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.

Risk factors for postpartum posttraumatic stress disorder after emergency admission.

BACKGROUND: Postpartum posttraumatic stress disorder (PTSD) can occur in women who give birth after emergency admission. The identification of risk factors for this condition is crucial for developing effective preventive measures. This retrospective study aimed to explore the incidence and risk factors for postpartum PTSD in women who give birth after emergency admission. METHODS: Medical records of women who gave birth after emergency admission were collected between March 2021 and April 2023. The patients' general conditions and perinatal clinical indicators were recorded. The puerperae were divided into PTSD group and control group based on symptom occurrence at six weeks postpartum. Multivariate logistic regression analysis was performed to identify risk factors. RESULTS: A total of 276 puerperae were included, with a PTSD incidence of 20.3% at six weeks postpartum. Multivariate logistic regression analysis identified emergency cesarean section (odds ratio [OR]=2.102; 95% confidence interval [CI]: 1.114-3.966, P=0.022), admission to the emergency department after midnight (12:00 AM) (OR=2.245; 95%CI: 1.170-4.305, P<0.001), and cervical dilation (OR=3.203; 95%CI: 1.670-6.141, P=0.039) as independent risk factors for postpartum PTSD. Analgesia pump use (OR= 0.500; 95%CI: 0.259-0.966, P=0.015) was found to be a protective factor against postpartum PTSD. CONCLUSION: Emergency cesarean section, admission to the emergency department after midnight, and cervical dilation were identified as independent risk factors for postpartum PTSD, while analgesic pump use was a protective factor. These findings provide insights for developing more effective preventive measures for women who give birth after emergency admission.

Nanoparticle-Exposure-Triggered Virus Reactivation Induces Lung Emphysema in Mice.

Nanoparticles (NPs) released from engineered materials or combustion processes as well as persistent herpesvirus infection are omnipresent and are associated with chronic lung diseases. Previously, we showed that pulmonary exposure of a single dose of soot-like carbonaceous NPs (CNPs) or fiber-shaped double-walled carbon nanotubes (DWCNTs) induced an increase of lytic virus protein expression in mouse lungs latently infected with murine γ-herpesvirus 68 (MHV-68), with a similar pattern to acute infection suggesting virus reactivation. Here we investigate the effects of a more relevant repeated NP exposure on lung disease development as well as herpesvirus reactivation mechanistically and suggest an avenue for therapeutic prevention. In the MHV-68 mouse model, progressive lung inflammation and emphysema-like injury were detected 1 week after repetitive CNP and DWCNT exposure. NPs reactivated the latent herpesvirus mainly in CD11b+ macrophages in the lungs. In vitro, in persistently MHV-68 infected bone marrow-derived macrophages, ERK1/2, JNK, and p38 MAPK were rapidly activated after CNP and DWCNT exposure, followed by viral gene expression and increased viral titer but without generating a pro-inflammatory signature. Pharmacological inhibition of p38 activation abrogated CNP- but not DWCNT-triggered virus reactivation in vitro, and inhibitor pretreatment of latently infected mice attenuated CNP-exposure-induced pulmonary MHV-68 reactivation. Our findings suggest a crucial contribution of particle-exposure-triggered herpesvirus reactivation for nanomaterial exposure or air pollution related lung emphysema development, and pharmacological p38 inhibition might serve as a protective target to alleviate air pollution related chronic lung disease exacerbations. Because of the required precondition of latent infection described here, the use of single hit models might have severe limitations when assessing the respiratory toxicity of nanoparticle exposure.

Effect of Dexmedetomidine on Posttraumatic Stress Disorder in Patients Undergoing Emergency Trauma Surgery: A Randomized Clinical Trial.

