Identify organic contaminants of high-concern based on non-targeted toxicity testing and non-targeted LC-HRMS analysis in tap water and source water along the Yangtze River.

Many organic pollutants were detected in tap water (TW) and source water (SW) along the Yangtze River. However, the potential toxic effects and the high-concern organics (HCOs) which drive the effect are still unknown. Here, a non-targeted toxicity testing method based on the concentration-dependent transcriptome and non-targeted LC-HRMS analysis combining tiered filtering were used to reveal the overall biological effects and chemical information. Subsequently, we developed a qualitative pathway-structure relationship (QPSR) model to effectively match the biological and chemical information and successfully identified HCOs in TW and SW along the Yangtze River by potential substructures of HCOs. Non-targeted toxicity testing found that the biological potency of both TW and SW was stronger in the downstream of the Yangtze River, and disruption of the endocrine system and cancer were the main drivers of the effect. In addition, non-targeted LC-HRMS analysis combined with retention time prediction results identified 3220 and 631 high-confidence compound structures in positive and negative ion modes, respectively. Then, QPSR model was further implied and identified a total of 103 HCOs, containing 35 industrial chemicals, 30 PPCPs, 26 pesticides, and 12 hormones in TW and SW, respectively. Among them, the neuroactive and hormonal compounds oxoamide, 8-iso-16-cyclohexyl-tetranor prostaglandin E2, E Keppra, and Tocris-0788 showed the highest frequency of detection, which were identified in more than 1/3 of the samples. The strategy of combining non-targeted toxicity testing and non-targeted LC-HRMS analysis will support comprehensive biological effect assessment, identification of HCOs, and risk control of mixtures.

Porcine Kidney Organoids Derived from Naïve-like Embryonic Stem Cells.

The scarcity of donor kidneys greatly impacts the survival of patients with end-stage renal failure. Pigs are increasingly becoming potential organ donors but are limited by immunological rejection. Based on the human kidney organoid already established with the CHIR99021 and FGF9 induction strategy, we generated porcine kidney organoids from porcine naïve-like ESCs (nESCs). The derived porcine organoids had a tubule-like constructure and matrix components. The porcine organoids expressed renal markers including AQP1 (proximal tubule), WT1 and PODO (podocyte), and CD31 (vascular endothelial cells). These results imply that the organoids had developed the majority of the renal cell types and structures, including glomeruli and proximal tubules. The porcine organoids were also identified to have a dextran absorptive function. Importantly, porcine organoids have a certain abundance of vascular endothelial cells, which are the basis for investigating immune rejection. The derived porcine organoids might serve as materials for immunosuppressor screening for xenotransplantation.

Immunotoxicity Evaluation of Trihalophenolic Disinfection By-Products in Mouse and Human Mononuclear Macrophage Systems: The Role of RNA Epitranscriptomic Modification in Mammalian Immunity.

