YAP/TAZ activation mediates PQ-induced lung fibrosis by sustaining senescent pulmonary epithelial cells.

Paraquat (PQ) is a widely used herbicide and a common cause of poisoning that leads to pulmonary fibrosis with a high mortality rate. However, the underlying mechanisms of PQ-induced pulmonary fibrosis and whether pulmonary epithelial cell senescence is involved in the process remain elusive. In this study, PQ-induced pulmonary epithelial cell senescence and Hippo-YAP/TAZ activation were observed in both C57BL/6 mice and human epithelial cells. PQ-induced senescent pulmonary epithelial cells promoted lung fibroblast transformation through secreting senescence-associated secretory phenotype (SASP) factors. Yap/Taz knockdown in mice lungs significantly decreased the expression of downstream profibrotic protein Ctgf and senescent markers p16 and p21, and alleviated PQ-induced pulmonary fibrosis. Interfering YAP/TAZ in senescent human pulmonary epithelial cells resulted in decreased expression of the anti-apoptosis protein survivin and elevated level of apoptosis. In conclusion, our findings reveal a novel mechanism by which the involvement of Hippo-YAP/TAZ activation in pulmonary epithelial cell senescence mediates the pathogenesis of PQ-induced pulmonary fibrosis, thereby offering novel insights and potential targets for the clinical management of PQ poisoning as well as providing the mechanistic insight of the involvement of Yap/Taz activation in cell senescence in pulmonary fibrosis and its related pulmonary disorders. The YIN YANG balance between cell senescence and apoptosis is important to maintain the homeostasis of the lung, the disruption of which will lead to disease.

Non-cytochrome P450 enzyme aldehyde oxidase is involved in the oxidative metabolic pathway of diquat and its detoxification effect.

Diquat (DQ) poisoning has garnered attention in recent years, primarily due to the rising incidence of cases worldwide, coupled with the absence of a viable antidote for its treatment. Despite the fact that diquat monopyridone (DQ-M) has been identified as a significant metabolite of DQ, the enzyme responsible for its formation remains unknown. In this study, we have identified aldehyde oxidase (AOX) as a vital enzyme involved in DQ oxidative metabolism. The metabolism of DQ to DQ-M was significantly inhibited by AOX inhibitors including raloxifene and hydralazine. The source of oxygen incorporated into DQ-M was proved to be from water through a H218O incubation experiment which further corroborated DQ-M formation via AOX metabolism. The product of DQ-M in vitro generated by fresh rat tissues co-incubation was consistent with its AOX expression. The result of the molecular docking analysis of DQ and AOX protein showed that DQ is capable of binding to AOX. Furthermore, the cytotoxicity of DQ was significantly higher than DQ-M at the same concentration tested in six cell types. This work is the first to uncover the involvement of aldehyde oxidase, a non-cytochrome P450 enzyme, in the oxidative metabolic pathway of diquat, thus providing a potential target for the development of detoxification treatment.

The value of toxicological analysis in acute poisoning patients with uncertain exposure histories: a retrospective and descriptive study from an institute of poisoning.

BACKGROUND: In clinical practice, some patients might not be able or unwilling to provide a thorough history of medication and poison exposure. The aim of this study was to use toxicological analysis to examine the clinical characteristics of patients with acute poisoning whose exposure history was uncertain from a toxicological analysis perspective. METHODS: This was a retrospective and descriptive study from an institute of poisoning. Patient registration information and test reports spanning the period from April 1, 2020 to March 31, 2022, were obtained. Patients with uncertain exposure histories and who underwent toxicological analysis were included. Clinical manifestations and categories of toxics were analyzed. RESULTS: Among the 195 patients with positive toxicological analysis results, the main causes of uncertain exposure history was disturbance of consciousness (62.6%), unawareness (23.6%) and unwillingness or lack of cooperation (13.8%). The predominant clinical manifestations were disturbed consciousness (62.6%), followed by vomiting and nausea (14.4%) and liver function abnormalities (8.7%). A comparison of clinical manifestations between patients with positive and negative (n=99) toxicological analyses results revealed significantly different proportions of disturbances in consciousness (63% vs. 21%), dizziness (1.5% vs. 5.1%), multi-organ failure (1.5% vs. 7.1%), and local pain (0 vs 4%). The main categories of substances involved were psychiatric medications (23.1%), sedatives (20.5%), insecticides (13.8%), and herbicides (12.8%). CONCLUSION: The clinical manifestations of acute poisoning in patients with an uncertain exposure history are diverse and nonspecific, and toxicological analysis plays a pivotal role in the diagnosis and differential diagnosis of such patients.

