues (0-100 µM exposure) significantly inhibited OGA activity and up-regulated protein O-GlcNAcylation level in PC12 cells. Inhibition of OGA by bisphenol analogues further induced intracellular calcium, ROS, inflammation, repressed proliferation, interfered with cell cycle, induced apoptosis. And especially, 10 µM tetrabromo bisphenol A (TBBPA) exposure could impair the growth and development of neurite in human neural stem cells (hNSCs). Molecular docking for OGA/bisphenol analogue complexes revealed the hydrophobicity-dominated inhibition potency. OGA, as a new cellular target of bisphenol analogues, would illuminate the molecular mechanism of bisphenol analogues neurotoxicity.

Embryonic stem cell- and transcriptomics-based in vitro analyses reveal that bisphenols A, F and S have similar and very complex potential developmental toxicities.

Bisphenol A (BPA) is a very versatile industrial chemical. Many reports have associated BPA with several health effects. Some bisphenol alternatives have been introduced to replace BPA in its many applications. However, comprehensive toxicological evaluations for these replacements are still lacking. In this study, we examined the potential effects of BPA, bisphenol F (BPF) and bisphenol S (BPS), on embryonic development with an in vitro stem cell toxicology system and transcriptomics analyses. Mouse embryonic stem cells (mESCs) were differentiated via embryoid body formation, either globally towards the three primary germ layers and their lineages, or specifically into neuroectoderm/neural progenitor cells. During the differentiation, cells were treated with BPA, BPF, BPS, or DMSO control. Samples were collected at different time points, for qRT-PCR and RNA-seq analyses. BPA, BPF and BPS disrupted many processes, during mESC global and neural differentiations, in very similar manners. In fact, at each time point the three chemicals differentially regulated analogous gene categories, particularly the ones involved in cell-matrix and cell-cell adhesion, signal transduction pathways, and medical conditions such as cardiovascular diseases and cancer. Our findings demonstrate once more then BPA substitutes may not be very safe. They potentially have a very complex developmental toxicity, similarly to BPA, and seem more toxic than BPA itself. In addition, our results reveal that stem cell-based developmental toxicity assays can be very comprehensive.

Metabolic and transcriptional alternations for defense by interfering OsWRKY62 and OsWRKY76 transcriptions in rice.

Metabolomic and transcriptomic approaches were used to dissect the enhanced disease resistance in the plants harbouring a RNA interfering construct of OsWRKY62 and OsWRKY76 (dsOW62/76) genes. The primary metabolic pathways were activated in dsOW62/76 compared with wild-type (ZH17) plants, revealed by increased accumulation of amino acids and constituents of citric acid cycle etc. Contents of phenolic acids derived from phenylpropanoid pathway were elevated in dsOW62/76 plants. Importantly, phenolamides, conjugates of the phenolic acids with amines, were detected in large number and mostly at higher levels in dsOW62/76 compared with ZH17 plants; however, the free pools of flavonoids were mostly decreased in dsOW62/76. Salicylic acid (SA) and jasmonic acid (JA)/JA-Ile contents were increased in dsOW62/76 and knockout lines of individual OsWRKY62 and OsWRKY76 genes. Transcription of isochorismate synthase (OsICS1) gene was suppressed in dsOW62/76 and in MeJA-treated rice plants, whereas the transcription level of cinnamoyl-CoA hydratase-dehydrogenase (OsCHD) gene for ß-oxidation in peroxisome was increased. The calli with OsCHD mutation showed markedly decreased SA accumulation. These results indicate that OsWRKY62 and OsWRKY76 function as negative regulators of biosynthetic defense-related metabolites and provide evidence for an important role of phenylpropanoid pathway in SA production in rice.

Alternative Splicing of Rice WRKY62 and WRKY76 Transcription Factor Genes in Pathogen Defense.

