Synaptotagmin-11 Inhibits Synaptic Vesicle Endocytosis via Endophilin A1.

Synaptic vesicle (SV) endocytosis is a critical and well-regulated process for the maintenance of neurotransmission. We previously reported that synaptotagmin-11 (Syt11), an essential non-Ca2+-binding Syt associated with brain diseases, inhibits neuronal endocytosis (Wang et al., 2016). Here, we found that Syt11 deficiency caused accelerated SV endocytosis and vesicle recycling under sustained stimulation and led to the abnormal membrane partition of synaptic proteins in mouse hippocampal boutons of either sex. Furthermore, our study revealed that Syt11 has direct but Ca2+-independent binding with endophilin A1 (EndoA1), a membrane curvature sensor and endocytic protein recruiter, with high affinity. EndoA1-knockdown significantly reversed Syt11-KO phenotype, identifying EndoA1 as a main inhibitory target of Syt11 during SV endocytosis. The N-terminus of EndoA1 and the C2B domain of Syt11 were responsible for this interaction. A peptide (amino acids 314-336) derived from the Syt11 C2B efficiently blocked Syt11-EndoA1 binding both in vitro and in vivo Application of this peptide inhibited SV endocytosis in WT hippocampal neurons but not in EndoA1-knockdown neurons. Moreover, intracellular application of this peptide in mouse calyx of Held terminals of either sex effectively hampered both fast and slow SV endocytosis at physiological temperature. We thus propose that Syt11 ensures the precision of protein retrieval during SV endocytosis by inhibiting EndoA1 function at neuronal terminals.SIGNIFICANCE STATEMENT Endocytosis is a key stage of synaptic vesicle (SV) recycling. SV endocytosis retrieves vesicular membrane and protein components precisely to support sustained neurotransmission. However, the molecular mechanisms underlying the regulation of SV endocytosis remain elusive. Here, we reported that Syt11-KO accelerated SV endocytosis and impaired membrane partition of synaptic proteins. EndoA1 was identified as a main inhibitory target of Syt11 during SV endocytosis. Our study reveals a novel inhibitory mechanism of SV endocytosis in preventing hyperactivation of endocytosis, potentially safeguarding the recycling of synaptic proteins during sustained neurotransmission.

Sirtuin-1 attenuates cadmium-induced renal cell senescence through p53 deacetylation.

Cadmium (Cd), the common environmental pollutant, primarily targets at renal proximal tubules and induces nephrotoxicity. Cellular senescence, a phenomenon of cell growth arrest and a characteristics of maladaptive cell self-repair, is associated with renal disease progression. However, whether and how Cd induces renal tubular cells premature senescence is unknown. In our study, we found that Cd induced kidney damage and dysfunctions, which correlated with exacerbated tubular cell senescence, evidenced by increased senescence-associated ß-galactosidase activity, the upregulated protein expression of p53 and p21Waf1/Cip1 proteins, and elevated expression and secretion of cytokines in human proximal tubular epithelial HK-2 cells in vitro and in Cd-treated mice in vivo. Moreover, a S-phase arrest and decrease in Edu positive rate were found in Cd-treated HK-2 cells. Mechanistically, Cd suppressed the expression and activity of Sirtuin-1 (SIRT1), an anti-senescence deacetylase, resulting in the accumulation of acetylated p53 and upregulation of p21Waf1/Cip1. Activation of SIRT1 significantly abolished Cd-induced premature senescence and S-phase arrest. Finally, silencing p21Waf1/Cip1 efficiently delayed premature senescence and recovered cell cycle progression. These findings indicate that Cd promotes tubular cells senescence and impairs tubular cells regeneration, resulting in kidney dysfunctions, which could be ameliorated by SIRT1 activation.

Identification of differential metabolic characteristics between tumor and normal tissue from colorectal cancer patients by gas chromatography-mass spectrometry.