BACKGROUND: 2,4,6-Trichlorophenol (TCP), 2,4,6-tribromophenol (TBP) and 2,4,6-triiodophenol (TIP) are three widely detected trihalophenolic disinfection by-products (DBPs). Previous studies have mainly focused on the carcinogenic risk and developmental toxicity of 2,4,6-trihalophenols. Very little is known about their immunotoxicity in mammals. OBJECTIVES: We investigated the effects of 2,4,6-trihalophenols on mammalian immunity using a mouse macrophage model infected with bacteria or intracellular parasites and aimed to elucidate the underlying mechanisms from an epitranscriptomic perspective. The identified mechanisms were further validated in human peripheral blood mononuclear cells (PBMCs). METHODS: The mouse macrophage cell line RAW264.7 and primary mouse peritoneal macrophages were exposed to different concentrations of TCP, TBP, and TIP. The pro-inflammatory marker Ly6C, the survival of the bacterium Escherichia coli (E. coli), and the parasite burden of Toxoplasma gondii (T. gondii) were assessed. Furthermore, the global gene expression profiling of macrophages following exposure to 2,4,6-trihalophenols was obtained through RNA-sequencing (RNA-seq). The effects of 2,4,6-trihalophenols on RNA N6-methyladenosine (m6A) methyltransferases and total RNA m6A levels were evaluated using Western blotting and dot blot, respectively. Transcriptome-wide m6A methylome was analyzed by m6A-seq. In addition, expression of m6A regulators and total RNA m6A levels in human PBMCs exposed to 2,4,6-trihalophenols were detected using quantitative reverse transcriptase polymerase chain reaction and dot blot, respectively. RESULTS: Mouse macrophages exposed to TCP, TBP, or TIP had lower expression of the pro-inflammatory marker Ly6C, with a greater difference from control observed for TIP-exposed cells. Consistently, macrophages exposed to such DBPs, especially TIP, were susceptible to infection with the bacterium E. coli and the intracellular parasite T. gondii, indicating a compromised ability of macrophages to defend against pathogens. Intriguingly, macrophages exposed to TIP had significantly greater m6A levels, which correlated with the greater expression levels of m6A methyltransferases. Macrophages exposed to each of the three 2,4,6-trihalophenols exhibited transcriptome-wide redistribution of m6A. In particular, the m6A peaks in genes associated with immune-related pathways were altered after exposure. In addition, differences in m6A were also observed in human PBMCs after exposure to 2,4,6-trihalophenols. DISCUSSION: These findings suggest that 2,4,6-trihalophenol exposure impaired the ability of macrophages to defend against pathogens. This response might be associated with notable differences in m6A after exposure. To the best of our knowledge, this study presents the first m6A landscape across the transcriptome of immune cells exposed to pollutants. However, significant challenges remain in elucidating the mechanisms by which m6A mediates immune dysregulation in infected macrophages after 2,4,6-trihalophenol exposure. https://doi.org/10.1289/EHP11329.

Choroidal vascular changes in silicone oil-filled eyes after vitrectomy for rhegmatogenous retinal detachments.

INTRODUCTION: The tamponade of silicone oil (SO) can affect both the structure and blood flow of the retina. However, there are few studies on the effect of SO tamponade on choroidal blood flow. Our study aimed to compare the effects of SO tamponade on the choroidal vascular index (CVI) and choroidal thickness (CT) in patients with unilateral rhegmatogenous retinal detachment (RRD) with operated eyes and fellow healthy eyes. METHODS: We retrospectively collected demographic and clinical data from 36 patients who underwent 23G pars plana vitrectomy and SO tamponade for unilateral complicated RRD. Enhanced depth imaging-optical coherence tomography (EDI-OCT) scans were performed both within 1 week before SO removal and at the last follow-up visit after SO removal. Using ImageJ software, images were binarized to segment the total choroidal area, luminal area, and stromal area, respectively. The CVI was calculated as CVI=(luminal area)/(total choroidal area), and CT was also evaluated. RESULTS: During SO tamponade, the CVI and luminal area in operated eyes were significantly lower compared to fellow eyes (57.616 ± 0.030 vs. 60.042 ± 0.019, P < 0.0001; 0.909 [0.694; 1.185] vs. 1.091 [0.785; 1.296], P = 0.007). Even after SO removal, the CVI remained lower in operated eyes than in fellow eyes (59.530 ± 0.018 vs. 60.319 ± 0.020, P = 0.031). Both CVI and luminal area were lower in operated eyes before SO removal than after SO removal (57.616 ± 0.030 vs. 59.530 ± 0.018, P = 0.0003; 0.909 [0.694; 1.185] vs. 0.994 [0.712; 1.348], P = 0.028). The duration of SO tamponade was positively correlated with the difference in CVI between fellow eyes and operated eyes during SO tamponade (P = 0.035). Total choroidal area, stromal area, and CT did not differ significantly between fellow eyes and operated eyes or between pre- and post-SO removal. CONCLUSIONS: SO tamponade reduces CVI and decreases choroidal blood circulation in patients with retinal detachments required vitrectomy combined with SO tamponade. The longer the SO tamponade time, the more CVI reduction. In future work, we will aim to reduce these side effects by shortening the duration of silicone oil filling.

Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2.

A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.

Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States.

