OBJECTIVES: Tiletamine, a veterinary anesthetic, has emerged as a novel psychoactive substance and has been abused in many parts of the world, causing great harm to public health. However, the sensitivity of existing detection methods cannot meet the needs of forensic practice. This study aims to establish an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of tiletamine and its metabolite desethyltiletamine in human biological samples, and to verify its applicability in forensic practice. METHODS: SKF_525A was used as the internal standard. Biological samples were extracted with acetonitrile containing 1 ng/mL SKF_525A, vortexed for 10 min, ultrasonicated for 20 min, centrifuged at 10 000 r/min for 10 min, and 500 μL of the supernatant was filtered through a 0.22 μm membrane. Analyses were performed using an ACQUITY UPLC H-Class PLUS system and an XEVO TQ-S Micro triple quadrupole mass spectrometer. An ACQUITY UPLC^® BEH C18 (1.7 µm, 2.1 mm×100 mm) column at a flow rate of 0.3 mL/min was used, and four mobile phase systems were tested to optimize separation. Detection used positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, with quantifier ion transitions of mass to charge 224.043→179.016 for tiletamine and mass to charge 196.08→151.06 for desethyltiletamine. Calibration curves were established over 0.1-200 ng/mL in spiked blood samples. The linear range, limit of detection (LOD), and limit of quantification (LOQ) were determined. Low (5 ng/mL), medium (20 ng/mL), and high (100 ng/mL) concentrations of tiletamine were spiked into blood, liver, and kidney to evaluate precision, accuracy, matrix effect, recovery, and stability. Finally, actual forensic case samples were tested to validate applicability. RESULTS: The established UPLC-MS/MS method achieved simultaneous detection of tiletamine and desethyltiletamine in human biological samples, with retention times of 3.42 min and 2.82 min, respectively. Using mobile phase A (20 mmol/L ammonium acetate and 0.1% formic acid in water) and mobile phase B (acetonitrile) produced the best separation. In blood, tiletamine showed good linearity from 0.1-200 ng/mL (r=0.992, R²=0.983), LOD 0.03 ng/mL, LOQ 0.1 ng/mL, recovery 92%-107%, and matrix effect 71%-99%. In liver and kidney, recoveries were 91%-98% and 93%-104%, and matrix effects were 69%-96% and 72%-100%, respectively. Intra- and inter-day precision [expressed as relative standard deviation (RSD)] and accuracy [expressed as relative error (RE)] were within 15%, and samples were stable at -20 ℃. Tiletamine was detected in actual case samples at 0.37 μg/mL (blood), 0.15 μg/g (liver), 0.11 μg/g (kidney) in case 1, and 8.75 ng/mL (blood) in case 2; desethyltiletamine was also detected in blood. CONCLUSIONS The UPLC-MS/MS method is efficient, accurate, and sensitive, and is suitable for detecting tiletamine and desethyltiletamine in human biological samples.
Tandem Mass Spectrometry/methods* ; Humans ; Chromatography, High Pressure Liquid/methods* ; Substance Abuse Detection/methods* ; Liquid Chromatography-Mass Spectrometry

ObjectiveTo investigate the relationship between serum 25(OH)D and gestational diabetes mellitus in the second trimester (GDM), to analyze the interaction effect of key risk factors, so as to provide a basis for clinical personalized vitamin D supplementation. MethodsA total of 266 pregnant women who registered and took regular obstetric check-ups in Shanghai Pudong New Area Health Care Hospital for Women and Children from June to December 2022, were selected as the research subjects. According to the results of oral glucose tolerance test (OGTT), the subjects were divided into the GDM group (131) and control group (135). The level of serum 25(OH)D at the time of OGTT were detected and other clinical indicators were followed up. ResultsThe age, systolic blood pressure in early pregnancy, pre-pregnancy BMI, FPG, OGTT 1-hour and 2-hour glucose, GHb, HOMA-IR, TG, AST, Cr, D-D, FDP and SF at 35 weeks’ gestation of the pregnant women were higher in the GDM group than that of the pregnant women in the normal group, while gestational weight gain and serum 25(OH)D level were significantly lower than that of the pregnant women in the normal group. Serum 25(OH)D was negatively correlated with HOMA-IR and WBS’s, but positively correlated with TG and ALT. Serum 25(OH)D was non-linearly correlated with the risk of GDM in an inverted J-shape, and there was an interaction effect of advanced age, pre-pregnancy obesity and vitamin D deficiency on the risk of GDM. ConclusionVitamin D is non-linearly associated with the risk of GDM. The risk of GDM is significantly reduced when serum 25(OH)D level ≥30 ng∙mL^-1.