Importance: Posttraumatic stress disorder (PTSD) is common in people who have experienced trauma, especially those hospitalized for surgery. Dexmedetomidine may reduce or reverse the early consolidation and formation of conditioned fear memory and prevent the occurrence of postoperative PTSD. Objective: To evaluate the effects of intraoperative and postoperative low-dose intravenous pumping dexmedetomidine on PTSD among patients with trauma undergoing emergency surgery. Design, Setting, and Participants: This double-blind, randomized clinical trial was conducted from January 22 to October 20, 2022, with follow-up 1 month postoperatively, in patients with trauma undergoing emergency surgery at 4 hospital centers in Jiangsu Province, China. A total of 477 participants were screened. The observers were blinded to patient groupings, particularly for subjective measurements. Interventions: Dexmedetomidine or placebo (normal saline) was administered at a maintenance dose of 0.1 µg/kg hourly from the start of anesthesia until the end of surgery and at the same rate after surgery from 9 pm to 7 am on days 1 to 3. Main Outcomes and Measures: The primary outcome was the difference in the incidence of PTSD 1 month after surgery in the 2 groups. This outcome was assessed with the Clinician-Administered PTSD Scale for Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) (CAPS-5). The secondary outcomes were the pain score within 48 hours and 1 month postoperatively; incidence of postoperative delirium, nausea, and pruritus; subjective sleep quality; anxiety; and occurrence of adverse events. Results: A total of 310 patients (154 in the normal saline group and 156 in the dexmedetomidine group) were included in the modified intention-to-treat analysis (mean [SD] age, 40.2 [10.3] years; 179 men [57.7%]). The incidence of PTSD was significantly lower in the dexmedetomidine group than in the control group 1 month postoperatively (14.1% vs 24.0%; P = .03). The participants in the dexmedetomidine group had a significantly lower CAPS-5 score than those in the control group (17.3 [5.3] vs 18.9 [6.6]; mean difference, 1.65; 95% CI, 0.31-2.99; P = .02). After adjusting for potential confounders, the patients in the dexmedetomidine group were less likely to develop PTSD than those in the control group 1 month postoperatively (adjusted odds ratio, 0.51; 95% CI, 0.27-0.94; P = .03). Conclusions and Relevance: In this randomized clinical trial, the administration of intraoperative and postoperative dexmedetomidine reduced the incidence of PTSD among patients with trauma. The findings of this trial support the use of dexmedetomidine in emergency trauma surgery. Trial Registration: Chinese Clinical Trial Register Identifier: ChiCTR2200056162.

Therapeutic effects and mechanism of human amnion-derived mesenchymal stem cells on hypercoagulability in a uremic calciphylaxis patient.

Calciphylaxis is a rare cutaneous vascular disease that manifests with intolerable pains, non-healing skin wounds, histologically characterized by calcification, fibrointimal hyperplasia, and microvessel thrombosis. Currently, there are no standardized guidelines for this disease. Recent studies have recognized a high prevalence of thrombophilias and hypercoagulable conditions in calciphylaxis patients. Here, we report a case of uremic calciphylaxis patient whom was refractory to conventional treatments and then received a salvage strategy with intravenous and local hAMSC application. In order to investigate the therapeutic mechanism of hAMSCs from the novel perspective of hypercoagulability, coagulation-related indicators, wound status, quality of life and skin biopsy were followed up. Polymerase chain reaction (PCR) was performed to determine the distribution of hAMSCs in multiple tissues including lung, kidney and muscle after infusion of hAMSCs for 24 h, 1 week and 1 month in mice aiming to investigate whether hAMSCs retain locally active roles after intravenous administration. Improvement of hypercoagulable condition involving correction of platelet, D-dimer and plasminogen levels, skin regeneration and pain alleviation were revealed after hAMSC administration over one-year period. Skin biopsy pathology suggested regenerative tissues after 1 month hAMSC application and full epidermal regeneration after 20 months hAMSC treatment. PCR analysis indicated that hAMSCs were homing in lung, kidney and muscle tissues of mice even until tail vein injection of hAMSCs for 1 month. We propose that hypercoagulability is a promising therapeutic target of calciphylaxis patients, which can be effectively improved by hAMSC treatment.

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.

RNA Binding Protein Quaking Promotes Hypoxia-induced Smooth Muscle Reprogramming in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a devastating disease characterized by progressive increases in pulmonary vascular resistance and remodeling, which eventually leads to right ventricular failure and death. The aim of this study was to identify novel molecular mechanisms involved in the hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) in PH. In this study, we first demonstrated that the mRNA and protein expression amounts of QKI (Quaking), an RNA-binding protein, were elevated in human and rodent PH lung and pulmonary artery tissues and hypoxic human PASMCs. QKI deficiency attenuated PASMC proliferation in vitro and vascular remodeling in vivo. Next, we elucidated that QKI increases STAT3 (signal transducer and activator of transcription 3) mRNA stability by binding to its 3' untranslated region. QKI inhibition reduced STAT3 expression and alleviated PASMC proliferation in vitro. Moreover, we also observed that the upregulated expression of STAT3 promoted PASMC proliferation in vitro and in vivo. In addition, as a transcription factor, STAT3 bound to microRNA (miR)-146b promoter to enhance its expression. We further showed that miR-146b promoted the proliferation of smooth muscle cells by inhibiting STAT1 and TET2 (Tet methylcytosine dioxygenase 2) during pulmonary vascular remodeling. This study has demonstrated new mechanistic insights into hypoxic reprogramming that arouses vascular remodeling, thus providing proof of concept for targeting vascular remodeling by directly modulating the QKI-STAT3-miR-146b pathway in PH.