The inner cell mass of the pre-implantation blastocyst consists of the epiblast and hypoblast from which embryonic stem cells (ESCs) and extra-embryonic endoderm (XEN) stem cells, respectively, can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of its in vivo tissue origin. We have developed a novel approach for deriving porcine XEN (pXEN) cells via culturing the blastocysts with a chemical cocktail culture system. The pXEN cells were positive for XEN markers, including Gata4, Gata6, Sox17, and Sall4, but not for pluripotent markers Oct4, Sox2, and Nanog. The pXEN cells also retained the ability to undergo visceral endoderm (VE) and parietal endoderm (PE) differentiation in vitro. The maintenance of pXEN required FGF/MEK+TGFß signaling pathways. The pXEN cells showed a stable phenotype through more than 50 passages in culture and could be established repeatedly from blastocysts or converted from the naïve-like ESCs established in our lab. These cells provide a new tool for exploring the pathways of porcine embryo development and differentiation and providing further reference to the establishment of porcine ESCs with potency of germline chimerism and gamete development.

Generation of Urine-Derived Induced Pluripotent Stem Cell Line from Patients with Acute Kidney Injury.

Acute kidney injury (AKI) is mainly characterized by rapid decline of renal function. Currently, the strategy of stem cells might be a therapy to treat AKI. The objective of this study was to obtain human urine-derived cells (HUCs) from patients with AKI, followed by establishing induced pluripotent stem (iPS) cell line. We isolated urine cells from patients with AKI and found that the cells could survive long term with epithelioid morphology and maintain a normal karyotype. The cell line had expression of renal-specific markers and renal development-related genes. After induction, the urine cells cotransfecting with TET-ON vectors were converted into iPS cells. The HUC-derived iPS (HUC-iPS) was positive for alkaline phosphatase staining, and had expression of pluripotency markers, consistent with human embryonic fibroblast-derived iPS cell. Notably, HUC-iPS could be induced to undergo directional kidney precursor cells (KPCs) differentiation under defined conditions, and transplantation of KPCs resulted in reducing kidney damage from ischemia-reperfusion injury in mice. Therefore, we successfully established HUC-iPS cell from patients with AKI and provided a novel stem cell resource for cell therapy in AKI.

SIX1 Activation Is Involved in Cell Proliferation, Migration, and Anti-inflammation of Acute Ischemia/Reperfusion Injury in Mice.

Nephrogenic proteins are re-expressed after ischemia/reperfusion (I/R) injury; however, the role of these proteins is still unknown. We found that sine oculis homeobox 1 (SIX1), a developmentally regulated homeoprotein, is reactivated in tubular epithelial cells after I/R injury associated with cell proliferation/migration and anti-inflammation. We demonstrated that SIX1 promoted cell proliferation by upregulating cyclin and glycolytic genes, and might increase cell migration by upregulating the expression of matrix metalloproteinase 9 (MMP9) directly or indirectly in the cell model. Notably, SIX1 targeted the promoters of the amino-terminal enhancer of split (AES) and fused in sarcoma (FUS), which are cofactors of nuclear factor-κB (NF-κB) subunit RELA, and then inhibited the transactivation function of RELA. The expression of monocyte chemotactic protein-1 (MCP-1) was decreased by the SIX1-mediated NF-κB pathway. Our results showed that the expression of cyclin, glycolytic genes, and MMP9 were significantly increased, and the infiltration of monocytes/macrophages (Mophs) was suppressed in SIX1 overexpression kidney at 1, 2, and 3 days after reperfusion. The overexpression of SIX1 resulted in reducing kidney damage from I/R injury in mice by promoting cell proliferation and migration and by inhibiting inflammation. Our study provides evidence that SIX1 involved in cell proliferation, migration, and anti-inflammation in the I/R model, which might be a potential therapeutic target that could be used to ameliorate kidney damage.

CRISPR/Cas9-Mediated α-ENaC Knockout in a Murine Pancreatic ß-Cell Line.