Objective:To explore the effectiveness and safety of focused ultrasound ablation surgery (FUAS) for abdominal wall endometriosis.Methods:From November 2019 to October 2022, a total of 34 patients with abdominal wall endometriosis who underwent FUAS were collected, and their clinical features, imaging features, intraoperative treatment and side effects after treatment were analyzed retrospectively, and the improvement of symptoms and re-intervention were followed up.Results:(1) Characteristics of clinical data: the average age of 34 patients with abdominal wall endometriosis was (32.8±3.8) years old. The largest diameter of the lesion was 48 mm, and the median lesion diameter was 24 mm. Thirty cases (88%, 30/34) had moderate to severe periodic pain in abdominal incision before FUAS. All patients were diagnosed by preoperative magnetic resonance imaging, including 19 cases (56%, 19/34) of superficial type, 8 cases (24%, 8/34) of intermediate type and 7 cases (21%, 7/34) of deep type. (2) FUAS treatment parameters: ablation was completed with average operation time of (64±18) minutes, average sonication time was (385±108) s, (103±11) W of average power, (38 819±16 309) J of average total energy, the average treatment area volume of (3.11±1.42) cm 3, and (377.79±106.34) s/h of average treatment intensity. (3) Efficiency: the pain of patients after FUAS was significantly relieved, and the pain scores of patients after 1 month, 3 months, 6 months and 1 year after FUAS were significantly decreased ( Z=-4.66, -5.13, -5.11 and -4.91, all P<0.01). One year after FUAS, the near relief and effective pain relief rate was 74% (25/34), and the clinical effective rate was 85% (29/34). Five patients recurred after one year, including 3 patients who underwent abdominal wall endometriosis lesion resection and 2 patients who received drug treatment. One month after FUAS, the size of the lesion did not change significantly compared with that before FUAS ( P>0.05), and the size of the lesion decreased significantly after FUAS at 3 months, 6 months and 1 year ( Z=-2.15, -2.67 and -3.41, all P<0.05). It has no difference in pain relief among different types ( P>0.05), but has significant difference in focus reduction among three types ( P<0.01). (4) Safety: there were 34 cases (100%, 34/34) of skin burning sensation, 19 cases (56%, 19/34) of pain in the treatment area and 2 cases (6%, 2/34) of hematuria. All patients got better after corresponding treatments. Conclusion:FUAS is safe and effective for the treatment of abdominal wall endometriosis, which has clinical application value.

OBJECTIVE: To explore the correlation between clinical manifestations of Limb-girdle muscular dystrophy autosomal recessive 9 FKRP-related (R9 FKRP-related) and variants of the FKRP gene. METHODS: Two children who had presented at the Children's Hospital of Nanjing Medical University respectively due to increased serum myocardial zymogram and hepatic dysfunction on September 30, 2018 and August 3, 2018 were selected as the study subjects. Clinical data of the children were collected. Both children were suspected for Duchenne or Becker muscular dystrophy for asymptomatic high creatine kinase (CK) levels. Peripheral blood samples of the children and their parents were collected for whole exome sequencing, and candidate variants were validated by Sanger sequencing. RESULTS: Genetic testing revealed that both children have carried compound heterozygous variants of the FKRP gene. The c.545A>G and c.941C>T variants in child 1 have been reported previously, among which the c.545A>G is a hot spot mutation in the Chinese population. Child 2 has carried c.602T>C and c.961G>A variants, both of which were unreported previously. CONCLUSION Both children have met the diagnostic criteria for LGMD R9 FKRP-related. Carriers of the c.545A>G variant may present milder symptoms. Compared with patients carrying null variants, carriers of compound heterozygous missense variants may present with a milder phenotype, manifesting as asymptomatic high CK level.
Humans ; Child ; Asian People/genetics* ; Genetic Testing ; Muscular Dystrophies, Limb-Girdle/genetics* ; Muscular Dystrophy, Duchenne ; Pentosyltransferases/genetics*

[This corrects the article DOI: 10.1016/j.apsb.2023.04.002.].

