Spatial distribution pattern of immune cells is associated with patient prognosis in colorectal cancer.

BACKGROUND: The spatial context of tumor-infiltrating immune cells (TIICs) is important in predicting colorectal cancer (CRC) patients' clinical outcomes. However, the prognostic value of the TIIC spatial distribution is unknown. Thus, we aimed to investigate the association between TIICs in situ and patient prognosis in a large CRC sample. METHODS: We implemented multiplex immunohistochemistry staining technology in 190 CRC samples to quantify 14 TIIC subgroups in situ. To delineate the spatial relationship of TIICs to tumor cells, tissue slides were segmented into tumor cell and microenvironment compartments based on image recognition technology, and the distance between immune and tumor cells was calculated by implementing the computational pipeline phenoptr. RESULTS: MPO+ neutrophils and CD68+IDO1+ tumor-associated macrophages (TAMs) were enriched in the epithelial compartment, and myeloid lineage cells were located nearest to tumor cells. Except for CD68+CD163+ TAMs, other cells were all positively associated with favorable prognosis. The prognostic predictive power of TIICs was highly related to their distance to tumor cells. Unsupervised clustering analysis divided colorectal cancer into three subtypes with distinct prognostic outcomes, and correlation analysis revealed the synergy among B cells, CD68+IDO1+TAMs, and T lineage cells in producing an effective immune response. CONCLUSIONS: Our study suggests that the integration of spatial localization with TIIC abundance is important for comprehensive prognostic assessment.

MK8617 inhibits M1 macrophage polarization and inflammation via the HIF-1α/GYS1/UDPG/P2Y14 pathway.

Background: Nonresolving inflammation is a major driver of disease and needs to be taken seriously. Hypoxia-inducible factor (HIF) is closely associated with inflammation. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs), as stabilizers of HIF, have recently been reported to have the ability to block inflammation. We used MK8617, a novel HIF-PHI, to study its effect on macrophage inflammation and to explore its possible mechanisms. Methods: Cell viability after MK8617 and lipopolysaccharide (LPS) addition was assessed by Cell Counting Kit-8 (CCK8) to find the appropriate drug concentration. MK8617 pretreated or unpretreated cells were then stimulated with LPS to induce macrophage polarization and inflammation. Inflammatory indicators in cells were assessed by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blot (WB) and immunofluorescence (IF). The level of uridine diphosphate glucose (UDPG) in the cell supernatant was measured by ELISA. Purinergic G protein-coupled receptor P2Y14, as well as hypoxia-inducible factor-1α (HIF-1α) and glycogen synthase 1 (GYS1) were detected by qRT-PCR and WB. After UDPG inhibition with glycogen phosphorylase inhibitor (GPI) or knockdown of HIF-1α and GYS1 with lentivirus, P2Y14 and inflammatory indexes of macrophages were detected by qRT-PCR and WB. Results: MK8617 reduced LPS-induced release of pro-inflammatory factors as well as UDPG secretion and P2Y14 expression. UDPG upregulated P2Y14 and inflammatory indicators, while inhibition of UDPG suppressed LPS-induced inflammation. In addition, HIF-1α directly regulated GYS1, which encoded glycogen synthase, an enzyme that mediated the synthesis of glycogen by UDPG, thereby affecting UDPG secretion. Knockdown of HIF-1α and GYS1 disrupted the anti-inflammatory effect of MK8617. Conclusions: Our study demonstrated the role of MK8617 in macrophage inflammation and revealed that its mechanism of action may be related to the HIF-1α/GYS1/UDPG/P2Y14 pathway, providing new therapeutic ideas for the study of inflammation.

Inventory, source and health risk assessment of nitrated and parent PAHs in agricultural soils over a rural river in Southeast China.