Colorectal cancer (CRC) is one of the most common human malignancies and encompasses cancers of the colon and rectum. Although the gold-standard colonoscopy screening method is effective in detecting CRC, this method is invasive and can result in severe complications for patients. The purpose of this study was to determine differences in metabolites between CRC and matched adjacent nontumor tissues from CRC patients, to identify potential biomarkers that may be informative and developed screening methods. Metabolomic analysis was performed on clinically localized CRC tissue and matched adjacent nontumor tissue from 20 CRC patients. Unsupervised analysis, supervised analysis, univariate analysis and pathway analysis were used to identify potential metabolic biomarkers of CRC. The levels of 25 metabolites in CRC tissues were significantly altered compared with the matched adjacent nontumor tissues. Four metabolites (lactic acid, alanine, phosphate and aspartic acid) demonstrated good area under the curve of receiver-operator characteristic with acceptable sensitivities and specificities, indicating their potential as important biomarkers for CRC. Alterations of amino acid metabolism and enhanced glycolysis may be major factors in the development and progression of CRC. Lactic acid, alanine, phosphate, and aspartic acid could be effective diagnostic indicators for CRC.

Efficacy and predictive factors of transarterial chemoembolization combined with lenvatinib plus programmed cell death protein-1 inhibition for unresectable hepatocellular carcinoma.

BACKGROUND: The efficacy and safety of transarterial chemoembolization (TACE) combined with lenvatinib plus programmed cell death protein-1 (PD-1) for unresectable hepatocellular carcinoma (HCC) have rarely been evaluated and it is unknown which factors are related to efficacy. AIM: To evaluate the efficacy and independent predictive factors of TACE combined with lenvatinib plus PD-1 inhibitors for unresectable HCC. METHODS: This study retrospectively enrolled patients with unresectable HCC who received TACE/lenvatinib/PD-1 treatment between March 2019 and April 2022. Overall survival (OS) and progression-free survival (PFS) were determined. The objective response rate (ORR) and disease control rate (DCR) were evaluated in accordance with the modified Response Evaluation Criteria in Solid Tumors. Additionally, the prognostic factors affecting the clinical outcome were assessed. RESULTS: One hundred and two patients were enrolled with a median follow-up duration of 12.63 months. The median OS was 26.43 months (95%CI: 17.00-35.87), and the median PFS was 10.07 months (95%CI: 8.50-11.65). The ORR and DCR were 61.76% and 81.37%, respectively. The patients with Barcelona Clinic Liver Cancer Classification (BCLC) B stage, early neutrophil-to-lymphocyte ratio (NLR) response (decrease), or early alpha-fetoprotein (AFP) response (decrease > 20%) had superior OS and PFS than their counterparts. CONCLUSION: This study showed that TACE/lenvatinib/PD-1 treatment was well tolerated with encouraging efficacy in patients with unresectable HCC. The patients with BCLC B-stage disease with early NLR response (decrease) and early AFP response (decrease > 20%) may achieve better clinical outcomes with this triple therapy.

MXRA7 is involved in monocyte-to-macrophage differentiation.

Macrophages are critical in mediating immune and inflammatory responses, while monocyte-to-macrophage differentiation is one of the main macrophage resources that involves various matrix proteins. Matrix remodeling associated 7 (MXRA7) was recently discovered to affect a variety of physiological and pathological processes related to matrix biology. In the present study, we investigated the role of MXRA7 in monocyte-to-macrophage differentiation in vitro. We found that knockdown of MXRA7 inhibited the proliferation of THP-1 human monocytic cells. Knockdown of MXRA7 increased the adhesion ability of THP-1 cells through upregulation the expression of adhesion molecules VCAM-1 and ICAM1. Knockdown of MXRA7 alone could promoted the differentiation of THP-1 cells to macrophages. Furthermore, the MXRA7-knockdown THP-1 cells produced a more significant upregulation pattern with M1-type cytokines (TNF-α, IL-1ß and IL-6) than with those M2-type molecules (TGF-ß1 and IL-1RA) upon PMA stimulation, indicating that knockdown of MXRA7 facilitated THP-1 cells differentiation toward M1 macrophages. RNA sequencing analysis revealed the potential biological roles of MXRA7 in cell adhesion, macrophage and monocyte differentiation. Moreover, MXRA7 knockdown promoted the expression of NF-κB p52/p100, while PMA stimulation could increase the expression of NF-κB p52/p100 and activating MAPK signaling pathways in MXRA7 knockdown cells. In conclusion, MXRA7 affected the differentiation of THP-1 cells toward macrophages possibly through NF-κB signaling pathways.

