The Effect of Erythromycin in Macrolide-Resistant Bordetella pertussis: Inhibitory Effect on Growth, Toxin Expression, and Virulence.

INTRODUCTION: The macrolide-resistant Bordetella pertussis (MRBp) has appeared in Asia and has even been prevalent in China. Since the antibiotic sensitivity test is not carried out in the clinical setting, macrolide is still the first choice of antibiotic in MRBp infection. Further, the macrolide therapy for pertussis needs to be revised. Macrolide has always shown a positive effect on other macrolide-resistant bacterium infections in clinical applications. However, the mechanism of macrolide on MRBp remains unclear. OBJECTIVE: The objective of this study was to investigate the effect of virulence of MRBp under the sub-MIC erythromycin. METHODS: This study evaluated a representative isolate BP19147 (ptxP1/fhaB3-MRBp) under a series of sub-inhibitory concentrations of erythromycin. We measured the growth curve, biofilm formation, and autoaggregation assay under Stainer and Scholte (SS) broth. The relative gene expression was detected by RT-qPCR. RESULTS: The proteomics was detected by label-fee DIA. The growth ability and virulence factors of MR isolate BP19147 were inhibited by sub-MIC of erythromycin and had a concentration- dependent effect. From the proteomics results, the pertussis toxin, filamentous haemagglutinin, and pertactin did not show a statistical difference (p >0.05). Other virulence factors (including dermonecrotic toxin, Invasive Adenylate cyclase/haemolysin. etc) showed a statistical difference (p <0.05). In the KEGG enrichment, the BvgAS system, biofilm formation, and some adaptive systems were inhibited by erythromycin. CONCLUSION: The sub-MIC of erythromycin may reduce the virulence of MRBp, which will provide a theoretical basis for the rational use of erythromycin for MRBp infection and help the development of new antibiotics.

Synthesis and characterization of the trans- and cis-isomers of N-acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine and their attempted detection in human urine.

1,3-Butadiene (BD) is a carcinogenic air pollutant. N-acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine (MHBMA3 or 4HBeMA), an urinary BD metabolite with unspecified configuration, is considered the most sensitive BD biomarker and has been used in routine biomonitoring since 2012. However, two issues remain unaddressed: why its concentrations are unusually high relative to other urinary BD biomarkers and why some authors reported no detection of the biomarker whereas other authors readily quantitated it. To address the issues, we synthesized and structurally characterized the authentic trans- and cis-isomers of MHBMA3 (designated NE and NZ, respectively), developed an isotope-dilution LC-MS/MS method for their quantification, and examined 67 urine samples from barbecue restaurant personnel (n = 47) and hotel administrative staff (n = 20). The restaurant personnel were exposed to barbecue fumes, which contain relatively high concentrations of BD. The results showed that NE and NZ had highly similar NMR spectra, and were difficult to be well separated chromatographically. The NMR data showed that the MHBMA3 isomer investigated in most previous studies was NE. We did not detect NE and NZ in any samples; however, an interfering peak with varying heights was observed in most samples. Notably, under the chromatographic conditions used in the literature, the peak exhibited indistinguishable retention time from that of NE. Thus, it is highly likely that the interfering peak has been mis-identified as NE in previous studies, providing a reasonable explanation for the high MHBMA3 concentration in urine. The contradiction in the presence of MHBMA3 in urine was also caused by the mis-identification, because the researchers who reported the absence of MHBMA3 were actually detecting NZ. Thus, we clarified the confusion on MHBMA3 in previous studies through correctly identifying the two MHBMA3 isomers. The presence of NE and NZ in human urine warrants further investigations.

Prediction of clear cell renal cell carcinoma prognosis based on an immunogenomic landscape analysis.