OBJECTIVE To study the effects of Dianxianqing granules on the tau protein in P301S mice by regulating mitophagy. METHODS Totally 36 P301S mice were randomly divided into model group， Dianxianqing granule group （12.48 g/kg）， donepezil hydrochloride group （positive control， 1.3 mg/kg）， with 12 mice in each group； another 10 C57BL6 mice were selected as control group. Administration groups were given relevant drug solutions intragastrically， and control group and model group were given constant volume of water intragastrically. The gavage volume was 20 mL/kg， once a day， for consecutive 5 months. During the experiment， the general condition of mice was observed in each group. After the last medication， the learning and memory ability was determined by Y maze test and Morris water maze test； HE staining was used to observe the morphological changes in brain tissue， and Nissl staining was used to observe the structure of neural cells and the number of Nissl bodies in cerebral tissue. Immunohistochemistry was used to detect the expressions of phospho-tau serine 202/threonine 205 （abbreviated as AT8） in brain tissue. Western blot assay was used to determine the expressions of mitophagy-associated proteins [PTEN-induced putative kinase-1 （PINK1）， Parkin， microtubule-associated protein 1 light chain 3B （LC3B）， p62]， synaptic-associated proteins [postsynaptic density protein-95 （PSD-95）， synaptophysin （SYP）， and growth-associated protein-43 （GAP-43）] and the phosphorylation of tau protein [expressed by the phosphorylation levels of serine 199 （Ser199） and Ser202] in brain tissue. RESULTS The mice in E-mail：lnzyxyqy2003@163.com model group showed symptoms such as white hair， decreased body mass， and lower limb paralysis， with incomplete hippocampal structures in their brain tissue， as well as incomplete cell membrane edges and cell structures； the spontaneous alternating response rate， the times of crossing platform， the number of Nissl bodies， the protein expressions of PINK1， Parkin， LC3B， SYP， GAP-43， and PSD-95 were decreased significantly， compared with control group； swimming latency （fourth and fifth day）， the protein expressions of AT8 and p62，the phosphorylation levels of Ser199 and Ser202 were increased or lengthened significantly， compared with control group （P＜0.05 or P＜0.01）. Compared with model group， the above symptoms and indexes of mice were improved significantly in administration groups （P＜0.05 or P＜0.01）. CONCLUSIONS Dianxianqing granules can effectively improve cognitive impairment in P301S mice，the mechanism of which may be associated with inducing mitochondrial autophagy， reducing the hyperphosphorylation of tau protein， up-regulating the expression of synaptic-associated proteins in brain tissue，and repairing damaged neural cells.

Lipids have been found to modulate tumor biology, including proliferation, survival, and metastasis. With the new understanding of tumor immune escape that has developed in recent years, the influence of lipids on the cancer-immunity cycle has also been gradually discovered. First, regarding antigen presentation, cholesterol prevents tumor antigens from being identified by antigen presenting cells. Fatty acids reduce the expression of major histocompatibility complex class I and costimulatory factors in dendritic cells, impairing antigen presentation to T cells. Prostaglandin E2 (PGE2) reduce the accumulation of tumor-infiltrating dendritic cells. Regarding T-cell priming and activation, cholesterol destroys the structure of the T-cell receptor and reduces immunodetection. In contrast, cholesterol also promotes T-cell receptor clustering and relative signal transduction. PGE2 represses T-cell proliferation. Finally, regarding T-cell killing of cancer cells, PGE2 and cholesterol weaken granule-dependent cytotoxicity. Moreover, fatty acids, cholesterol, and PGE2 can improve the activity of immunosuppressive cells, increase the expression of immune checkpoints and promote the secretion of immunosuppressive cytokines. Given the regulatory role of lipids in the cancer-immunity cycle, drugs that modulate fatty acids, cholesterol and PGE2 have been envisioned as effective way in restoring antitumor immunity and synergizing with immunotherapy. These strategies have been studied in both preclinical and clinical studies.

