An epileptic encephalopathy associated GABRG2 missense mutation leads to pre- and postsynaptic defects in zebrafish.

Mutations in the γ-aminobutyric acid type A (GABAA) receptor γ2 subunit gene, GABRG2, have been associated with a variety of epilepsy syndromes. A de novo mutation (c.T1027C, p.F343L) in GABRG2 was identified in a patient with early onset epileptic encephalopathy. Zebrafish overexpressing mutant human GABRG2 (F343L) subunits displayed spontaneous seizure activity and convulsive behaviors. In this study, we demonstrated that Tg (hGABRG2F343L) zebrafish displayed hyperactivity during light phase with normal circadian rhythm, as well as increased drug-induced locomotor activity. Real-time quantitative PCR, whole mount in situ hybridization and western blotting showed that Tg(hGABRG2F343L) zebrafish had altered expression of GABAA receptor subunits. Furthermore, investigation of synaptic protein expression and synapse ultrastructure uncovered a robust synaptic phenotype that is causally linked to GABRG2(F343L) mutation. Strikingly, Tg(hGABRG2F343L) zebrafish not only had postsynaptic defects, but also displayed an unanticipated deficit at the presynaptic level. Overall, our Tg(hGABRG2F343L) overexpression zebrafish model has expanded the GABAergic paradigm in epileptic encephalopathy from channelopathy to synaptopathy.

Risk factors and predictors of recurrence of febrile seizures in children in Nantong, China: a retrospective cohort study.

BACKGROUND: Although most children with febrile seizures (FS) have a favorable prognosis, some experience recurrence within 1-3 years. Age, peak temperature, and family history are now recognized as important risk factors for FS recurrence, yet studies in this area are lacking in China. This study aimed to investigate the risk factors for FS recurrence in children in Nantong, China, and to develop a prediction model. METHODS: This retrospective cohort study analyzed 463 children diagnosed with febrile seizures (FS) who presented to the Affiliated Hospital of Nantong University between January 2015 and June 2020. Basic information, disease characteristics, and laboratory and imaging data were collected. A follow-up survey was conducted one year post-discharge to assess the recurrence status of FS in children. Univariate logistic regression and random forest models were used to identify and rank the predictive ability of risk factors for recurrence. RESULTS: Of the 463 children with FS, 70 experienced recurrences within 1 year of discharge, resulting in a one-year recurrence rate of 15%. Age (OR = 0.61, 95% CI: 0.46, 0.80, P < 0.001), duration of the first episode (OR = 1.03, 95% CI: 1.00, 1.06, P = 0.040), and peak temperature (OR = 0.68, 95% CI: 0.47, 0.98, P = 0.036) were identified as independent risk factors for FS recurrence. Age had the highest relative importance in predicting FS recurrence, followed by the duration of the first episode, with an area under the ROC curve of 0.717. CONCLUSION: Young age and duration of the first seizure are important independent risk factors for FS recurrence and are key considerations for predicting recurrence. Further research is needed to confirm the potential use of Neutrophil-lymphocyte ratio (NLR) as a predictor of FS recurrence.

Determination of Multiple Neurotransmitters through LC-MS/MS to Confirm the Therapeutic Effects of Althaea rosea Flower on TTX-Intoxicated Rats.

Tetrodotoxin (TTX) inhibits neurotransmission in animals, and there is no specific antidote. In clinical practice in China, Althaea rosea (A. rosea flower) extract has been used to treat TTX poisoning. In this work, the efficacy of the ethyl acetate fraction extract of A. rosea flower in treating TTX poisoning in rats was investigated. A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine nine neurotransmitters in rat brain tissue, including γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT), noradrenaline (NE), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindole-3-acetic acid (5-HIAA), epinephrine (E), and tyramine (Tyn). The detoxifying effect of A. rosea flower was verified by comparing the changes in neurotransmitters' content in brain tissue before and after poisoning in rats. The assay was performed in multiple reaction monitoring mode. The quantification method was performed by plotting an internal-standard working curve with good linearity (R2 > 0.9941) and sensitivity. Analyte recoveries were 94.04-107.53% (RSD < 4.21%). Results indicated that the levels of 5-HT, DA, E, and NE in the brains of TTX-intoxicated rats decreased, whereas the levels of GABA, Tyn, and 5-HIAA showed an opposite trend, and HVA and DOPAC were not detected. The levels of all seven neurotransmitters returned to normal after the gavage administration of ethyl acetate extract of A. rosea flower to prove that the ethyl acetate extract of A. rosea flower had a therapeutic effect on TTX poisoning. The work provided new ideas for studies on TTX detoxification.