Many ion channels participate in controlling insulin synthesis and secretion of pancreatic ß-cells. Epithelial sodium channel (ENaC) expressed in human pancreatic tissue, but the biological role of ENaC in pancreatic ß-cells is still unclear. Here, we applied the CRISPR/Cas9 gene editing technique to knockout α-ENaC gene in a murine pancreatic ß-cell line (MIN6 cell). Four single-guide RNA (sgRNA) sites were designed for the exons of α-ENaC. The sgRNA1 and sgRNA3 with the higher activity were constructed and co-transfected into MIN6 cells. Through processing a series of experiment flow included drug screening, cloning, and sequencing, the α-ENaC gene-knockout (α-ENaC-/-) in MIN6 cells were obtained. Compared with the wild-type MIN6 cells, the cell viability and insulin content were significantly increased in α-ENaC-/- MIN6 cells. Therefore, α-ENaC-/- MIN6 cells generated by CRISPR/Cas9 technology added an effective tool to study the biological function of α-ENaC in pancreatic ß-cells.

LncRNA NEAT1 Regulates 5-Fu Sensitivity, Apoptosis and Invasion in Colorectal Cancer Through the MiR-150-5p/CPSF4 Axis.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent malignancies in the world. Long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) is involved in the development of many cancers. However, its role and mechanism in CRC progression still need further exploration. METHODS: The expression levels of lnc-NEAT1, microRNA-150-5p (miR-150-5p) and cleavage and polyadenylation specific factor 4 (CPSF4) were determined by quantitative real-time PCR (qRT-PCR). The sensitivity of cells to 5-fluorouracil (5-Fu) was measured by 3-(4,5-dimethyl-2 thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell apoptosis and invasion were evaluated by flow cytometry and transwell assays, respectively. Western blot (WB) analysis was used to assess the levels of resistance-related proteins and CPSF4 protein. Besides, dual-luciferase reporter assay was used to verify the interactions among lnc-NEAT1, miR-150-5p and CPSF4. Also, mice xenograft models were used to determine the effect of lnc-NEAT1 on CRC tumor growth in vivo. RESULTS: In CRC, the expression of lnc-NEAT1 was upregulated and miR-150-5p was downregulated, and the expression of both was negatively correlated. Silencing of lnc-NEAT1 promoted the 5-Fu sensitivity, enhanced the apoptosis and suppressed the invasion of CRC cells. MiR-150-5p could be sponged by lnc-NEAT1, and its inhibitors could partially reverse the effect of lnc-NEAT1 silencing on CRC progression. Besides, CPSF4 could be targeted by miR-150-5p, and its overexpression also could invert the effect of lnc-NEAT1 knockdown on CRC progression. Further, CPSF4 expression was regulated by lnc-NEAT1 and miR-150-5p. In addition, interference of lnc-NEAT1 reduced tumor volume and improved the sensitivity of CRC to 5-Fu in vivo. CONCLUSION: Lnc-NEAT1 acted as an oncogene in CRC through regulating CPSF4 expression by sponging miR-150-5p. The discovery of lnc-NEAT1/miR-150-5p/CPSF4 axis provided a novel approach for CRC genomic therapy strategy.

Preparation of Poloxamer188-b-PCL and Study on in vitro Radioprotection Activity of Curcumin-Loaded Nanoparticles.

A novel polymer of poloxamer188-b-PCL was synthesized via a ring-opening polymerization. Fourier transform infrared spectroscopy (FTIR), Raman, and 1H nuclear magnetic resonance (1H NMR) spectra were used to study the structures of obtained poloxamer188-b-PCL. The thermo-stability of poloxamer188 -b-PCL was carried out with a thermal gravimetric analyzer (TGA), and cytotoxicity was obtained using the CCK8 method. Cargo-free and curcumin (CUR)-loaded poloxamer188-b-PCL NPs were fabricated via the solvent evaporation method. The morphology, particle size distribution, and stability of cargo-free NPs were studied with a scanning electron microscope (SEM) and laser particle analyzer. The in vitro radioprotection activity of CUR-loaded NPs was performed. FTIR, Raman, and 1H NMR spectra confirmed that poloxamer188-b-PCL was obtained. TGA curves suggested poloxamer188-b-PCL had better thermo-stability than original poloxamer188. Cell tests suggested that the cargo-free NPs had no cytotoxicity. SEM image showed that the cargo-free NPs were spherical with a diameter of 100 nm. Free radical scavenging experiments proved that CUR-loaded NPs had better antioxidant activity than CUR solutions. CUR-loaded NPs could be detected in all tissues, including liver, kidneys and lung. In summary, this work demonstrated a feasibility of developing an injective formulation of CUR and provided a protection agent in caner radiotherapy.