Dual-Mode Separation and SERS Detection of Carbaryl with PA-6/AuNRs@ZIF-8 Films.

Appropriate separation and enrichment steps can enhance the performance of SERS assays. For rapid, in-situ detection of carbaryl, a novel PA-6/AuNRs@ZIF-8 film that can be applied to dual-mode separation and SERS detection, has been developed. In the film, PA-6 was used as a TLC substrate for the initial separation of the substance to be measured. ZIF-8 provides chemical enhancement in SERS as well as enrichment and secondary separation of the analytes. Utilizing this film, we have successfully implemented a TLC-SERS rapid detection scheme, resulting in a detection limit for carbaryl as low as 1 × 10-9 M in lake water in 15 min, which is significantly lower than existing standards. Additionally, the manufacturing cost of one PA-6/AuNRs@ZIF-8 film can be kept within the range of $0.20-$0.40 economically, presenting substantial financial advantages. The method is highly promising for pesticide detection as well as forensic in-situ testing.

Genome-wide association studies combined with k-fold cross-validation identify rs17822931 as an ancestry-informative marker in Han Chinese population.

DNA-based ancestry inference has long been a research hot spot in forensic science. The differentiation of Han Chinese population, such as the northern-to-southern substructure, would benefit forensic practice. In the present study, we enrolled participants from northern and southern China, each participant was genotyped at ∼400 K single-nucleotide polymorphisms (SNPs) and data of CHB and CHS from 1000 Genomes Project were used to perform genome-wide association analyses. Meanwhile, a new method combining genome-wide association study (GWAS) analyses with k-fold cross-validation in a small sample size was introduced. As a result, one SNP rs17822931 emerged with a p-value of 7.51E - 6. We also simulated a huge dataset to verify whether k-fold cross-validation could reduce the false-negative rate of GWAS. The identified ABCC11 rs17822931 has been reported to have allele frequencies varied with the geographical gradient distribution in humans. We also found a great difference in the allele frequency distributions of rs17822931 among five different cohorts of the Chinese population. In conclusion, our study demonstrated that even small-scale GWAS can also have potential to identify effective loci with implemented k-fold cross-validation method and shed light on the potential maker of rs17822931 in differentiating the north-to-south substructure of the Han Chinese population.

Multi-Omics Analyses Reveal the Mechanisms of Early Stage Kidney Toxicity by Diquat.

Diquat (DQ), a widely used bipyridyl herbicide, is associated with significantly higher rates of kidney injuries compared to other pesticides. However, the underlying molecular mechanisms are largely unknown. In this study, we identified the molecular changes in the early stage of DQ-induced kidney damage in a mouse model through transcriptomic, proteomic and metabolomic analyses. We identified 869 genes, 351 proteins and 96 metabolites that were differentially expressed in the DQ-treated mice relative to the control mice (p < 0.05), and showed significant enrichment in the PPAR signaling pathway and fatty acid metabolism. Hmgcs2, Cyp4a10, Cyp4a14 and Lpl were identified as the major proteins/genes associated with DQ-induced kidney damage. In addition, eicosapentaenoic acid, linoleic acid, palmitic acid and (R)-3-hydroxybutyric acid were the major metabolites related to DQ-induced kidney injury. Overall, the multi-omics analysis showed that DQ-induced kidney damage is associated with dysregulation of the PPAR signaling pathway, and an aberrant increase in Hmgcs2 expression and 3-hydroxybutyric acid levels. Our findings provide new insights into the molecular basis of DQ-induced early kidney damage.