Extraction, derivatization, and determination of metabolome in human macrophages.

A GC/TOF-MS was applied to the determination of metabolites in human macrophages. The extraction conditions and quenching conditions were investigated and optimized. The results indicated that 0.9% w/v sodium chloride at 4°C was the most favorable condition to quench macrophage, 1 mL 50% ACN for 2 min in ice bath was the optimal condition to extract 5 × 10(6) cells. Two hundred six peaks could be detectable with peak area over 50 using this method. Among these peaks, 45 peaks with the similarity over 700 were identified using standard compounds for endogenous metabolites. Thirty-seven out of 45 metabolites could be quantified directly by this method. Twenty metabolites were selected randomly, and 15 amino acids were used for method validation. The correlation coefficients (r) ranging from 0.9902 to 0.9977 were obtained for 15 amino acids in the range of 2.35-150.20 µg/mL. The intraday and interday precisions were lower than 19.90% for the randomly selected 20 endogenous metabolites. Using this development method and multivariate statistical technique, several potential biomarkers were found from human macrophages infected by different Mycobacterium tuberculosis (M. tuberculosis) strains. The results suggest that the method could be applied to the investigation of the pathogenicity of tuberculosis.

Optimization of Douchi fibrinolytic enzyme production by statistical experimental methods.

Thrombus disease, one of the common cardiovascular diseases, has attracted worldwide attention for its rising mortality and morbidity. Due to the distinct shortages of current fibrinolytic drugs, new fibrinolytic agents warrant investigation. In this study, 8 fibrinolytic enzyme-producing strains were isolated from Douchi-a traditional Chinese food, and strain XY-1 which produced the largest amount of the enzyme was chosen for the following experiments. The enzyme produced by strain XY-1 was named Douchi fibrinolytic enzyme (DFE). We optimized the liquid culture medium of strain XY-1 for enzyme production using Plackett-Burman and Box-Behnken design. The predicted maximal DFE yield was 19.78 FU/mL with 11.4 g/L peptone, 0.5 g/L magnesium sulfate and 1 g/L sodium chloride. However, we acquired maximal production of 21.33 FU/mL in actual experiments, equal to 107.84% of the theoretical value, and the yield had been increased by 79.55% as compared to the yield of un-optimized culture. It was demonstrated that the combined use of Plackett-Burman design and response surface methodology in fermentation optimization can effectively and rapidly increase DFE production.

TSRL-Net: Target-aware supervision residual learning for stroke segmentation.

Accurate stroke segmentation is a crucial task in establishing a computer-aided diagnostic system for brain diseases. However, reducing false negatives and accurately segmenting strokes in MRI images is often challenging because of the class imbalance and intraclass ambiguities problems. To address these issues, we propose a novel target-aware supervision residual learning framework for stroke segmentation. Considering the problem of imbalance of positive and negative samples, a creatively target-aware loss function is designed to dilate strong attention regions, pay high attention to the positive sample losses, and compensate for the loss of negative samples around the target. Then, a coarse-grained residual learning module is developed to gradually fix the lost residual features during the decoding phase to alleviate the problem of high number of false negatives caused by intraclass ambiguities. Here, our reverse/positive attention unit suppresses redundant target/background noise and allows relatively more focused highlighting of important features in the target residual region. Extensive experiments were performed on the Anatomical Tracings of Lesions After Stroke and Ischemic Stroke Lesion Segmentation public datasets, with results suggesting the effectiveness of our proposed method compared to several state-of-the-art methods.

[Effect of MXRA7 on the Biological Functions of Acute B Lymphoblastic Leukemia Cell Line REH].