Nitrated polycyclic aromatic hydrocarbons (NPAHs) have become a concerning topic because of their widespread occurrence and carcinogenicity. However, studies on NPAHs in soils, especially in agricultural soils, are still limited. In this study, a systematic monitoring campaign of 15 NPAHs and 16 polycyclic aromatic hydrocarbons (PAHs) was performed in agricultural soils from the Taige Canal basin in 2018, which is a typical agricultural activity area of the Yangtze River Delta. The total concentration of NPAHs and PAHs ranged from 14.4 to 85.5 ng g-1 and 118-1108 ng g-1, respectively. Among the target analytes, 1,8-dinitropyrene and fluoranthene were the most predominant congeners accounting for 35.0% of ∑15NPAHs and 17.2% of ∑16PAHs, respectively. Four-ring NPAHs and PAHs were predominant, followed by three-ring NPAHs and PAHs. NPAHs and PAHs had a similar spatial distribution pattern with high concentrations in the northeastern Taige Canal basin. The soil mass inventory of ∑16PAHs and ∑15NPAHs was evaluated to be 31.7 and 2.55 metric tons, respectively. Total organic carbon had a significant impact on the distribution of PAHs in soils. The correlation between PAH congeners in agricultural soils was higher than that between NPAH congeners. Based on diagnostic ratios and principal component analysis-multiple linear regression model, vehicle exhaust emission, coal combustion, and biomass combustion were the predominant sources of these NPAHs and PAHs. According to the lifetime incremental carcinogenic risk model, the health risk posed by NPAHs and PAHs in agricultural soils of the Taige Canal basin was virtually negligible. The total health risk in soils of the Taige Canal basin to adults was slightly higher than that to children.

Single-cell profiling of the copy-number heterogeneity in colorectal cancer.

BACKGROUND: With functionally heterogeneous cells, tumors comprise a complex ecosystem to promote tumor adaptability and evolution under strong selective pressure from the given microenvironment. Diversifying tumor cells or intra-tumor heterogeneity is essential for tumor growth, invasion, and immune evasion. However, no reliable method to classify tumor cell subtypes is yet available. In this study, we introduced the single-cell sequencing combined with copy number characteristics to identify the types of tumor cells in microsatellite stable (MSS) colorectal cancer (CRC). METHODS: To characterize the somatic copy number alteration (SCNA) of MSS CRC in a single cell profile, we analyzed 26 tissue samples from 19 Korean patients (GSE132465, the Samsung Medical Center [SMC] dataset) and then verified our findings with 15 tissue samples from five Belgian patients (GSE144735, the Katholieke Universiteit Leuven 3 [KUL3] dataset). The Cancer Genome Atlas (TCGA) cohort, GSE39582 cohort, and National Cancer Center (NCC) cohort (24 MSS CRC patients were enrolled in this study between March 2017 and October 2017) were used to validate the clinical features of prognostic signatures. RESULTS: We employed single cell RNA-sequencing data to identify three types of tumor cells in MSS CRC by their SCNA characteristics. Among these three types of tumor cells, C1 and C3 had a higher SCNA burden; C1 had significant chromosome 13 and 20 amplification, whereas C3 was the polar opposite of C1, which exhibited deletion in chromosome 13 and 20. The three types of tumor cells exhibited various functions in the tumor microenvironment and harbored different mutations. C1 and C2 were linked to the immune response and hypoxia, respectively, while C3 was critical for cell adhesion activity and tumor angiogenesis. Additionally, one gene ( OLFM4 ) was identified as epithelium-specific biomarker of better prognosis of CRC (TCGA cohort: P  = 0.0110; GSE39582 cohort: P  = 0.0098; NCC cohort: P  = 0.0360). CONCLUSIONS: On the basis of copy number characteristics, we illustrated tumor heterogeneity in MSS CRC and identified three types of tumor cells with distinct roles in tumor microenvironment. By understanding heterogeneity in the intricate tumor microenvironment, we gained an insight into the mechanisms of tumor evolution, which may support the development of therapeutic strategies.