Swietenine Alleviates Vascular Remodelling by Enhancing Mitophagy of Pulmonary Arterial Smooth Muscle Cells in Experimental Pulmonary Hypertension.

BACKGROUND: Vascular remodelling during pulmonary hypertension (PH) is characterized by the phenotypic transformation of pulmonary arterial smooth muscle cells (PASMCs). Swietenine (Swi), extracted from the seeds of traditional medicine Swietenia mahagoni, has been used to treat cardiac remodelling, but the effect of Swi on PH is unknown. This study aims to evaluate the effect of Swi on hypoxia-induced phenotypic transformation of PASMCs in experimental PH. METHODS: In our research, C57BL/6 mice were treated with SU5416 and exposed to hypoxia for 4 weeks to establish HySu-PH model. Mice in the Swi treatment group were subjected to HySu with daily administration of Swi. Hemodynamic parameters, echocardiography, and degree of vascular muscularization were measured to evaluate the PH model. Proliferation of PASMC was assessed by Ki67 and EdU assay. Cell migration was detected by wound-healing assay. Mitophagy levels were evaluated by mito-tracker and lyso-tracker, autophagic flux, and protein expression of Pink1 and Lc3 II. The molecular docking was used to validate the interaction of Swi with Nrf2. Immunofluorescence and immunohistochemical staining were applied to determine the subcellular localization of Nrf2. RESULTS: The results showed that Swi attenuated hypoxia-induced increase of right ventricle systolic pressure, Fulton index, and vascular remodelling and decreased PASMC proliferation, migration, and enhanced mitophagy. Furthermore, the interaction of Swi with Nrf2 promoted the translocation of Nrf2 into the nucleus, resulting in the induction of Pink1. CONCLUSIONS: This study demonstrates that Swi prevents vascular remodelling in experimental PH through inhibition of phenotypic transformation and hyperproliferation of PASMCs caused by reversing hypoxia-induced inhibition of mitophagy.

Evaluation of a SNP-STR haplotype panel for forensic genotype imputation.

Short tandem repeat polymorphism (STR)-based individual identification is a popular and reliable method in many forensic applications. However, STRs still frequently fail to find any matched records. In such cases, if known STRs could provide more information, it would be very helpful to solve specific problems. Genotype imputation has long been used in the study of single nucleotide polymorphisms (SNPs) and has recently been introduced into forensic fields. The idea is that, through a reference haplotype panel containing SNPs and STRs, we can obtain unknown genetic information through genotype imputation based on known STR or SNP genotypes. Several recent studies have already demonstrated this exciting idea, and a 1000 Genomes SNP-STR haplotype panel has also been released. To further study the performance of genotype imputation in forensic fields, we collected STR, microhaplotype (MH) and SNP array genotypes from Chinese Han population individuals and then performed genotype imputation analysis based on the released reference panel. As a result, the average locus imputation accuracy was ∼83 % (or ∼70 %) when SNPs in the SNP array (or MH SNPs) were imputed from STRs, and was ∼30 % when highly polymorphic markers (STRs and MHs) were imputed from each other. When STRs were imputed from SNP array, the average locus imputation accuracy increased to ∼48 %. After analyzing the match scores between real STRs and the STRs imputed from SNPs, ∼80 % of studied STR records can be connected to corresponding SNP records, which may help for individual identification. Our results indicate that genotype imputation has great potential for forensic applications.

Simultaneous determination of diquat and its two primary metabolites in rat plasma by ultraperformance liquid chromatography-tandem mass spectrometry and its application to the toxicokinetic study.