Study on Quality Control of Compound Anoectochilus roxburghii (Wall.) Lindl. by Liquid Chromatography-Tandem Mass Spectrometry.

Compound Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii) oral liquid (CAROL) is a hospital preparation of A. roxburghii and Ganoderma lucidum (G. lucidum), which have hepatoprotective effects. Eight active components (five nucleosides/nucleobases and three triterpenoid acids) in CAROL, A. roxburghii, and G. lucidum were simultaneously detected by high-performance liquid chromatography−tandem mass spectrometry (LC−MS/MS). The multiple reaction monitoring (MRM) mode was applied for the detection of analytes. These eight compounds were separated well within 12 min and quantified using the internal standard working curve method. The method showed good linearity (R2 > 0.9935) and high sensitivity (limit of detection = 0.29 ng/mL). The analyte recovery ranged from 85.07% to 97.50% (relative standard deviation < 3.31%). The content of the target analytes in four batches of CAROL, and the raw materials of G. lucidum and A. roxburghii from the five regions was determined using this method. The contents of guanosine and ganoderic acid A in four batches of oral liquid were high and stabilized and could be recommended as quality markers (Q-marker) for CAROL. Simultaneous qualitative and quantitative analysis of nucleosides and triterpenoid acids in CAROL, A. roxburghii, and G. lucidum by LC−MS/MS based on the MRM model was reported for the first time. The proposed method provides a sensitive, rapid, and reliable approach for the quality control of Chinese medicinal products.

A de novo nonsense mutation of STXBP1 causes early-onset epileptic encephalopathy.

Mutations in syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to multiple neurodevelopmental disorders, including severe early-onset epileptic encephalopathies (EOEEs). A de novo nonsense mutation of STXBP1 (c. 863G > A, p. W288X) was found in a patient diagnosed with EOEE at the age of 17 days. The electroencephalogram (EEG) showed sharp waves and spikes, while brain magnetic resonance imaging was normal. We generated a zebrafish EOEE model by overexpressing mutant STXBP1(W288X) and studied the behavioral changes further to understand the mechanism of W288X mutation in epileptogenesis. In addition, effective antiepileptic drugs were screened in the zebrafish model. Zebrafish STXBP1 homologs were highly conserved and prominently expressed in the larval zebrafish brain. The Tg(hSTXBP1W288X) zebrafish larvae exhibited hyperactivity compared with the wild-type (WT) controls. The expression of STXBP1 decreased during the development course from 1 to 5 days post fertilization. Spontaneous seizures and increased c-fos expression were observed in the mutant zebrafish larvae. The susceptibility of Tg(hSTXBP1W288X) zebrafish to pentylenetetrazol challenge also dramatically increased. Levetiracetam, clonazepam, and topiramate showed antiepileptic effects in the Tg(hSTXBP1W288X) larvae to different extents. Our findings in the newly generated mutant line of zebrafish suggested that zebrafish recapitulated clinical phenotypes associated with human STXBP1 mutation, which provided an appropriate in vivo model for epilepsy research.

Three-phase hollow fiber liquid-phase microextraction combined with HPLC for determination of three trace acidic plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl.

The analysis of plant growth regulators presents a challenge due to their trace quantities and complex matrices. A novel, simple, and effective analytical method for the determination of three trace acidic plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl was developed to address this issue. Three-phase hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography was applied for the enrichment, purification, and determination of three acidic plant growth regulators, namely, indole-3-acetic-acid, indole-3-butyric-acid, and (+)-abscisic acid. The factors affecting extraction performance, including extractant species, pH of donor and acceptor phases, salt addition dosage, extraction time, temperature, and stirring rate, were investigated and optimized. Under optimum conditions, the proposed method provided good linearity (R2 , 0.9994-0.9999), low limit of detection (0.038-0.12 ng/mL), and acceptable relative recoveries (56.7-117.6%). The enrichment factors were between 153 and 328. The developed method was successfully applied to the enrichment and determination of plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl and exhibited increased purification capacity, higher sensitivity, and decreased organic solvent consumption compared with conventional sample preparation methods. This method may provide a testing platform for the monitoring of plant growth regulator residues, ensuring the safe and effective use of traditional Chinese medicine.