Mevalonate metabolism plays an important role in regulating tumor growth and progression; however, its role in immune evasion and immune checkpoint modulation remains unclear. Here, we found that non-small cell lung cancer (NSCLC) patients with higher plasma mevalonate response better to anti-PD-(L)1 therapy, as indicated by prolonged progression-free survival and overall survival. Plasma mevalonate levels were positively correlated with programmed death ligand-1 (PD-L1) expression in tumor tissues. In NSCLC cell lines and patient-derived cells, supplementation of mevalonate significantly up-regulated the expression of PD-L1, whereas deprivation of mevalonate reduced PD-L1 expression. Mevalonate increased CD274 mRNA level but did not affect CD274 transcription. Further, we confirmed that mevalonate improved CD274 mRNA stability. Mevalonate promoted the affinity of the AU-rich element-binding protein HuR to the 3'-UTR regions of CD274 mRNA and thereby stabilized CD274 mRNA. By in vivo study, we further confirmed that mevalonate addition enhanced the anti-tumor effect of anti-PD-L1, increased the infiltration of CD8⁺ T cells, and improved cytotoxic function of T cells. Collectively, our findings discovered plasma mevalonate levels positively correlated with the therapeutic efficacy of anti-PD-(L)1 antibody, and provided the evidence that mevalonate supplementation could be an immunosensitizer in NSCLC.

Extracellular vesicles (EVs) can carry a variety of bioactive components including nucleic acids, proteins and small molecule metabolites, and their value in tumor diagnosis and treatment has been widely recognized. However, current studies on EV inclusions mainly focus on RNA and protein, and the role of small molecule metabolites that can most directly reflect the cell state in EV remains unclear. EV metabolomics in cancer research has gradually gained traction in recent years. There are still many challenges in EV metabolomics research due to the complexity of pretreatment and low content of metabolite, but its value in regulating tumor progression and serving as tumor markers has gradually emerged, which is expected to provide new targets for tumor diagnosis and treatment.

Objective:To retrospectively analyze the pregnancy outcomes of patients with adenomyosis requiring fertility in a single center under real world condition.Methods:From June 2015 to May 2020, 231 cases of pregnancy complicated with adenomyosis diagnosed by ultrasound with fertility requirements were treated in the Women′s and Children′s Hospital Affiliated to Qingdao University with complete clinical data. And they were divided into three groups according to the treatment of adenomyosis before pregnancy: expectation group, drug group and operation group. The relevant data before pregnancy of the three groups were analyzed, and the pregnancy outcomes of the patients were summarized. According to whether the early pregnancy was treated with medication, the patients who were naturally conceived without symptoms of threatened abortion were divided into observation group and fetus protection group, and the pregnancy outcomes of the two groups were compared.Results:(1) Compared with the expectation group, the ages of patients in the drug group and the operation group were larger [(31.5±1.8) vs (34.1±3.7) vs (36.9±3.6) years old], and the difference was statistically significant ( P<0.05). Only 9 patients (11.5%, 9/78) had clinical symptoms in the expectation group, while the patients in the drug group and the operation group had a higher proportion of dysmenorrhea and increased menstrual volume. The uterine volume of the drug group and the operation group were larger than that of the expectation group [(151±46) vs (166±27) vs (97±18) cm 3], the difference was statistically significant ( P<0.05). 78.6% (33/42) of the operation group were focal adenomyosis. The proportion of natural pregnancy in the expectation group was 97.4% (76/78), and in vitro fertilization and embryo transfer was mainly used in the drug group and the operation group. (2) The abortion rates of the three groups were 48.7% (26/111), 4/17, 67.5% (27/78) respectively. Compared with the drug group and the operation group, the preterm birth rate was lower [55.9% (33/111) vs 11/17 vs 12.5% (5/78)] and the natural delivery rate was higher [44.1% (26/111) vs 4/17 vs 67.5% (27/78)] in the expectation group. (3) There were 89 cases of spontaneous pregnancy without threatened abortion symptoms, including 31 cases in the observation group and 58 cases in the fetus protection group. Compared with the observation group, the abortion rate of patients in the fetus protection group was lower [41.9% (13/31) vs 34.5% (20/58)], and the difference was statistically significant ( P<0.05). Conclusions:Patients with adenomyosis who have fertility requirements should be comprehensively evaluated and individualized treatment plans should be given. Pregnancy patients with adenomyosis have a high rate of miscarriage, and they should be included in the management of high-risk pregnant women. Active fetal protection treatment during early pregnancy might improve pregnancy outcomes.