Screening and identification of key biomarkers of papillary renal cell carcinoma by bioinformatic analysis.

BACKGROUND: Papillary renal cell carcinoma (PRCC) is the most common type of renal cell carcinoma after clear cell renal cell carcinoma (ccRCC). Its pathological classification is controversial, and its molecular mechanism is poorly understood. Therefore, the identification of key genes and their biological pathways is of great significance to elucidate the molecular mechanisms of PRCC occurrence and progression. METHODS: The PRCC-related datasets GSE7023, GSE48352 and GSE15641 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified, and gene ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. Cytoscape and STRING were used to construct the protein-protein interaction network (PPI) and perform module analysis to identify hub genes and key pathways. A heatmap of hub genes was constructed using the UCSC cancer genomics browser. Overall survival and recurrence-free survival of patients stratified by the expression levels of hub genes were analysed using Kaplan-Meier Plotter. The online database UALCAN was applied to analyse gene expression based on tissue type, stage, subtype and race. RESULTS: A total of 214 DEGs, specifically, 205 downregulated genes and 9 upregulated genes, were identified. The DEGs were mainly enriched in angiogenesis, kidney development, oxidation-reduction process, metabolic pathways, etc. The 17 hub genes identified were mainly enriched in the biological processes of angiogenesis, cell adhesion, platelet degranulation, and leukocyte transendothelial migration. Survival analysis showed that EGF, KDR, CXCL12, REN, PECAM1, CDH5, THY1, WT1, PLAU and DCN might be related to the carcinogenesis, metastasis or recurrence of PRCC. UALCAN analysis showed that low expression of PECAM1 and PLAU in PRCC tissues was related to stage, subtype and race. CONCLUSIONS: The DEGs and hub genes identified in the present study provide insight into the specific molecular mechanisms of PRCC occurrence and development and may be potential molecular markers and therapeutic targets for the accurate classification and efficient diagnosis and treatment of PRCC.

[Determination of diethyl aminoethyl hexanoate residues in apples by gas chromatography-mass spectrometry combined with improved QuEChERS].

A method was developed for the determination of diethyl aminoethyl hexanoate (DA-6) residues in apples by gas chromatography-mass spectrometry (GC-MS) combined with improved QuEChERS. The samples were first extracted with warm water, then, the extract was subjected to liquid-liquid re-extraction with dichloromethane, followed by rotary evaporation concentration. The residues were cleaned up with primary secondary amine (PSA). The analytes were separated on an HP-5 capillary column and detected using an EI source in the SIM mode. The recoveries ranged from 74.1% to 84.2% at three spiked levels, with relative standard deviations (RSDs) between 1.5% and 4.1%. The limit of detection and limit of quantification were 0.0024 mg/kg and 0.008 mg/kg, respectively. The developed method is sensitive, and practically applicable for the determination of DA-6 residues in apple samples.

MALDI-TOF Mass Array Analysis of Nell-1 Promoter Methylation Patterns in Human Gastric Cancer.

Mass spectrometry (MS) enables rapid and sensitive qualitative and quantitative analyses of biomolecules (proteins, peptides, oligosaccharides, lipids, DNA, and RNA), drugs, and metabolites. MS has become an essential tool in modern biomedical research, including the analysis of DNA methylation. DNA methylation has been reported in many cancers, suggesting that it can be utilized as an early biomarker to improve the early diagnosis rate. Using matrix-assisted laser desorption/ionization time-of-flight MS and MassCLEAVE reagent, we compared Nell-1 hypermethylation levels among tumor tissues, paracarcinoma tissues, and normal tissues from gastric cancer patients. Almost 80% of the CpG sites in the amplicons produced were covered by the analysis. Our results indicate a significant difference in methylation status between gastric cancer tissue (a higher level) and normal tissue. The same trend was identified in gastric cancer tissue versus paracarcinoma tissue. We also detected lower relative expression of Nell-1 by real-time PCR. Furthermore, immunohistochemical analyses revealed that Nell-1 staining was less intense in cancer tissue relative to normal tissue and that the tumor cells had spread to the muscle layer. These findings may serve as a guide for the early diagnosis of gastric cancer.