Immune cell infiltration and tumor-related immune molecules play key roles in tumorigenesis and tumor progression. The influence of immune interactions on the molecular characteristics and prognosis of clear cell renal cell carcinoma (ccRCC) remains unclear. A machine learning algorithm was applied to the transcriptome data from The Cancer Genome Atlas database to determine the immunophenotypic and immunological characteristics of ccRCC patients. These algorithms included single-sample gene set enrichment analyses and cell type identification. Using bioinformatics techniques, we examined the prognostic potential and regulatory networks of immune-related genes (IRGs) involved in ccRCC immune interactions. Fifteen IRGs (CCL7, CHGA, CMA1, CRABP2, IFNE, ISG15, NPR3, PDIA2, PGLYRP2, PLA2G2A, SAA1, TEK, TGFA, TNFSF14, and UCN2) were identified as prognostic IRGs associated with overall survival and were used to construct a prognostic model. The area under the receiver operating characteristic curve at 1 year was 0.927; 3 years, 0.822; and 5 years, 0.717, indicating good predictive accuracy. Molecular regulatory networks were found to govern immune interactions in ccRCC. Additionally, we developed a nomogram containing the model and clinical characteristics with high prognostic potential. By systematically examining the sophisticated regulatory mechanisms, molecular characteristics, and prognostic potential of ccRCC immune interactions, we provided an important framework for understanding the molecular mechanisms of ccRCC and identifying new prognostic markers and therapeutic targets for future research.

Comparing the diagnostic value of 68Ga-prostate-specific membrane antigen PET/CT and multiparametric MRI in pelvic lymph node metastasis of locally advanced prostate cancer.

Background: Multiparametric magnetic resonance imaging (mpMRI) is a commonly used method to diagnose pelvic lymph node metastasis (PLNM) in prostate cancer (PCa) patients, but there are few comparative studies on mpMRI and 68Ga-prostate-specific membrane antigen (PSMA) positron emission tomography (PET)/computed tomography (CT) in locally advanced PCa (LAPC) patients. Therefore, we designed a retrospective study to compare the diagnostic value of 68Ga-PSMA PET/CT and mpMRI for PLNM of LAPC. Methods: A retrospective study was performed on 50 patients with LAPC who underwent radical prostatectomy (RP) in Tongji Hospital from 2021 to 2023. All patients underwent PET/CT and mpMRI examination, and were diagnosed as LAPC before surgery, followed by robot-assisted laparoscopic prostatectomy or laparoscopic RP and extended pelvic lymph node dissection (ePLND). Routine postoperative pathological examination was performed. According to the results, the sensitivity, specificity, positive predictive value, and negative predictive value of 68Ga-PSMA PET/CT and mpMRI for the diagnosis of PLNM of LAPC were compared. Results: Among the 50 patients, the mean age was 65.5±10.3 years, the preoperative total serum prostate-specific antigen (PSA) was 30.7±12.3 ng/mL, and the Gleason score was 7 [7, 8]. The difference in diagnostic efficacy between 68Ga-PSMA PET/CT and mpMRI in the preoperative diagnosis of PLNM of PCa was determined by postoperative pathological results. Based on the number of patients who developed PLNM, the sensitivity, specificity, positive predictive value, and negative predictive value of 68Ga-PSMA PET/CT were as follows: 93.75%, 100.00%, 100.00%, 97.14%, and 68.75%, 97.06%, 91.67%, 86.84% for mpMRI, respectively. Based on the number of pelvic metastatic lymph nodes, the sensitivity, specificity, positive predictive value, and negative predictive value of 68Ga-PSMA PET/CT were 95.24%, 100.00%, 100.00%, 99.48%, and 65.08%, 99.13%, 89.13%, 96.30% for mpMRI, respectively. It turned out that PET/CT was more sensitive than mpMRI in detecting PLNM of PCa, and the difference was statistically significant. Conclusions: 68Ga-PSMA PET/CT is more sensitive than mpMRI in the detection of PLNM in patients with LAPC. It is a promising method in the diagnosis and preoperative assessment of PLNM in LAPC.

Open versus robot-assisted retroperitoneal tumors resection involving inferior vena cava, abdominal aorta, and renal hilum: a comparative study.