OBJECTIVE: To discover the relationship between matrix remodeling associated 7 (MXRA7) and acute B lymphoblastic leukemia (B-ALL), and explore the effect of MXRA7 on the biological functions of B-ALL cell line REH. METHODS: The expression of MXRA7 in blood diseases was searched and analyzed through BloodSpot database. Real-time qPCR was used to detect the expression level of MXRA7 in B-ALL cell line 697 and REH cells. Lentivirus-mediated shRNA interference technology was utilized to knock down the expression of MXRA7 in REH cells. The effects of MXRA7 on the biological functions of REH cells were studied by in vitro experiments. Cell proliferation was detected by CCK-8 assay, cell cycle was detected by PI staining, cell apoptosis was detected by Annexin V and 7-AAD staining, and the expression of apoptosis pathway related proteins was detected by Western blot. RESULTS: Database analysis showed that MXRA7 was highly expressed in B-ALL patients, and real-time qPCR results showed that MXRA7 was also highly expressed in cell lines 697 and REH cells. Knockdown of MXRA7 in REH cells inhibited the cell proliferation and increased the percentage of G0/G1 phase cells. After treatment with cytarabine, the apoptotic ratio was increased in MXRA7-impaired REH cells, and the activation of caspase-3 and caspase-9 were also increased. CONCLUSION: Knockdown of MXRA7 can reduce the malignancy of REH cells by inhibiting the cell proliferation and increasing the sensitivity of REH cells to cytarabine. These results indicate MXRA7 may be as a novel target for the treatment of B-ALL, and the potential usefulness of MXRA7 in B-ALL deserves further investigation.

MXRA7 is involved in megakaryocyte differentiation and platelet production.

Matrix remodeling is a critical process in hematopoiesis. The biology of MXRA7, as a matrix remodeling associated gene, has still not been reported in hematopoietic process. Public databases showed that MXRA7 expressed in hematopoietic stem cells, suggesting that it may be involved in hematopoiesis. We found that the amounts of megakaryocytes were lower in bone marrow and spleen from Mxra7-/- mice compared with that from wild-type mice. Knock-out of MXRA7 also reduced the amount of platelet in peripheral blood and affected the function of platelets. Knock-out of MXRA7 inhibited hematopoietic stem/progenitor cells differentiate to megakaryocytes possibly through down-regulating the expression of GATA-1 and FOG-1. Moreover, knockdown of MXRA7 in MEG-01 cells could inhibit the cell proliferation and cell apoptosis. Knockdown of MXRA7 inhibited the differentiation of MEG-01 cells and proplatelet formation through suppressing the ERK/MAPK signaling pathway and the expression of ß-tubulin. In conclusion, the current study demonstrated the potential significance of MXRA7 in megakaryocyte differentiation and platelet production. The novel findings proposed a new target for the treatment of platelet-related diseases, and much more investigations are guaranteed to dissect the mechanisms of MXRA7 in megakaryocyte differentiation and platelet production.

N-methyl-d-aspartate receptor 1 activation mediates cadmium-induced epithelial-mesenchymal transition in proximal tubular cells.

Cadmium (Cd) is a commonly found environmental pollutant and is known to damage multiple organs with kidneys being the most common one. N-methyl-d-aspartate receptor 1 (NMDAR1) is a ligand-gated ion channel that is highly permeable to calcium ion (Ca2+). Because Cd2+ and Ca2+ have structural and physicochemical similarities, whether and how Cd could interfere NMDAR1 function to cause renal epithelial cells dysfunction remains unknown. In this study, we investigated the role of NMDAR1 in Cd-induced renal damage and found that Cd treatment upregulated NMDAR1 expression and promoted epithelial-mesenchymal transition (EMT) in mouse kidneys in vivo and human proximal tubular epithelial HK-2 cells in vitro, which were accompanied with activation of the inositol-requiring enzyme 1 (IRE-1α) / spliced X box binding protein-1 (XBP-1s) pathway, an indicative of endoplasmic reticulum (ER) stress. Mechanistically, NMDAR1 upregulation by Cd promoted Ca2+ channel opening and Ca2+ influx, resulting in ER stress and subsequently EMT in HK-2 cells. Inhibition of NMDAR1 by pharmacological antagonist MK-801 significantly attenuated Cd-induced Ca2+ influx, ER stress, and EMT. Pretreatment with the IRE-1α/XBP-1s pathway inhibitor STF-083010 also restored the epithelial phenotype of Cd-treated HK-2 cells. Therefore, our findings suggest that NMDAR1 activation mediates Cd-induced EMT in proximal epithelial cells likely through the IRE-1α/XBP-1s pathway, supporting the idea that NMDAR1 could be a potential therapeutic target for Cd-induced renal damage.