Nucleophilic substitution as a mechanism of atrazine sequestration in soil.

Formation of nonextractable residue was widely observed as a sink of atrazine (ATZ) in soil. However, the mechanisms by which ATZ binds to soil organic matter remain unclear. In this study, we demonstrated that neucleophilic substitution could serve an important pathway causing ATZ sequestration. The carbon bonded to the chlorine in ATZ molecule is partially positively charged due to the strong electronegativity of chlorine and is susceptible to the attack of nucleophiles such as aniline. Since aromatic amines are relatively rare in natural soils, amino acids/peptides were hypothesized to act as the main nucleophiles in real environment. However, substantially ATZ transformation was only observed in the presence of those species containing thiol functionality. Thus, we speculated that it was the thiol group in amino acids/peptides acting as the nucleophile. Nitrogen in amino acids was in fact not an active nucleophile toward ATZ. In addition to the sulfur-containing amino acids, other thiol compounds, and sulfide were also proved to be reactive to ATZ. Thus, the sequestration potential of ATZ probably correlates to the availability of thiol compounds in soil.

Development of a New Decision Tree to Rapidly Screen Chemical Estrogenic Activities of Xenopus laevis.

During the last past decades, there is an increasing number of studies about estrogenic activities of the environmental pollutants on amphibians and many determination methods have been proposed. However, these determination methods are time-consuming and expensive, and a rapid and simple method to screen and test the chemicals for estrogenic activities to amphibians is therefore imperative. Herein is proposed a new decision tree formulated not only with physicochemical parameters but also a biological parameter that was successfully used to screen estrogenic activities of the chemicals on amphibians. The biological parameter, CDOCKER interaction energy (Ebinding ) between chemicals and the target proteins was calculated based on the method of molecular docking, and it was used to revise the decision tree formulated by Hong only with physicochemical parameters for screening estrogenic activity of chemicals in rat. According to the correlation between Ebinding of rat and Xenopus laevis, a new decision tree for estrogenic activities in Xenopus laevis is finally proposed. Then it was validated by using the randomly 8 chemicals which can be frequently exposed to Xenopus laevis, and the agreement between the results from the new decision tree and the ones from experiments is generally satisfactory. Consequently, the new decision tree can be used to screen the estrogenic activities of the chemicals, and combinational use of the Ebinding and classical physicochemical parameters can greatly improves Hong's decision tree.

Microcystin-LR induced reactive oxygen species mediate cytoskeletal disruption and apoptosis of hepatocytes in Cyprinus carpio L.

Microcystins (MCs) are a group of cyclic hepatotoxic peptides produced by cyanobacteria. Microcystin-LR (MC-LR) contains Leucine (L) and Arginine (R) in the variable positions, and is one of the most common and potently toxic peptides. MC-LR can inhibit protein phosphatase type 1 and type 2A (PP1 and PP2A) activities and induce excessive production of reactive oxygen species (ROS). The underlying mechanism of the inhibition of PP1 and PP2A has been extensively studied. The over-production of ROS is considered to be another main mechanism behind MC-LR toxicity; however, the detailed toxicological mechanism involved in over-production of ROS in carp (Cyprinus carpio L.) remains largely unclear. In our present study, the hydroxyl radical (•OH) was significantly induced in the liver of carp after a relatively short-term exposure to MC-LR. The elevated reactive oxygen species (ROS) production may play an important role in the disruption of microtubule structure. Pre-injection of the antioxidant N-acetyl-cysteine (NAC) provided significant protection to the cytoskeleton, however buthionine sulfoximine (BSO) exacerbated cytoskeletal destruction. In addition, the elevated ROS formation induced the expression of apoptosis-related genes, including p38, JNKa, and bcl-2. A significant increase in apoptotic cells was observed at 12-48 hours. Our study further supports evidence that ROS are involved in MC-LR induced damage to liver cells in carp, and indicates the need for further study of the molecular mechanisms behind MC-LR toxicity.