INTRODUCTION: Surgery is currently the only effective treatment for retroperitoneal tumors that do not involve any specific organ. The use of robots for removing both benign and malignant retroperitoneal tumors is considered safe and feasible. However, there is insufficient evidence to determine whether robotic retroperitoneal tumor resection (RMBRs) is superior to open retroperitoneal malignant resection (OMBRs). This study compares the short-term outcomes of robotic excision of benign and malignant retroperitoneal tumors with open excision of the same-sized tumors. METHODS: The study compared demographics and outcomes of patients who underwent robotic resection (n = 54) vs open resection (n = 54) of retroperitoneal tumors between March 2018 and December 2022. A 1:1 matching analysis was conducted to ensure a fair comparison. RESULTS: The study found that RBMRs resulted in reduced operative time (OT), estimated blood loss (EBM), and postoperative hospital stay (PSH) when compared to OBMRs. Additionally, RBMRs reduced EBL, PHS, and OT for patients with malignant tumor involvement in major vessels. No significant differences were found in tumor size, blood transfusion rate, and morbidity rate between the RBMRs and OBMRs groups. CONCLUSION: When comparing RMBRs to OMBRs, it was observed that RMBR was associated with lower (EBL), shorter postoperative hospital stays (PHS), and reduced operative time (OT) in a specific group of patients with both benign and malignant tumors.

Identification and validation of a novel anoikis-related prognostic model for prostate cancer.

BACKGROUND: Anoikis resistance is a hallmark characteristic of oncogenic transformation, which is crucial for tumor progression and metastasis. The aim of this study was to identify and validate a novel anoikis-related prognostic model for prostate cancer (PCa). METHODS: We collected a gene expression profile, single nucleotide polymorphism mutation and copy number variation (CNV) data of 495 PCa patients from the TCGA database and 140 PCa samples from the MSKCC dataset. We extracted 434 anoikis-related genes and unsupervised consensus cluster analysis was used to identify molecular subtypes. The immune infiltration, molecular function, and genome alteration of subtypes were evaluated. A risk signature was developed using Cox regression analysis and validated with the MSKCC dataset. We also identify potential drugs for high-risk group patients. RESULTS: Two subtypes were identified. C1 exhibited a higher level of CNV amplification, immune score, stromal score, aneuploidy score, homologous recombination deficiency, intratumor heterogeneity, single-nucleotide variant neoantigens, and tumor mutational burden compared to C2. C2 showed a better survival outcome and had a high level of gamma delta T cell and activated B cell infiltration. The risk signature consisting of four genes (HELLS, ZWINT, ABCC5, and TPSB2) was developed (area under the curve = 0.780) and was found to be an independent prognostic factor for overall survival in PCa patients. Four CTRP-derived and four PRISM-derived compounds were identified for high-risk patients. CONCLUSIONS: The anoikis-related prognostic model developed in this study could be a useful tool for clinical decision-making. This study may provide a new perspective for the treatment of anoikis-related PCa.

Deep learning model for the detection of prostate cancer and classification of clinically significant disease using multiparametric MRI in comparison to PI-RADs score.

BACKGROUND: The Prostate Imaging Reporting and Data System (PI-RADS) is an established reporting scheme for multiparametric magnetic resonance imaging (mpMRI) to distinguish clinically significant prostate cancer (csPCa). Deep learning (DL) holds great potential for automating csPCa classification on mpMRI. METHOD: To compare the performance between a DL algorithm and PI-RADS categorization in PCa detection and csPCa classification, we included 1,729 consecutive patients who underwent radical prostatectomy or biopsy in Tongji hospital. We developed DL models by integrating individual mpMRI sequences and employing an ensemble approach for distinguishing between csPCa and CiSPCa (specifically defined as PCa with a Gleason group 1 or benign prostate disease, training cohort: 1,285 patients vs. external testing cohort: 315 patients). RESULTS: DL-based models exhibited higher csPCa detection rates than PI-RADS categorization (area under the curve [AUC]: 0.902; sensitivity: 0.728; specificity: 0.906 vs. AUC: 0.759; sensitivity: 0.761; specificity: 0.756) (P < 0.001) Notably, DL networks exhibited significant strength in the prostate-specific antigen (PSA) arm < 10 ng/ml compared with PI-RADS assessment (AUC: 0.788; sensitivity: 0.588; specificity: 0.883 vs. AUC: 0.618; sensitivity: 0.379; specificity: 0.763) (P = 0.041). CONCLUSIONS: We developed DL-based mpMRI ensemble models for csPCa classification with improved sensitivity, specificity, and accuracy compared with clinical PI-RADS assessment. In the PSA-stratified condition, the DL ensemble model performed better than PI-RADS in the detection of csPCa in both the high PSA group and the low PSA group.