Report on Cardiac Gross Pathologic Measurements of Sudden Cardiac Death in Adults.

OBJECTIVES: To analyze the gross pathological data of sudden cardiac death (SCD) with different causes, to provide data support for the identification of sudden cardiac death with unknown causes. METHODS: A total of 167 adult SCD cases in the archive of the Forensic Expertise Institute of Nanjing Medical University from 2010 to 2020 were collected. The gross pathological data of SCD cases were summarized and the characteristics of different causes of death were statistically analyzed. RESULTS: The ratio of male to female SCD cases was 3.4∶1. Coronary heart disease was the leading cause of SCD, and mainly distributed in people over 40 years old. SCD caused by myocarditis was mainly distributed in young people and the mean age of death was (34.00±9.55) years. By analyzing the differences in cardiac pathological parameters of SCD with different causes, it was found that the aortic valve circumference was significantly dilated in the SCD caused by aortic aneurysm or dissection (P<0.05). The heart weight of SCD caused by aortic aneurysm or dissection and combined factors was greater, and both pulmonary and tricuspid valvular rings were dilated in the SCD caused by combined factors in adult males (P<0.05). CONCLUSIONS: Various gross pathological measures of SCD with different causes are different, which has reference value in the cause of death identification of SCD.

Whole-exome sequencing and electrophysiological study reveal a novel loss-of-function mutation of KCNA10 in epinephrine provoked long QT syndrome with familial history of sudden cardiac death.

Congenital long QT syndrome (LQTS) is one type of inherited fatal cardiac arrhythmia that may lead to sudden cardiac death (SCD). Mutations in more than 16 genes have been reported to be associated with LQTS, whereas the genetic causes of about 20% of cases remain unknown. In the present study, we investigated a four-generation pedigree with familial history of syncope and SCD. The proband was a 33-year-old young woman who experienced 3 episodes of syncope when walking at night. The electrocardiogram revealed a markedly epinephrine-provoked prolonged QT interval (QT = 468 ms, QTc = 651 ms) but no obvious arrhythmia in the resting state. Three family members have died of suspected SCD. Whole-exome sequencing and bioinformatic analysis based on pedigree revealed that a novel missense mutation KCNA10 (c.1397G＞A/Arg466Gln) was the potential genetic lesion. Sanger sequencing was performed to confirm the whole-exome sequencing results. This mutation resulted in the KV1.8 channel amino acid residue 466 changing from arginine to glutamine, and the electrophysiological experiments verified it as a loss-of-function mutation of KV1.8, which reduced the K+ currents of KV1.8 and might result in the prolonged QT interval. These findings suggested that KCNA10 (c.1397G＞A) mutation was possibly pathogenic in this enrolled LQTS family, and may provide a new potential genetic target for diagnosis and counseling of stress-related LQTS families as well as the postmortem diagnosis of SCD.

Plasma extracellular vesicles microRNA-208b-3p and microRNA-143-3p as novel biomarkers for sudden cardiac death prediction in acute coronary syndrome.

Acute coronary syndrome (ACS) occurs as a result of myocardial ischemia that can give rise to a variety of acute cardiovascular events, including arrhythmia, heart failure and sudden cardiac death (SCD). Currently, there are challenges and insufficient innovations regarding early diagnosis and therapeutic approaches within ACS patients experiencing SCD. Plasma extracellular vesicles (EVs) might serve as biomarkers of many diseases depending on the biological molecules of their cargo, such as miRNAs. This study aims to identify the plasma EVs containing miRNAs as novel biomarkers for the prediction of SCD in ACS patients. A total of 39 ACS patients experiencing SCD and 39 healthy control individuals (HC) were enrolled, among which 9 samples in each group were randomly selected as testing groups for miRNA sequencing in plasma EVs, and the remaining samples were assigned to the validation group. The top 10 significant expression miRNAs were verified by the real-time quantitative polymerase chain reaction. Upregulation of miR-208b-3p, miR-143-3p, miR-145-3p and miR-152-3p, and down-regulation of miR-183-5p were further validated in the validation group. Spearman's correlation analysis and the receiver operating characteristic (ROC) curve showed that both miR-208b-3p and miR-143-3p levels were positively correlated with myoglobin (MYO), and their predictive power for SCD was confirmed. In conclusion, our findings indicate that plasma EVs miR-208b-3p and miR-143-3p may serve as promising biomarkers in predicting SCD in patients with ACS, as well as postmortem forensic diagnosis of the cause of death due to ACS.