[Removal of estrogens in laccase catalyzed oxidative processes].

The removal of 5 estrogens (bisphenol A, estradiol, ethinylestradiol, estrone and octylphenol) in laccase catalyzed oxidative processes was studied. The effects of pH and NOM on the removal of EDCs were discussed in detail, as well as the kinetics of EE2 removal and the reaction products. The results indicated that laccase was capable of removing estrogens efficiently. The optimal pH was between 4 and 6. NOM significantly inhibited the removal of estrogens at the initial stage of the reaction. However, the adverse effect of NOM was not obvious after 24 h treatment. The removal of EE2 obeyed the second-order kinetics. The activity of laccase remained stable during the reactions and the stability was higher than that of peroxidase. MS analysis demonstrated that the EE2 dimer formed through radical coupling mechanism was the main reaction product.

Erythropoietin protects against cisplatin-induced nephrotoxicity by attenuating endoplasmic reticulum stress-induced apoptosis.

BACKGROUND: The anticancer drug cisplatin (CP)-induced nephrotoxicity associated with apoptosis plays crucial roles in tumor patients. Erythropoietin (EPO) has recently been shown to enhance recovery from CP-induced acute kidney injury (AKI) in rats by exerting anti-apoptotic effects. However, the molecular mechanisms of Erythropoietin protects against CP-induced AKI are not very clear. The present study investigated the protective effects of erythropoietin (EPO) against CP-induced nephrotoxicity and the possible mechanism in rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into four groups: (1) Control group (n=16): which received a single intraperitoneal injection of vehicle (0.9% saline; 5 mL/kg); (2) CP group (n=16): which received a single intraperitoneal injection of 10.0 mg/kg CP (previously dissolved at 2.0 mg/mL in 0.9% saline solution); (3) CP+rHuEPO group (n=16): which received rHuEPO (5000 U/kg) with co-injection of the LY294002 vehicle dimethyl sulfoxide (DMSO, 33.3 µL/kg; Sigma, St. Louis, MO, USA) by tail vein injection 2 days before CP administration, 15 min before CP administration, and 2 days after CP administration; (4) CP+rHuEPO+LY group (n=16): which received LY294002 (0.3 mg/kg) 10 min before rHuEPO administration by three injections into the tail vein at 2-day intervals beginning 2 days before CP administration. Apoptosis was assessed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. The expressions of C/EBP-homologous protein (CHOP), glucose-regulated protein 78 (GRP78), caspase-12, the phosphorylation of Akt, cleaved-caspase-3 and EPO receptor (EPOR) were measured after CP-treated. In addition, light microscopy and immunohistochemistry examinations were performed. RESULTS: The levels of serum urea and creatinine were increased at 96 h after CP-administered group. The eHuEPO-administered group had significantly lower serum creatinine levels. CP caused an increase in TUNEL-positive cells that was accompanied and apoptotic cell death induced by CP was significantly abrogated by rHuEPO at 96h by morphological evidence of apoptosis. The over-expression of CP-induced endoplasmic reticulum (ER) stress markers (CHOP and GRP78) and activation of caspase-12 were suppressed by rHuEPO, which also attenuated the CP-induced suppression of phosphatidylinositol-3kinase/Akt (PI3K/Akt) signaling in rat kidneys. In addition, LY294002 diminished the effect of rHuEPO on renal protection and antiapoptosis. CONCLUSIONS: Injection of rHuEPO enhance recovery from CP-induced AKI in rats by ameliorating renal functional impairment and exerting important anti-apoptotic effects. However, rHuEPO inhibited CP-induced AKI by a possible mechanism involving PI3K/Akt activation and the inhibition of ER stress-mediated apoptosis.