PURPOSE: This study aimed to develop and validate an ultraperformance liquid chromatography-tandem mass spectrometry to simultaneously determine diquat (DQ) and its two primary metabolites in rat plasma and its application to the toxicokinetic study. METHOD: The chromatographic separation of DQ and its two primary metabolites was performed with hydrophilic interaction chromatography column by adding formic acid and ammonium acetate in mobile phase in stepwise elution mode. DQ and its two primary metabolites were detected by liquid chromatography-tandem mass spectrometry in positive mode. RESULTS: The lower limit of quantification ranging from 0.3 to 3.0 ng/mL for DQ and its two primary metabolites was achieved by using only 50 µL of rat plasma. The maximum concentration (Cmax) was 977 ng/mL, half-life (t1/2) was 13.1 h, and area under the plasma concentration-time curve (AUC0-t) was 2770 h*ng/mL for DQ, Cmax was 47.1 ng/mL, t1/2 was 25.1 h, and AUC0-t was 180 h·ng/mL for diquat monopyridone (DQ-M) and Cmax was 246 ng/mL, t1/2 was 8.2 h, and AUC0-t was 2430 h·ng/mL for diquat dipyridone (DQ-D), respectively. CONCLUSIONS: The validated method was shown to be suitable for simultaneous determination of diquat and its two primary metabolites in rat plasma. This study is the first to study the toxicokinetics of DQ and its two primary metabolites.

Development and validation of a sensitive and high throughput UPLC-MS/MS method for determination of paraquat and diquat in human plasma and urine: application to poisoning cases at emergency departments of hospitals.

PURPOSE: Paraquat and diquat are well-known toxic herbicides, at least responsible for hundreds of fatal poisoning events worldwide. However, the determination of diquat and paraquat in plasma and urine is very challenging because of their high polarity and double charge characteristics. In this study, we aim to develop a rapid and reliable method for the determination of paraquat and diquat in human plasma and urine by ultraperformance liquid chromatography-tandem mass spectrometry. METHOD: The chromatographic separation of paraquat and diquat was tested with different chromatographic columns and different mobile phase conditions. The mass parameters were optimized by product ions, source gas flow, cone flow, desolvation temperature, and capillary voltage. The isocratic elution mode gave rapid appearance of peak of paraquat and diquat. RESULTS: The sharp peak shapes for paraquat and diquat were achieved with CORTECS® UPLC® HILIC (100 × 2.1 mm, 1.6 µm) column by adding formic acid and ammonium acetate in mobile phase in isocratic elution mode. The lower limit of quantification of 1.0 ng/mL for paraquat and diquat were achieved using only 50 µL of human plasma or urine. The running time for analysis of both paraquat and diquat was as short as 3.5 min per sample. CONCLUSIONS: A rapid and reliable method for the determination of paraquat and diquat was developed and applied to 387 clinical poisoning cases and 22 poisoning cases were found to be paraquat or diquat poisoning.

Rapid detection of α-amanitin and ß-amanitin in rat plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to the toxicokinetic study of Lepiota brunneoincarnata.

PURPOSE: Lepiota brunneoincarnata is a well-known poisonous mushroom and is responsible for fatal mushroom poisoning cases worldwide. α-Amanitin and ß-amanitin are the main amatoxin compounds of Lepiota brunneoincarnata. However, there are no published toxicokinetic studies of Lepiota brunneoincarnata. To study the toxicokinetics of Lepiota brunneoincarnata, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determination of α-amanitin and ß-amanitin in rat plasma. METHODS: UPLC-MS/MS analyses were performed with a triple quadrupole mass spectrometer in positive-ion mode. The sensitivity of α-amanitin and ß-amanitin detection was increased by inhibiting the production of [M + Na]+ adducts. α-Amanitin and ß-amanitin were separated and quantified on an UPLC octadecyl silyl column in only 2.5 min. RESULTS: The linear ranges were 3.0-3000 ng/mL for α-amanitin and 1.8-1800 ng/mL for ß-amanitin with a correlation coefficient r > 0.99 for both analytes. The lower limit of quantification of 3.0 ng/mL for α-amanitin and 1.8 ng/mL for ß-amanitin was achieved using only 50 µL of rat plasma. The accuracy of α-amanitin and ß-amanitin was between - 9.5 and 7.0% with the precision ranged from 2.2 to 12.5%. The developed method was then applied for Lepiota brunneoincarnata toxicokinetic study after intravenous administration of Lepiota brunneoincarnata extracts. CONCLUSIONS: Establishing UPLC-MS/MS method for quantifying amanitines in rat plasma successfully enabled toxicokinetic study of Lepiota brunneoincarnata extracts.