Phylloseptin-PBa1, -PBa2, -PBa3: Three novel antimicrobial peptides from the skin secretion of Burmeister's leaf frog (Phyllomedusa burmeisteri).

Skin secretions are known as a highly-complex mixture of abundant and diverse bioactive molecules and its study has attracted increasing attention over recent years. Phylloseptin is a unique family of antimicrobial peptides which have been only isolated from frogs of the Phyllomedusinae subfamily. Here, three novel peptide precursors were successfully cloned from a cDNA library, which was constructed from the skin secretion of Phyllomedusa burmeisteri, as pair of primers (one nested universal primer and a designed degenerate sense primer) were employed for "shotgun" cloning. The encoded mature peptides were validated by MS/MS sequencing, and subsequently termed as Phylloseptin-PBa1, -PBa2 and -PBa3. Phylloseptin-PBa1 and -PBa2 were demonstrated to possess potent antimicrobial activities against Gram-positive bacteria and yeast, as well as broad-spectrum anticancer activities, while they possess varying haemolytic activity at the effective concentration. In contrast, Phylloseptin-PBa3 was found to exhibit a strong haemolytic activity even though it was only found to possess a weak antimicrobial activity and inconspicuous anticancer activity.

Profiling neuron-autonomous lncRNA changes upon ischemia/reperfusion injury.

Extensive changes of neuronal transcriptome occur post ischemic stroke and during the following reperfusion. Although numerous studies focused on transcriptome changes of mRNAs associated with ischemic stroke, little is known about whether and how long non-coding RNAs (lncRNAs), which play critical roles in cellular homeostasis, are involved in this process. In this study, we performed high throughput screening to analyze expression changes of lncRNAs in primarily cultured hippocampal neurons under an oxygen-glucose deprivation/reperfusion (OGD/R) condition at 0 h, 6 h, 12 h, and 18 h, respectively. Knock down of one validated lncRNAs (Tnxa-ps1) promoted neuronal survival by inhibiting apoptosis. Coding non-coding co-expression network analysis revealed that the expression of Tnxa-ps1 was highly correlated with changes of a particular group of genes, many of which are associated with neural protection. Finally, we showed that down-regulation of Tnxa-ps1 reversed the expression changes of four mRNAs post OGD/R, revealing a regulatory effect between Tnxa-ps1 and selected genes. Together, our data revealed possible participation of lncRNAs in the pathophysiology of OGD/R and thereby provided new insights into the studies of potential therapeutic targets for ischemic stroke.

APRIL is Involved in the Proliferation and Metastasis of Acute Lymphoblastic Leukemia Cells.

Our previous work showed that a proliferation-inducing ligand (APRIL) was involved in the development of acute lymphoblastic leukemia (ALL) in children. However, the precise role of APRIL in ALL remains unknown. To investigate this issue, we silenced and overexpressed APRIL in Nalm-6 ALL cells using short hairpin RNA targeting the APRIL gene and recombinant human APRIL, respectively, and evaluated the effects on cell proliferation, apoptosis, and migration. APRIL mRNA and APRIL and matrix metalloproteinase-2 protein levels were evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blott, respectively. We found that APRIL expression was reduced by shRNA-mediated knockdown in Nalm-6 cells; this was associated with a decrease in cell proliferation (P<0.05). APRIL knockdown increased apoptosis (P<0.01) but suppressed cell migration along with matrix metalloproteinase-2 protein level. Overexpressing recombinant human APRIL had the opposite effects in each case (P<0.05). These results demonstrate a link between APRIL expression and ALL development and suggest that APRIL is a potential therapeutic target for ALL treatment.

Six-month efficacy of the Ketogenic diet is predicted after 3 months and is unrelated to clinical variables.