Critical role of MXRA7 in differentiation blockade in human acute promyelocytic leukemia cells.

The biology of the matrix remodeling-associated 7 (MXRA7) gene has been ill defined. Bioinformatic analysis of public data sets revealed that MXRA7 messenger RNA (mRNA) was highly expressed in acute myeloid leukemia (AML), especially acute promyelocytic leukemia (APL). High expression of MXRA7 was associated with poor overall survival of patients with AML. We confirmed that MXRA7 expression was upregulated in patients with APL and cell lines. Knockdown or overexpression of MXRA7 did not affect the proliferation of NB4 cells directly. Knockdown of MXRA7 in NB4 cells promoted drug-induced cell apoptosis, whereas overexpression of MXRA7 had no obvious influence on drug-induced cell apoptosis. Lowering MXRA7 protein levels in NB4 cells promoted all-trans retinoic acid (ATRA)-induced cell differentiation possibly through decreasing the PML-RARα level and increasing PML and RARα levels. Correspondingly, overexpression of MXRA7 showed consistent results. We also demonstrated that MXRA7 altered the expression of genes involved in leukemic cell differentiation and growth. Knockdown of MXRA7 upregulated the expression levels of C/EBPB, C/EBPD, and UBE2L6, and downregulated the expression levels of KDM5A, CCND2, and SPARC. Moreover, knockdown of MXRA7 inhibited the malignancy of NB4 cells in a non-obese diabetic-severe combined immune-deficient mice model. In conclusion, this study demonstrated that MXRA7 influences the pathogenesis of APL via regulation of cell differentiation. The novel findings about the role of MXRA7 in leukemia not only shed light on the biology of this gene but also proposed this gene as a new target for APL treatment.

AGMR-Net: Attention-guided multiscale recovery framework for stroke segmentation.

Automatic and accurate lesion segmentation is critical to the clinical estimation of the lesion status of stroke diseases and appropriate diagnostic systems. Although existing methods have achieved remarkable results, their further adoption is hindered by: (1) intraclass inconsistency, i.e., large variability between different areas of the lesion; and (2) interclass indistinction, in which normal brain tissue resembles the lesion in appearance. To meet these challenges in stroke segmentation, we propose a novel method, namely attention-guided multiscale recovery framework (AGMR-Net) in this paper. Firstly, a coarse-grained patch attention (CPA) module in the encoding is adopted to obtain a patch-based coarse-grained attention map in a multistage, explicitly supervised way, enabling target spatial context saliency representation with a patch-based weighting technique that eliminates the effect of intraclass inconsistency. Secondly, to obtain more detailed boundary partitioning to meet the challenge of interclass indistinction, a newly designed cross-dimensional feature fusion (CFF) module is used to capture global contextual information to further guide the selective aggregation of 2D and 3D features, which can compensate for the lack of boundary learning capability of 2D convolution. Lastly, in the decoding stage, an innovative designed multiscale deconvolution upsampling (MDU) is used for enhanced recovery of target spatial and boundary information. AGMR-Net is evaluated on the open-source dataset Anatomical Tracings of Lesions After Stroke, achieving the highest Dice similarity coefficient of 0.594, Hausdorff distance of 27.005 mm, and average symmetry surface distance of 7.137 mm, which demonstrates that our proposed method outperforms state-of-the-art methods and has great potential for stroke diagnosis.

[The Effect of Matrix Remodeling Associated 7 (MXRA7) Expression on the Biological Function of SHI-1 Cells].