Application of next-generation sequencing in the detection of low-abundance mutations.

Mutation accumulation in somatic cells contributes to cancer development, aging and many non-malignant diseases. The true mutation frequency in normal cells is extremely low, which presents a challenge in detecting these mutations at such low frequencies. The emergence of next-generation sequencing (NGS) technology enables direct detection of rare mutations across the entire genome of any species. This breakthrough overcomes numerous limitations of traditional mutation detection techniques that rely on specific detection models and sites. However, conventional NGS is limited in its application for detecting low-frequency mutations due to its high sequencing error rate. To address this challenge, high-accuracy NGS sequencing techniques based on molecular consensus sequencing strategies have been developed. These techniques have the ability to correct sequencing errors, resulting in error rates lower than 10-7, are expected to serve as effective tools for low-frequency mutation detection. Error-corrected NGS (ecNGS) techniques hold great potential in various areas, including safety evaluation and research on environmental mutagens, risk assessment of cell and gene therapy drugs, population health risk monitoring, and fundamental research in life sciences. This review highlights a comprehensive review of the research progress in low-frequency mutation detection techniques based on NGS, and provides a glimpse into their potential applications. It also offers an outlook on the potential applications of these techniques, thereby providing valuable insights for further development, research, and application of this technology in relevant fields.

Global fungal-host interactome mapping identifies host targets of candidalysin.

Candidalysin, a cytolytic peptide toxin secreted by the human fungal pathogen Candida albicans, is critical for fungal pathogenesis. Yet, its intracellular targets have not been extensively mapped. Here, we performed a high-throughput enhanced yeast two-hybrid (HT-eY2H) screen to map the interactome of all eight Ece1 peptides with their direct human protein targets and identified a list of potential interacting proteins, some of which were shared between the peptides. CCNH, a regulatory subunit of the CDK-activating kinase (CAK) complex involved in DNA damage repair, was identified as one of the host targets of candidalysin. Mechanistic studies revealed that candidalysin triggers a significantly increased double-strand DNA breaks (DSBs), as evidenced by the formation of γ-H2AX foci and colocalization of CCNH and γ-H2AX. Importantly, candidalysin binds directly to CCNH to activate CAK to inhibit DNA damage repair pathway. Loss of CCNH alleviates DSBs formation under candidalysin treatment. Depletion of candidalysin-encoding gene fails to induce DSBs and stimulates CCNH upregulation in a murine model of oropharyngeal candidiasis. Collectively, our study reveals that a secreted fungal toxin acts to hijack the canonical DNA damage repair pathway by targeting CCNH and to promote fungal infection.

Genome-wide direct quantification of in vivo mutagenesis using high-accuracy paired-end and complementary consensus sequencing.

Error-corrected next-generation sequencing (ecNGS) is an emerging technology for accurately measuring somatic mutations. Here, we report paired-end and complementary consensus sequencing (PECC-Seq), a high-accuracy ecNGS approach for genome-wide somatic mutation detection. We characterize a novel 2-aminoimidazolone lesion besides 7,8-dihydro-8-oxoguanine and the resulting end-repair artifacts originating from NGS library preparation that obscure the sequencing accuracy of NGS. We modify library preparation protocol for the enzymatic removal of end-repair artifacts and improve the accuracy of our previously developed duplex consensus sequencing method. Optimized PECC-Seq shows an error rate of <5 × 10-8 with consensus bases compressed from approximately 25 Gb of raw sequencing data, enabling the accurate detection of low-abundance somatic mutations. We apply PECC-Seq to the quantification of in vivo mutagenesis. Compared with the classic gpt gene mutation assay using gpt delta transgenic mice, PECC-Seq exhibits high sensitivity in quantitatively measuring dose-dependent mutagenesis induced by Aristolochic acid I (AAI). Moreover, PECC-Seq specifically characterizes the distinct genome-wide mutational signatures of AAI, Benzo[a]pyrene, N-Nitroso-N-ethylurea and N-nitrosodiethylamine and reveals the mutational signature of Quinoline in common mouse models. Overall, our findings demonstrate that high-accuracy PECC-Seq is a promising tool for genome-wide somatic mutagenesis quantification and for in vivo mutagenicity testing.