OBJECTIVE: The aim of this study was to evaluate the 6-month efficacy of a Ketogenic diet (KD) in children with drug-resistant epilepsy and to analyze the associated factors that affect the efficacy of a KD. METHODS: Eighty-seven pediatric patients with drug-resistant epilepsy who followed a KD for at least 6 months were included in this study. The efficacy of a KD was assessed based upon the seizure frequency, as recorded by parents and caregivers. The number of cases and the degree of efficacy in different age ranges were also considered. The effects of gender, age, seizure type, etiology, blood glucose and ketone levels, seizure frequency before the diet, and cognition on the length of time on a KD were analyzed. RESULTS: (1) There was no significant correlation between the length of time on a KD and efficacy (χ(2)=2.31, P=0.51). The 3-month efficacy of a KD was 51%, which did not further increase when the course was extended to 6 months. (2) There was a positive correlation between increased cognition and the efficacy of a KD after 3 months (γ=0.31, P=0.003). (3) The efficacy analysis of 3-month treatment with a KD revealed, with respect to seizure types, that there were 37 patients with multiple seizure phenotypes and 50 patients with a single seizure phenotype. The overall efficacy of a KD in the group with multiple seizure phenotypes was 61%. The efficacy of a KD was not statistically associated with a coexisting syndrome or a type of syndrome; however, the efficacy of a KD had a tendency to be increased in certain types of syndromes. The overall efficacy in the group with a single seizure phenotype was 87%, and the efficacy was not associated with seizure type. (4) The 3-month efficacy of a KD was not correlated with age, gender, etiology, blood glucose or ketone levels, or the seizure frequency before treatment. CONCLUSION: An observation time of 3 months is appropriate for assessing the efficacy of a KD in treating children with drug-resistant epilepsy. The factors that likely influence the efficacy of a KD are unclear, but our study suggests that incorporating more patient samples will help determine whether patients with certain syndromes can benefit from a KD.

Pharmacokinetics, Tissue Distribution and Protein Binding Studies of Chrysocauloflavone I in Rats.

Chrysocauloflavone I, an unfrequent biflavonoid, was purified from Selaginella doederleinii in this study. It showed cytotoxic effects on three human cancer cells, NCI-H1975, A549, and HepG-2, in vitro. In silico assessment of the physicochemical properties was performed for predicting the permeability and intestinal absorption of the tested compound. Subsequently, a rapid, sensitive, and specific high-performance liquid chromatography method was developed for determination of the compound in different biological samples to ascertain the pharmacokinetics, tissue distribution, and protein binding profiles of this active ingredient in rats. After intravenous dosing of chrysocauloflavone I at different levels (10 and 20 mg/kg), the elimination half-life was approximately 85 min, and the AUC0-∞ increased with the dose from 148.52 mg/L × min for 10 mg/kg to 399.01 mg/L × min for 20 mg/kg. After single intravenous dosing (20 mg/kg), chrysocauloflavone I was detected in all tissues studied with higher levels in the heart, blood, and lungs. The results of equilibrium dialysis indicated a very high protein binding degree (over 97%) for chrysocauloflavone I. After intragastric administration of 100 mg/kg chrysocauloflavone I to rats, no parent drug was detected in the rat plasma. This is the first report of the favorable bioactivities, plasma pharmacokinetics, tissue distribution, and protein binding profiles of the rare biflavone chrysocauloflavone I.

Raised expression of APRIL in Chinese children with acute lymphoblastic leukemia and its clinical implications.