Predicting the structure of unexplored novel fentanyl analogues by deep learning model.

Fentanyl and its analogues are psychoactive substances and the concern of fentanyl abuse has been existed in decades. Because the structure of fentanyl is easy to be modified, criminals may synthesize new fentanyl analogues to avoid supervision. The drug supervision is based on the structure matching to the database and too few kinds of fentanyl analogues are included in the database, so it is necessary to find out more potential fentanyl analogues and expand the sample space of fentanyl analogues. In this study, we introduced two deep generative models (SeqGAN and MolGPT) to generate potential fentanyl analogues, and a total of 11 041 valid molecules were obtained. The results showed that not only can we generate molecules with similar property distribution of original data, but the generated molecules also contain potential fentanyl analogues that are not pretty similar to any of original data. Ten molecules based on the rules of fentanyl analogues were selected for NMR, MS and IR validation. The results indicated that these molecules are all unreported fentanyl analogues. Furthermore, this study is the first to apply the deep learning to the generation of fentanyl analogues, greatly expands the exploring space of fentanyl analogues and provides help for the supervision of fentanyl.

In Situ Identification of Unknown Crystals in Acute Kidney Injury Using Raman Spectroscopy.

Raman spectroscopy is a well-established and powerful tool for in situ biomolecular evaluation. Type 2 crystal nephropathies are characterized by the deposition of crystalline materials in the tubular lumen, resulting in rapid onset of acute kidney injury without specific symptoms. Timely crystal identification is essential for its diagnosis, mechanism exploration and therapy, but remains challenging. This study aims to develop a Raman spectroscopy-based method to assist pathological diagnosis of type 2 crystal nephropathies. Unknown crystals in renal tissue slides from a victim suffered extensive burn injury were detected by Raman spectroscopy, and the inclusion of crystals was determined by comparing Raman data with established database. Multiple crystals were scanned to verify the reproducibility of crystal in situ. Raman data of 20 random crystals were obtained, and the distribution and uniformity of substances in crystals were investigated by Raman imaging. A mouse model was established to mimic the crystal nephropathy to verify the availability of Raman spectroscopy in frozen biopsy. All crystals on the human slides were identified to be calcium oxalate dihydrate, and the distribution and content of calcium oxalate dihydrate on a single crystal were uneven. Raman spectroscopy was further validated to be available in identification of calcium oxalate dihydrate crystals in the biopsy specimens. Here, a Raman spectroscopy-based method for in situ identification of unknown crystals in both paraffin-embedded tissues and biopsy specimens was established, providing an effective and promising method to analyze unknown crystals in tissues and assist the precise pathological diagnosis in both clinical and forensic medicine.

Could routine forensic STR genotyping data leak personal phenotypic information?

The application of forensic genetic markers must comply with privacy rights and legal policies on a premise that the markers do not expose phenotypic information. The most widely-used short tandem repeats (STRs) are generally viewed as 'junk' DNA because most STRs are located in non-coding regions and therefore refrain from leaking phenotypic traits. But with a deepening understanding of phenotypes and underlying genetic structure, whether STRs could potentially reflect any phenotypic information may need re-examining. Therefore, we performed the following analyses. First, we analyzed the association between 15 STRs and three facial characteristics (single or double eyelid, with or without epicanthus, unattached or attached earlobe) on 721 unrelated Han Chinese individuals. Then, we collected 27199 individuals' STRs and geographic data from the literature to investigate the association between STRs and bio-geographic information, and predict geographic information by STRs on additional 1993 unrelated individuals. We found that there was scarcely any association between STRs with studied facial characteristics. Although allele19 in D2S1338 and allele 18 in FGA (P = 0.0032, P = 0.0030, respectively after Bonferroni correction) showed statistical significance, the prediction effectiveness was very low. For the STRs and bio-geographic information, the principal component analysis showed the first three components could explain 87.7% of the variance, but the prediction accuracy only reached 25.2%. We demonstrated that the forensic phenotypes are usually complex traits, it is hardly possible to uncover phenotypic information by testing only dozens of STR loci.