Clinical Significance of 4L Lymph Node Dissection in Left Lung Cancer.

PURPOSE: To investigate the prognostic impact of 4L lymph node (LN) dissection in left lung cancer and to analyze the relative risk factors for 4L LN metastasis. PATIENTS AND METHODS: We retrospectively collected data from 657 patients with primary left lung cancer who underwent surgical pulmonary resection from January 2005 to December 2009. One hundred thirty-nine patients underwent 4L LN dissection (4LD+ group); the other 518 patients did not receive 4L LN dissection (4LD- group). Propensity score weighting was applied to reduce the effects of observed confounding between the two groups. Study end points were disease-free survival (DFS) and overall survival (OS). RESULTS: The metastasis rate of station 4L was 20.9%, which was significantly higher than those of station 7 (14.0%; P = .048) and station 9 (9.8%; P < .001). Station 4L metastasis was associated with most other LN station metastases in univariate analysis, but only station 10 LN metastasis was an independent risk factor for 4L LN metastasis (odds ratio, 0.253; 95% CI, 0.109 to 0.588; P = .001) in multivariate logistic analysis. The 4LD+ group had a significantly better survival than the 4LD- group (5-year DFS, 54.8% v 42.7%; P = .0376; 5-year OS, 58.9% v 47.2%; P = .0200). After allowing potential confounders in multivariate survival analysis, dissection of 4L LN retained its independent favorable effect on DFS (hazard ratio, 1.502; 95% CI, 1.159 to 1.947; P = .002) and OS (hazard ratio, 1.585; 95% CI, 1.222 to 2.057; P = .001). Propensity score weighting further confirmed that the 4LD+ group had a more favorable DFS ( P = .0014) and OS ( P < .001) than the 4LD- group. CONCLUSION: Station 4L LN involvement is not rare in left lung cancer, and dissection of the 4L LN station seems to be associated with a more favorable prognosis as compared with those who did not undergo this dissection.

Identification of preoperative prediction factors of tumor subtypes for patients with solitary ground-glass opacity pulmonary nodules.

BACKGROUND: Recent wide spread use of low-dose helical computed tomography for the screening of lung cancer have led to an increase in the detection rate of very faint and smaller lesions known as ground-glass opacity nodules. The purpose of this study was to investigate the clinical factors of lung cancer patients with solitary ground-glass opacity pulmonary nodules on computed tomography. METHODS: A total of 423 resected solitary ground-glass opacity nodules were retrospectively evaluated. We analyzed the clinical, imaging and pathological data and investigated the clinical differences in patient with adenocarcinoma in situ / minimally invasive adenocarcinoma and those with invasive adenocarcinoma. RESULTS: Three hundred and ninety-three adenocarcinomas (92.9%) and 30 benign nodules were diagnosed. Age, the history of family cancer, serum carcinoembryonic antigen level, tumor size, ground-glass opacity types, and bubble-like sign in chest CT differed significantly between adenocarcinoma in situ / minimally invasive adenocarcinoma and invasive adenocarcinoma (p:0.008, 0.046, 0.000, 0.000, 0.000 and 0.001). Receiver operating characteristic curves and univariate analysis revealed that patients with more than 58.5 years, a serum carcinoembryonic antigen level > 1.970 µg/L, a tumor size> 13.50 mm, mixed ground-glass opacity nodules and a bubble-like sign were more likely to be diagnosed as invasive adenocarcinoma. The combination of five factors above had an area under the curve of 0.91, with a sensitivity of 82% and a specificity of 87%. CONCLUSION: The five-factor combination helps us to distinguish adenocarcinoma in situ / minimally invasive adenocarcinoma from invasive adenocarcinoma and to perform appropriate surgery for solitory ground-glass opacity nodules.