This study was to determine the expression of a proliferation-inducing ligand (APRIL) and its receptors, B-cell maturation antigen (BCMA) and transmembrane activator and calcium-modulating cyclophilin ligand interactor in childhood acute lymphoblastic leukemia (ALL). The correlation between the plasma APRIL levels and clinical status was also evaluated. Plasma samples from 20 untreated children with ALL, 23 children with ALL in remission, and 15 normal controls were assayed for APRIL plasma concentration by enzyme-linked immunosorbent assay. Real-time quantitative polymerase chain reaction was performed to determine the mRNA expression of APRIL and its receptors in blood mononuclear cells in 20 untreated ALL children and 15 normal controls. The untreated ALL patients had higher plasma APRIL levels than the remission group and the normal controls (P<0.001, respectively). No significant difference was found between the remission group and the normal controls in the plasma APRIL levels (P=0.339). The plasma APRIL levels in the untreated patients correlated with white blood cell count at diagnosis (P=0.002) and risk category (P=0.013). The mRNA expression of both APRIL and BCMA in blood mononuclear cells of the ALL patients were higher than those of the normal controls (both P<0.001). No significant difference was found between the patients and the normal controls in the transmembrane activator and calcium-modulating cyclophilin ligand interactor expression (P>0.05). These findings indicate that APRIL and BCMA are over expressed in untreated ALL children. The levels of APRIL correlate with the progression of childhood ALL, which may provide certain clues for monitoring ALL clinically.

Effects of compound Anoectochilus roxburghii (Wall.) Lindl. oral liquid on relative metabolic enzymes and various biochemical indices in Wistar rats with isoniazid-induced liver injury.

Isoniazid (INH) is the first-line anti-tuberculosis drug in clinical practice, and its main adverse effect is drug-induced liver injury (DILI). This study aimed to investigate the hepatoprotective effect of Compound Anoectochilus roxburghii (Wall.) Lindl. Oral Liquid (CAROL) and to provide a new strategy for the search of potential drugs against INH-induced liver injury in Wistar rats. Animal experiment was based on INH (100 mg/kg) induced liver injury to explore the intervention effects of CAROL at doses of 1.35, 2.70, and 5.40 mL/kg. LC-QTOF-MS/MS was used to identify hepatoprotective components in CAROL and its' exposed components in rat serum. The hepatoprotective effect of CAROL was evaluated by pathological observation of rat liver tissue and changes in levels of biochemical indices and cytokines in serum or liver tissue. Of the 58 hepatoprotective components identified, 15 were detected in the serum of rats with liver-injured treated by high-dose CAROL. Results of animal experiments showed that the levels of various biochemical indexes and cytokines were significantly reversed with CAROL intervention. In particular, the expression level of cytokeratin-18 and high-mobility group box 1, as specific and sensitive indicators of DILI, was significantly reduced in the serum of rats with CAROL intervention compared with the INH model group. The same reversal was observed in the levels of TBIL, ALP, ALT, and AST in serum, as well as in the levels of TNF-α, IL-6, SOD, and MDA in liver tissue. For INH-metabolizing enzymes, an evident expression inhibition was observed in N-acetyltransferase 2 and glutathione S-transferases with CAROL intervention, which may be the key to controlling INH hepatotoxicity. CAROL has a favorable hepatoprotective effect on INH-induced liver injury. This study takes the first step in studying the hepatoprotective mechanism of CAROL against INH hepatotoxicity and provides reference for wider clinical applications.

Corrigendum to "Anti-tumor effect and mechanism of the total biflavonoid extract from S doederleinii on human cervical cancer cells in vitro and in vivo" [Heliyon 10(2) January 2024 e24778].

[This corrects the article DOI: 10.1016/j.heliyon.2024.e24778.].

Anti-tumor effect and mechanism of the total biflavonoid extract from S doederleinii on human cervical cancer cells in vitro and in vivo.

In this study, the therapeutic effect and possible mechanism of the total biflavonoid extract of Selaginella doederleinii Hieron (SDTBE) against cervical cancer were originally investigated in vitro and in vivo. First, the inhibition of SDTBE on proliferation of cervical cancer HeLa cells was evaluated, followed by morphological observation with AO/EB staining, Annexin V/PI assay, and autophagic flux monitoring to evaluate the possible effect of SDTBE on cell apoptosis and autophagy. Cell cycle, as well as mitochondrial membrane potential (ΔÑ°m), was detected with flow cytometry. Further, the apoptosis related protein expression and the autophagy related gene LC3 mRNA transcription level were analyzed by Western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. Finally, the anti-cervical cancer effect of the SDTBE was also validated in vivo in HeLa cells grafts mice. As results, SDTBE inhibited HeLa cells proliferation with the IC50 values of 49.05 ± 6.76 and 44.14 ± 4.75 µg/mL for 48 and 72 h treatment, respectively. The extract caused mitochondrial ΔÑ° loss, induced cell apoptosis by upregulating Bax, downregulating Bcl-2, activating Caspase-9 and Caspase-3, promoting cell autophagy and blocking the cell cycle in G0/G1 phase. Furthermore, 100, 200, and 300 mg/kg SDTBE suppressed the growth of HeLa cells xenografts in mice with the mean inhibition rates, 25.3 %, 57.5 % and 62.9 %, respectively, and the change of apoptosis related proteins and microvascular density was confirmed in xenografts by immunohistochemistry analysis. The results show that SDTBE possesses anti-cervical cancer effect, and the mechanism involves in activating Caspase-dependent mitochondrial apoptosis pathway.