Ythdf2 promotes pulmonary hypertension by suppressing Hmox1-dependent anti-inflammatory and antioxidant function in alveolar macrophages.

Pulmonary hypertension (PH) is a devastating disease characterized by irreversible pulmonary vascular remodeling (PVR) that causes right ventricular failure and death. The early alternative activation of macrophages is a critical event in the development of PVR and PH, but the underlying mechanisms remain elusive. Previously we have shown that N6-methyladenosine (m6A) modifications of RNA contribute to phenotypic switching of pulmonary artery smooth muscle cells and PH. In the current study, we identify Ythdf2, an m6A reader, as an important regulator of pulmonary inflammation and redox regulation in PH. In a mouse model of PH, the protein expression of Ythdf2 was increased in alveolar macrophages (AMs) during the early stages of hypoxia. Mice with a myeloid specific knockout of Ythdf2 (Ythdf2Lyz2 Cre) were protected from PH with attenuated right ventricular hypertrophy and PVR compared to control mice and this was accompanied by decreased macrophage polarization and oxidative stress. In the absence of Ythdf2, heme oxygenase 1 (Hmox1) mRNA and protein expression were significantly elevated in hypoxic AMs. Mechanistically, Ythdf2 promoted the degradation of Hmox1 mRNA in a m6A dependent manner. Furthermore, an inhibitor of Hmox1 promoted macrophage alternative activation, and reversed the protection from PH seen in Ythdf2Lyz2 Cre mice under hypoxic exposure. Together, our data reveal a novel mechanism linking m6A RNA modification with changes in macrophage phenotype, inflammation and oxidative stress in PH, and identify Hmox1 as a downstream target of Ythdf2, suggesting that Ythdf2 may be a therapeutic target in PH.

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.

Inferring bio-geographical ancestry with 35 microhaplotypes.

During the past decades, with the blooming of large-scale human genetic studies, we are beginning to understand how bio-geographical information could be reflected by genetic variations. And the technological advance in massively parallel sequencing gives advantages to some novel forensic markers such as microhaplotype (BIM). In the present study, we selected and characterized 35 novel bio-geographical informative BIMs based on the 1000 Genomes Project (1KG). All loci had short lengths less than 100 bp, high effective allele numbers (Ae) from 1.875 to 3.980 with an average of 2.798, and high informativeness (In) value from 0.701 to 0.865 with an average of 0.748, which indicates the 35 BIMs possessed great discriminating power. Using these 35 BIMs, the individuals from 1KG could be successfully differentiated into five supergroups defined by 1KG (AFR, AMR, EAS, EUR, and SAS). We also included some individuals from the Simons Genome Diversity Project (SGDP) for further validation. As a result, most individuals could be accurately predicted except for those from super-populations that do not exist in 1KG dataset. In conclusion, the present novel 35 BIMs could be a useful tool for bio-geographical ancestry inference.

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.

Construction and Application of YOLOv3-Based Diatom Identification Model of Scanning Electron Microscope Images.

OBJECTIVES: To construct a YOLOv3-based model for diatom identification in scanning electron microscope images, explore the application performance in practical cases and discuss the advantages of this model. METHODS: A total of 25 000 scanning electron microscopy images were collected at 1 500× as an initial image set, and input into the YOLOv3 network to train the identification model after experts' annotation and image processing. Diatom scanning electron microscopy images of lung, liver and kidney tissues taken from 8 drowning cases were identified by this model under the threshold of 0.4, 0.6 and 0.8 respectively, and were also identified by experts manually. The application performance of this model was evaluated through the recognition speed, recall rate and precision rate. RESULTS: The mean average precision of the model in the validation set and test set was 94.8% and 94.3%, respectively, and the average recall rate was 81.2% and 81.5%, respectively. The recognition speed of the model is more than 9 times faster than that of manual recognition. Under the threshold of 0.4, the mean recall rate and precision rate of diatoms in lung tissues were 89.6% and 87.8%, respectively. The overall recall rate in liver and kidney tissues was 100% and the precision rate was less than 5%. As the threshold increased, the recall rate in all tissues decreased and the precision rate increased. The F1 score of the model in lung tissues decreased with the increase of threshold, while the F1 score in liver and kidney tissues with the increase of threshold. CONCLUSIONS: The YOLOv3-based diatom electron microscope images automatic identification model works at a rapid speed and shows high recall rates in all tissues and high precision rates in lung tissues under an appropriate threshold. The identification model greatly reduces the workload of manual recognition, and has a good application prospect.