PIG-A gene mutation as a mutagenicity biomarker among coke oven workers.

PIG-A gene mutations can be detected in humans, and PIG-A assays can potentially predict the risk of exposure to carcinogens. However, extensive, population-based studies to validate this are lacking. We studied a cohort of occupational coke oven workers with chronic high exposure to carcinogenic polycyclic aromatic hydrocarbons, which are well-studied genotoxins classified by the IARC as carcinogenic to humans. Peripheral blood erythrocytes of workers were assessed for gene mutations using a PIG-A assay, and chromosome damage using the cytokinesis-block micronucleus test with lymphocytes. Two sample populations from a non-industrialized city and new employees in industrial plants were selected as controls. We observed a significantly elevated PIG-A mutation frequency (MF) and increased frequencies of micronuclei (MN) and nuclear buds (NBUDs) in coke oven workers, compared with levels in the control groups. We found that the coke oven workers with different lengths of service had a relatively high mutation frequency. Overall, the study findings showed that occupational exposure of coke oven workers increases the genetic damage and the PIG-A MF could be a potential biomarker for risk assessment of carcinogen exposure.

Benchmark dose estimation for benzene-exposed workers in China: Based on quantitative and multi-endpoint genotoxicity assessments.

Based on previous exposure studies, benzene (BZ) has been classified as a human carcinogen and occupational exposure limit (OELs) for BZ has been set to be about 1 ppm around the world. However, health hazards have still been reported with exposure below the OEL. Thus, the OEL needs to be updated to reduce health risk. The overall aim of our study was therefore to generate new OEL for BZ via a benchmark dose (BMD) approach and based on quantitative and multi-endpoint genotoxicity assessments. Genotoxicities were determined using the novel human PIG-A gene mutation assay, the micronucleus (MN) test and the COMET assay in benzene-exposed workers. Among the 104 workers with below current OELs, they exhibited significantly higher PIG-A mutant frequencies (MFs) (15.96 ± 14.41 × 10-6) and MN frequencies (11.55 ± 6.83‰) than those among the controls (PIG-A MFs: 5.46 ± 4.56 × 10-6, MN frequencies: 4.51 ± 1.58 ‰), but no difference in the COMET assay. A significant association was also observed between BZ exposure doses and PIG-A MFs and MN frequencies (P < 0.001). Our results indicate that health hazards were induced among workers with below OEL exposures. Based on results from the PIG-A and MN assays, the lower confidence limit of the BMD (BMDL) were calculated to be 8.71 mg/m3-year and 0.44 mg/m3-year, respectively. Based on these calculations, the OEL for BZ was determined to be lower than 0.07 ppm. This value can be considered by regulatory agencies to set new exposure limits and to better protect workers.

The whole-cell proteome shows the characteristics of macrolides-resistant Bordetella pertussis in China linked to the biofilm formation.