Analysis of EphA5 receptor in the developing rat brain: an in vivo study in congenital hypothyroidism model.

UNLABELLED: The EphA5 receptor has recently been known to play an important role in the initiation of the early phase of synaptogenesis, during which irreparable harm would be done to the developing brain in the absence of sufficient thyroid hormone (TH). In the present article, we aimed to analyze the characteristics of EphA5 receptor expression in the brain of congenital hypothyroid rats. The results showed that the levels of the EphA5 receptor were downregulated by TH deficiency in the developing rat brain with remarkable spatial and temporal characteristics. In the hypothyroid rats, the mRNA and protein levels of EphA5 receptor decreased significantly in the hippocampus (27.92-53.26%), cerebral cortex (12.52-47.16%), and cerebellum (8.72-31.69%) compared with those in the normal rats from postnatal day 0 (P0) to P21 (p < 0.01). The expression of EphA5 receptor was highest and declined most as much as 53% in the hippocampus with TH deficiency. At P7, the EphA5 receptor decreased most prominently during all the observed time point. CONCLUSION: The EphA5 receptor plays actively in the brain development in congenital hypothyroid rats. Our study highlights the high expression of EphA5 receptor protein in hippocampus and dramatic changes at P7 in condition of TH deficiency, which may provide important basis for further investigations in manipulating congenital hypothyroidism.

A novel nanostructured organic framework sensor for selective and sensitive detection of doxycycline based on fluorescence enhancement.

It is critical for human health to develop sensitive and rapid analytical methods for detecting doxycycline (DOX) residues in food. This paper presents a novel metal-organic framework nanomaterial (Zn-MOF) based on dithiodiglycolic acid and its application in DOX detection by fluorescent probe method. Zn-MOF itself does not fluoresce. When DOX is added, the system exhibits strong fluorescence (100-fold) at 530 nm. The fluorescence intensity displayed an excellent linear relationship with DOX concentration with a detection limit of 2.7 nM. The reaction solution's fluorescence displayed a visible color shift from colorless to yellow that was concentration-dependent. A smartphone was used to detect DOX by recognizing the red, green, and blue values of the reaction solution and the corresponding test paper. The use of smartphones can speed up the detection process and streamline operations, offering a sensitive and visible method for the quantitative detection of DOX residues in actual samples. Interestingly, Zn-MOF can discriminate DOX from other tetracyclines with high selectivity. This material has been used successfully as a fluorescent probe to determine DOX in fish samples with an average spiked recovery of 94.6 % ∼ 95.1 %. The DOX levels in the measured perch samples were 1.25 âˆ¼ 157 µg/kg. There are 2 batches of DOX exceeding the standard in 14 batches.

Simultaneous analysis of paralytic shellfish toxins and tetrodotoxins in human serum by liquid chromatography coupled to Q-Exactive high-resolution mass spectrometry.