Simultaneous determination of pesticides, polycyclic aromatic hydrocarbons, polychlorinated biphenyls and phthalate esters in human adipose tissue by gas chromatography-tandem mass spectrometry.

This paper describes a method for the simultaneous determination of 284 environmental contaminants, including 57 pesticides, 15 polycyclic aromatic hydrocarbons (PAHs), 209 polychlorinated biphenyls (PCBs) and 3 phthalate esters (PAEs), in adipose tissue samples. For the first time, a gas chromatography-tandem mass spectrometry (GC-MS/MS) method following a homogenised extraction using acetonitrile and purification by gel permeation chromatography (GPC) was used. Various performance characteristics, such as the limit of detection (LOD), limit of quantification (LOQ), linear range, recovery and precision, were determined for each analyte. The LOD for most analytes was below 0.01 mg/kg. The recoveries and relative standard deviations (RSDs) were determined by spiking untreated samples with the analytes at the LOQ, 2×LOQ and 4×LOQ levels. The average recovery for most pesticides was between 70% and 120% and the precision values, expressed as RSD, were all below 20.4% (n=6). This method may provide an efficient tool for evaluating the extent of exposure to organic contaminants using human adipose tissue.

Pollution level and human health risk assessment of some pesticides and polychlorinated biphenyls in Nantong of Southeast China.

Food consumption is one of the key exposure routes of humans to contaminants. This article evaluated the residue levels of 51 pesticides and 16 polychlorinated biphenyls (PCBs) in selected fish and food items which were commonly consumed in the Nantong area of Jiangsu Province, Southeast China. The 51 pesticides and 16 PCBs were analyzed by highly sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS). The results showed that organochlorine pesticides such as dichlorodiphenyltrichloroethanes (DDTs), hexachlorocyclohexanes (HCHs), hexachlorobenzene (HCB) and mirex and other pesticides including chlorpyrifos, pyrethroid pesticides, metolachlor, pyridaben and trifluralin were frequently detected in the samples, which was consistent with the accumulation level and characteristics of these toxic chemicals in human adipose tissue of people living in Nantong. Meanwhile, correlation of the residue level of toxic chemicals with their physical chemical properties and historic use pattern in Nantong area was observed. Combined with dietary survey results at the same sampling locations, human health risk assessment of ingestion through the dietary route was performed. The results suggested that the non-cancer risks of the chemicals investigated can be considered negligible in the Nantong area, however, the cancer risks from lifetime dietary exposure to DDTs and HCB have exceeded the acceptable levels.

Accumulation levels and characteristics of some pesticides in human adipose tissue samples from Southeast China.

This paper presents a comprehensive study of pesticide levels and bio-accumulation characteristics in human adipose tissues among residents of Southeast China. A large number of adipose samples (n=633) were selected for 58 pesticides and were analyzed by high sensitive Gas Chromatography-Tandem Mass Spectrometry (GC-MS/MS). The results showed that POPs pesticides were frequently detected, including 2,4'-DDD, 2,4'-DDE, 2,4'-DDT, 4,4'-DDD, 4,4'-DDE, 4,4'-DDT, α-HCH, ß-HCH, γ-HCH, δ-HCH, hexachlorobenzene (HCB), and mirex. Other detected pesticide species were dicofol, methamidophos and chlordimeform, which have rarely been reported. Comparing to different countries, the concentrations of total DDT and HCH in these three Chinese southeastern sites were in the middle range, whereas the HCB and mirex were in the lower end. A significant correlation was observed between region as well as age and POPs pesticide levels. Some pesticide residue levels were also found significantly correlated to occupation. However, there was no significant correlation between gender and pesticides. Meanwhile, it is interesting to find that mortality of malignant tumors tends to associate with the pesticides levels in human adipose tissue. More importantly, the measured data presented in this study provide realistic information which is useful for assessing human exposure to pesticides in the general population of Southeast China.