The macrolides-resistant Bordetella pertussis (MR-Bp) isolates in China evolved from the ptxP1/fhaB3 allele and rapidly became predominant, suggestive of an adaptive transmission ability. This was different from the global prevalent ptxP3 strains, in which MR-Bp was rarely reported. The study aimed to determine the underlying mechanism responsible for fitness and resistance in these two strains. We identify proteomic differences between ptxP1/fhaB3 and ptxP3/fhaB1 strains using tandem mass tag (TMT)-based proteomics. We then performed in-depth bioinformatic analysis to determine differentially expressed genes (DEGs), followed by gene ontology (GO), and protein-protein interaction (PPI) network analysis. Further parallel reaction monitoring (PRM) analysis confirmed the expression of four target proteins. Finally, the crystal violet method was used to determine biofilm-forming ability. The results showed that the main significantly different proteins between the two represent isolates were related to biofilm formation. Furthermore, we have confirmed that ptxP1/fhaB3 showed hyperbiofilm formation in comparison with ptxP3/fhaB1. It is suggested that the resistance and adaptability of ptxP1/fhaB3 strains may be related to the formation of biofilm through proteomics. In a word, we determined the significantly different proteins between the ptxP1/fhaB3 and ptxP3/fhaB1 strains through whole-cell proteome, which were related to biofilm formation.

FTO regulates the DNA damage response via effects on cell-cycle progression.

The fat mass and obesity-associated protein FTO is an "eraser" of N6-methyladenosine, the most abundant mRNA modification. FTO plays important roles in tumorigenesis. However, its activities have not been fully elucidated and its possible involvement in DNA damage - the early driving event in tumorigenesis - remains poorly characterized. Here, we have investigated the role of FTO in the DNA damage response (DDR) and its underlying mechanisms. We demonstrate that FTO responds to various DNA damage stimuli. FTO is overexpressed in mice following exposure to the promutagens aristolochic acid I and benzo[a]pyrene. Knockout of the FTO gene in TK6 cells, via CRISPR/Cas9, increased genotoxicity induced by DNA damage stimuli (micronucleus and TK mutation assays). Cisplatin- and diepoxybutane-induced micronucleus frequencies and methyl methanesulfonate- and azathioprine-induced TK mutant frequencies were also higher in FTO KO cells. We investigated the potential roles of FTO in DDR. RNA sequencing and enrichment analysis revealed that FTO deletion disrupted the p38 MAPK pathway and inhibited the activation of nucleotide excision repair and cell-cycle-related pathways following cisplatin (DNA intrastrand cross-links) treatment. These effects were confirmed by western blotting and qRT-PCR. FTO deletion impaired cell-cycle arrest at the G2/M phase following cisplatin and diepoxybutane treatment (flow cytometry analysis). Our findings demonstrated that FTO is involved in several aspects of DDR, acting, at least in part, by impairing cell cycle progression.

De novo assembly of a chromosome-level reference genome of the ornamental butterfly Sericinus montelus based on nanopore sequencing and Hi-C analysis.

Sericinus montelus (Lepidoptera, Papilionidae, Parnassiinae) is a high-value ornamental swallowtail butterfly species widely distributed in Northern and Central China, Japan, Korea, and Russia. The larval stage of this species feeds exclusively on Aristolochia plants. The Aristolochia species is well known for its high levels of aristolochic acids (AAs), which have been found to be carcinogenic for numerous animals. The swallowtail butterfly is among the few that can feed on these toxic host plants. However, the genetic adaptation of S. montelus to confer new abilities for AA tolerance has not yet been well explored, largely due to the limited genomic resources of this species. This study aimed to present a chromosome-level reference genome for S. montelus using the Oxford Nanopore long-read sequencing, Illumina short-read sequencing, and Hi-C technology. The final assembly was composed of 581.44 Mb with an expected genome size of 619.27 Mb. Further, 99.98% of the bases could be anchored onto 30 chromosomes. The N50 of contigs and scaffolds was 5.74 and 19.12 Mb, respectively. Approximately 48.86% of the assembled genome was suggested to be repeat elements, and 13,720 protein-coding genes were predicted in the current assembly. The phylogenetic analysis indicated that S. montelus diverged from the common ancestor of swallowtails about 58.57-80.46 million years ago. Compared with related species, S. montelus showed a significant expansion of P450 gene family members, and positive selections on eloa, heatr1, and aph1a resulted in the AA tolerance for S. montelus larva. The de novo assembly of a high-quality reference genome for S. montelus provided a fundamental genomic tool for future research on evolution, genome genetics, and toxicology of the swallowtail butterflies.

Quinoline is more genotoxic than 4-methylquinoline in hiHeps cells and rodent liver.