Paralytic shellfish toxins (PSTs) and tetrodotoxins (TTXs) are powerful neurotoxins. Previous research reported that PSTs and TTXs are found together in seafoods and may pose a serious hazard to public health. In this study, a new analytical method combining modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) with high-performance liquid chromatography coupled to Q-Exactive Orbitrap high-resolution mass spectrometry was developed and validated for the quantification of 10 PSTs and 2 TTXs in human serum. Chromatographic separation was achieved using the HILIC TSK-Gel Amide-80 column. The mass spectrometer was operated in full scan/dd-MS2(data-dependent MS2) mode, and for quantification analysis. The dd-MS2 resolution was set to 17,500 fullwidthat halfmaximum （FWHM）. Results showed that methanol with 1 % (v/v) acetic acid extraction combined with 50 mg graphitized carbon black (GCB) and 50 mg octadecyl bonded silica gel (C18) was most suitable for purification. The mean recovery for all toxins ranged from 85.3 % to 118.2 % (RSD < 12 %). The limits of detection and quantification for human serum were in the ranges of 0.67-2.61 and 2.23-8.69 ng mL-1, respectively. The method was applied to analyze toxins in serum samples obtained from three poisoned patients in a case of poisoning caused by consumption of toxin-contaminated gastropoda (Bullacta exerata). The study has important application for rapid and accurate diagnosis of PSTs and TTXs toxin poisoning patients in clinic.

Uncovering the protective mechanism of Pien-Tze-Huang in rat with alcoholic liver injury based on cytokines analysis and untargeted metabonomics.

Pien-Tze-Huang (PTH) is a well-known traditional Chinese patent medicine with excellent liver-protection effect. However, the mechanism of hepatoprotective action has not yet been entirely elucidated. The aim of this study was to investigate the mechanism of protective effect of PTH on alcohol-induced liver injury in rats using cytokine analysis and untargeted metabolomics approaches. An alcoholic liver disease (ALD) model with SD rats was established, and PTH was administered according to the prescribed dose. The hepatoprotective effect of PTH was evaluated by pathological observation of liver tissue and changes in biochemical index activity and cytokines in serum. Serum samples were analyzed by ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS), and differentially expressed metabolites were screened by multivariate statistical analysis. KEGG combined with metabolic pathway analysis were used to evaluate the underlying metabolic pathways. Results showed liver histopathology injury was attenuated. The levels of IL-6, TNF-α and NF-κB were significantly decreased in rats intervened with PTH groups, suggesting that it may alleviate inflammation via suppressing the inflammatory cytokines signaling pathway. Eighty differentially expressed metabolites were found and identified. Pathway analysis indicated that the hepatoprotective effects of PTH occurred through the regulation of inflammatory cytokines signaling pathway, primary bile acid biosynthesis, vitamin B6 metabolism pathway, cholesterol metabolism, and tyrosine metabolism. PTH showed favorable hepatoprotective effect through multiple pathways. This study has great importance in fully revealing the mechanism of hepatoprotective action and can help improve the clinical application of PTH.

An "ON-OFF" fluorescent sensor based on a novel zinc-based flower-like structured metal-organic framework for sequential detection of deferasirox and tigecycline.

Deferasirox (DEF) is essential for patients with thalassemia requiring long-term transfusion therapy. Tigecycline (TIGE) is a first-line drug for the clinical treatment of complex, severe bacterial infections. The two drugs can be coordinated to treat Pseudomonas aeruginosa infections. Easy and efficient techniques for monitoring these two drugs in biological samples are few. Metal-organic framework (Zn-MOF) prepared from zinc nitrate hexahydrate and dithioglycolic acid has a flower structure. Interestingly, Zn-MOF can cause DEF to aggregate on it and induce DEF luminescence. The principle may be that Zn-MOF limits the vibration and rotation of DEF to avoid its nonradiative jump, which triggers aggregation-induced emission (AIE) and exhibits intense fluorescence. Further investigation revealed that TIGE could decompose Zn-MOF, thus alleviating the inhibitory effect of Zn-MOF on DEF and reducing the fluorescence intensity of DEF@Zn-MOF. A DEF/TIGE detection biosensor was created based on the fluorescence "turn-on" effect of Zn-MOF on DEF and the fluorescence "turn-off" effect of TIGE on DEF@Zn-MOF. The proposed technique was subsequently used to identify DEF/TIGE levels in pharmaceuticals and human plasma. The mean values for the percentage of the labeled amount of DEF/TIGE in DEF dispersible tablets/TIGE injection were 104.5 and 104.9%, respectively. The detection limits for the fluorescence detection of DEF and TIGE were 3.6 and 1.2 nM, respectively. This fluorescence assay is the first application of MOF to the simultaneous detection of DEF and TIGE and has the advantages of rapid sensitivity and high selectivity, providing a new strategy for drug detection.