The WRKY family of transcription factors (TFs) functions as transcriptional activators or repressors in various signaling pathways. In this study, we discovered that OsWRKY62 and OsWRKY76, two genes of the WRKY IIa subfamily, undergo constitutive and inducible alternative splicing. The full-length OsWRKY62.1 and OsWRKY76.1 proteins formed homocomplexes and heterocomplexes, and the heterocomplex dominates in the nuclei when analyzed in Nicotiana benthamiana leaves. Transgenic overexpression of OsWRKY62.1 and OsWRKY76.1 in rice (Oryza sativa) enhanced plant susceptibility to the blast fungus Magnaporthe oryzae and the leaf blight bacterium Xanthomonas oryzae pv oryzae, whereas RNA interference and loss-of-function knockout plants exhibited elevated resistance. The dsOW62/76 and knockout lines of OsWRKY62 and OsWRKY76 also showed greatly increased expression of defense-related genes and the accumulation of phytoalexins. The ratio of full-length versus truncated transcripts changed in dsOW62/76 plants as well as in response to pathogen infection. The short alternative OsWRKY62.2 and OsWRKY76.2 isoforms could interact with each other and with full-length proteins. OsWRKY62.2 showed a reduced repressor activity in planta, and two sequence determinants required for the repressor activity were identified in the amino terminus of OsWRKY62.1. The amino termini of OsWRKY62 and OsWRKY76 splice variants also showed reduced binding to the canonical W box motif. These results not only enhance our understanding of the DNA-binding property, the repressor sequence motifs, and the negative feedback regulation of the IIa subfamily of WRKYs but also provide evidence for alternative splicing of WRKY TFs during the plant defense response.

The evolution and pathogenic mechanisms of the rice sheath blight pathogen.

Rhizoctonia solani is a major fungal pathogen of rice (Oryza sativa L.) that causes great yield losses in all rice-growing regions of the world. Here we report the draft genome sequence of the rice sheath blight disease pathogen, R. solani AG1 IA, assembled using next-generation Illumina Genome Analyser sequencing technologies. The genome encodes a large and diverse set of secreted proteins, enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, which probably reflect an exclusive necrotrophic lifestyle. We find few repetitive elements, a closer relationship to Agaricomycotina among Basidiomycetes, and expand protein domains and families. Among the 25 candidate pathogen effectors identified according to their functionality and evolution, we validate 3 that trigger crop defence responses; hence we reveal the exclusive expression patterns of the pathogenic determinants during host infection.

Rapid detection of vip1-type genes from Bacillus cereus and characterization of a novel vip binary toxin gene.

A PCR-restriction fragment length polymorphism (PCR-RFLP) method for identifying vegetative insecticidal protein (vip) 1-type genes from Bacillus cereus was developed by designing specific primers based on the conserved regions of the genes to amplify vip1-type gene fragments. PCR products were digested with endonuclease AciI, and four known vip1-type genes were identified. Vip1Ac and vip1Aa-type genes appeared in 17 of 26 B. cereus strains. A novel vip1-type gene, vip1Ac1, was identified from B. cereus strain HL12. The vip1Ac1 and vip2Ae3 genes were co-expressed in Escherichia coli strain BL21 by vector pCOLADuet-1. The binary toxin showed activity only against Aphis gossypii (Homoptera), but not for Coleptera (Tenebrio molitor, Holotrichia oblita), Lepidoptera (Spodoptera exigua, Helicoverpa armigera, and Chilo suppressalis), Diptera (Culex quinquefasciatus). The LC(50) of this binary toxin for A. gossypii is 87.5 (34.2-145.3) ng mL(-1) . This is probably only the second report that Vip1 and Vip2 binary toxin shows toxicity against homopteran pests. The PCR-RFLP method developed could be very useful for identifying novel Vip1-Vip2-type binary toxins, and the novel binary toxins, Vip1Ac1 and Vip2Ae3, identified in this study may have applications in biological control of insects, thus avoiding potential problems of resistance.