Environmental pollutants, such as quinoline (QN) and 4-methylquinoline (4-MeQ), may be genotoxic and carcinogenic. Earlier studies, including in vitro genotoxicity tests, indicated that 4-MeQ is more mutagenic than QN. However, we hypothesized that the methyl group of 4-MeQ favors detoxication over bioactivation, and this factor may be overlooked in in vitro tests that do not incorporate supplementation with cofactors for enzymes that catalyze conjugation reactions. We used human induced hepatocyte cells (hiHeps), which express such enzymes, and compared the genotoxicity of 4-MeQ and QN. We also carried out an in vivo micronucleus (MN) test in rat liver, since 4-MeQ is not genotoxic in rodent bone marrow. In the Ames test and the Tk gene mutation assay, with rat S9 activation, 4-MeQ was more mutagenic than QN. However, QN induced significantly higher MN frequencies in hiHeps and rat liver than did 4-MeQ. Furthermore, QN upregulated genotoxicity marker genes much more than did 4-MeQ. We also investigated the roles of two important detoxication enzymes, UDP-glucuronosyltransferases (UGTs) and cytosolic sulfotransferases (SULTs). When hiHeps were preincubated with hesperetin (UGT inhibitor) and 2,6-dichloro-4-nitrophenol (SULT inhibitor), MN frequencies were elevated approximately 1.5-fold for 4-MeQ, whereas no significant effects were seen for QN. This study shows that QN is more genotoxic than 4-MeQ, when the roles of SULTs and UGTs in detoxication are considered and our results may improve understanding the structure-activity relationships of quinoline derivatives.

Inflammation Intensifies Monocrotaline-Induced Liver Injury.

Pyrrolizidine alkaloids (PAs) are the most common toxins of plant origin, and it is evident that PAs pollute soil, water, nearby plants, and derived foods. Cases of human poisoning due to ingestion of PA-contaminated foods have been reported in several countries. Monocrotaline (MCT) is a pyrrolizidine alkaloid from the plants of Crotalaria genus that causes hepatic and cardiopulmonary toxicities, and the exhibition of the toxicities requires the metabolic activation by CYP3A4 to form electrophilic dehydro-monocrotaline (DHM). The present study demonstrated that myeloperoxidase (MPO) also participated in the bioactivation of MCT. N-Chloromonocrotaline was detected in both HClO/MCT incubations and MPO/H2O2/MgCl2/MCT incubations. DHM-derived N-acetylcysteine (NAC) conjugates were detected in the above incubations fortified with NAC. Lipopolysaccharide-induced inflammation in mice resulted in an elevated level of hepatic MPO activity, increased metabolic activation of MCT, and intensified elevation of serum ALT and AST activity induced by MCT. MPO inhibitor 4-aminobenzoic acid hydrazide was found to reverse these alterations. Mpo-KO mice were resistant to the observed potentiating effect of inflammation on MCT-induced liver injury. In conclusion, inflammation intensified MCT-induced liver injury. MPO participated in the observed potentiating effect of inflammation on the hepatotoxicity induced by MCT.

UBC9 deficiency enhances immunostimulatory macrophage activation and subsequent antitumor T cell response in prostate cancer.

The role of tumor-associated macrophages (TAMs), along with the regulatory mechanisms underlying distinct macrophage activation states, remains poorly understood in prostate cancer (PCa). Herein, we report that PCa growth in mice with macrophage-specific Ubc9 deficiency is substantially suppressed compared with that in wild-type littermates, an effect partially ascribed to the augmented CD8+ T cell response. Biochemical and molecular analyses revealed that signal transducer and activator of transcription 4 (STAT4) is a crucial UBC9-mediated SUMOylation target, with lysine residue 350 (K350) as the major modification site. Site-directed mutation of STAT4 (K350R) enhanced its nuclear translocation and stability, thereby facilitating the proinflammatory activation of macrophages. Importantly, administration of the UBC9 inhibitor 2-D08 promoted the antitumor effect of TAMs and increased the expression of PD-1 on CD8+ T cells, supporting a synergistic antitumor efficacy once it combined with the immune checkpoint blockade therapy. Together, our results demonstrate that ablation of UBC9 could reverse the immunosuppressive phenotype of TAMs by promoting STAT4-mediated macrophage activation and macrophage-CD8+ T cell crosstalk, which provides valuable insights to halt the pathogenic process of tumorigenesis.

Engineered Adipose-Derived Stem Cells Overexpressing RXFP1 via CRISPR Activation Ameliorate Erectile Dysfunction in Diabetic Rats.

Due to the high incidence of diabetes mellitus (DM) and poor response to the first-line treatment of DM-induced erectile dysfunction (DMED), new therapeutic strategies for DMED are needed. Adipose-derived stem cell (ADSC) transplantation is considered a promising treatment modality for DMED but is limited by poor survival and efficacy after transplantation. In this study, we aimed to increase the therapeutic effect of DMED by overexpressing the relaxin family peptide receptor 1 (RXFP1) using a clustered regularly interspaced short palindromic repeats activation (CRISPRa) system in ADSCs. Two lentiviruses carrying the CRISPRa system transfected ADSCs to overexpress RXFP1 (RXFP1-ADSCs). The intracavernous injection of ADSCs was performed in DMED rats induced by the intraperitoneal injection of streptozotocin. Four weeks after transplantation, we measured erectile function and collected specimens of the corpus cavernosum for follow-up detection. The results showed that ADSCs improved erectile function in diabetic rats, and the RXFP1-ADSCs were more significant. We detected reduced levels of oxidative stress, apoptosis and fibrosis together with relative normalization of endothelial and smooth muscle cell function in the penis after ADSC transplantation. RXFP1-ADSCs had more potent efficacy in the above alterations compared to negative control ADSCs due to the high levels of survival and paracrine capacity in RXFP1-ADSCs. The results revealed that RXFP1-ADSC transplantation could partially preserve erectile function in DMED rats associated with the regulation of oxidative stress, apoptosis, fibrosis and endothelial and smooth muscle cell dysfunction. RXFP1 may be the new target for the genetic modification of ADSCs, which benefits the management of DMED.

GAS1RR, an immune-related enhancer RNA, is related to biochemical recurrence-free survival in prostate cancer.

Prostate cancer (PCa) is one of the malignant tumors of urinary system with a high morbidity. Enhancer RNA is a subclass of long non-coding RNA transcribed from active enhancer regions, which plays a critical role in gene transcriptional regulation. However, the role of enhancer RNA (eRNA) in PCa remains extremely mysterious. This study is aimed at exploring key prognostic eRNAs in PCa. First, we downloaded gene expression data and clinical data of 33 cancer types from UCSC Xena platform. Second, we selected reported putative eRNA-target pairs and performed the Kaplan-Meier survival and correlation analysis to determine the crucial eRNAs most related to biochemical recurrence (BCR)-free survival. Third, we explored the clinical characteristics with the key eRNA GAS1 adjacent regulatory RNA (GAS1RR) and performed a computational difference algorithm and the Cox regression analysis. Next, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to explore the underlying mechanisms. Finally, we used the pan-cancer data from The Cancer Genome Atlas (TCGA) and performed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of 18 pairs of specimens to prove the results we acquired. Among all 2695 putative eRNAs, 6 pairs of eRNA-target genes were prominently related to BCR-free survival. Growth arrest-specific protein 1 (GAS1) was a target gene of GAS1RR (r = 0.86, P < 0.001). Patients with low GAS1RR expression were likely to have unfavorable clinical characteristics. The result of computational Cox regression analysis demonstrated that GAS1RR may predict the prognosis of PCa independently. RT-qPCR results illuminated that GAS1RR and GAS1 were both downregulated in PCa tissues, and they show a strong positive correlation. GO and KEGG analyses revealed biological processes that GAS1RR was mainly associated with. Immune infiltration analysis indicated that GAS1RR expression is correlated with the infiltration level of six kinds of immune cells. Our results suggest that GAS1RR may be clinically useful in the prediction of PCa prognosis. Moreover, it may also be a prognostic predictor and theoretic target with great promise in PCa.