OBJECTIVE: To express matrix remodeling associated 7 (MXRA7) in the human acute myeloid leukemia SHI-1 cell line and to assess the role of MXRA7 in the biological function of SHI-1 cells. METHODS: The full-length cDNA sequence of human MXRA7 was synthesized and subcloned into the lentivirus shuttle vector pRRL-Venus. SHI-1 cells were transfected with the lentivirus which was packaged with 293T cells. The YFP-positive cells were sorted by flow cytometry and the stable cell lines were obtained by expanded culture. The expression and distribution of MXRA7 in SHI-1 cells were verified by real-time qPCR, Western blot and laser confocal techniques. Cell proliferation and cell cycle were measured by flow cytometry, and apoptosis was determined by Annexin V and 7-AAD staining. The expression of apoptosis related proteins were detected by Western blot. RESULTS: The stable SHI-1 cell line overexpressing MXRA7 was established successfully. Laser confocal analysis confirmed that MXRA7 was expressed in the cytoplasm of SHI-1 cells. Compared with the control cell line, the overexpression of MXRA7 showed no effect on the cell proliferation and cell cycle, but reduced the percentage of apoptosis cells induced by methotrexate. Moreover, the expression of BCL-2 protein was increased by overexpression of MXRA7, which can inhibit cell apoptosis. CONCLUSION: The SHI-1 stable cell line overexpressing MXRA7 was established successfully, and MXRA7 could inhibit drug-induced apoptosis through increasing the expression of BCL-2 protein.

IL-39 promotes chronic graft-versus-host disease by increasing T and B Cell pathogenicity.

BACKGROUND: Chronic graft-versus-host disease (cGVHD) remains a major complication during the late phase of allogeneic hematopoietic stem cell transplantation (allo-HSCT). IL-39, a newly described pro-inflammatory cytokine belonging to the IL-12 family, plays a role in lupus development. Recently, IL-39 has been identified as a pathogenic factor in acute GVHD (aGVHD). However, the role of IL-39 in the pathogenesis of cGVHD remains unclear. METHODS: We constructed a recombinant IL-39 plasmid and established scleroderma and lupus-like cGVHD models. Quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to detect IL-39 expression in mice and patients post transplantation, respectively. Hydrodynamic gene transfer (HGT) was performed to achieve IL-39 overexpression in vivo. Multiparameter flow cytometry, western blotting, and assays in vitro were performed to investigate the effect of IL-39 on cGVHD. RESULTS: The relative expression of IL-23p19 and EBi3 was significantly increased in the intestine of cGVHD mice on day 40 post allo-HSCT, and IL-39 levels were significantly elevated in the serum of patients following allo-HSCT. Overexpression of IL-39 significantly aggravated the severity of cGVHD. Increased IL-39 levels promoted T-cell activation and germinal center responses, and may exacerbate thymic damage. Consistently, blocking IL-39 markedly ameliorated immune dysregulation in the cGVHD mice. Furthermore, we found that IL-39 was produced by B cells, CD11b+ cells, and CD8+T cells after activation. Stimulation of IL-39 led to upregulation of the IL-39 receptor on CD4+T cells and further caused activation of the STAT1/STAT3 pathway, through which IL-39 may exert its pro-inflammatory effects. CONCLUSION: Our study reveals a critical role for IL-39 in cGVHD pathogenesis and indicates that IL-39 may serve as a potential therapeutic target for cGVHD prevention.

Dual role of inositol-requiring enzyme 1α (IRE-1α) in Cd-induced apoptosis in human renal tubular epithelial cells: Endoplasmic reticulum stress and STAT3 signaling activation.

Cadmium (Cd) is a nephrotoxicant that primarily damages renal proximal tubular cells. Endoplasmic reticulum (ER) stress is mechanistically linked to Cd-induced renal injury. Inositol-requiring enzyme 1 (IRE-1α) is the most conserved ER stress transducer protein, which has both kinase and endonuclease activities. This study aimed to investigate whether the two enzymatic activities of IRE-1α have different effects in its regulation of Cd-induced apoptosis. Human proximal tubular (HK-2) cells were treated with 20 µM CdCl2 for 0-24 h, and mice were fed with Cd-containing drinking water (100-400 mg/L) for 24 weeks. We found that Cd increased cell apoptosis in HK-2 cells and mouse kidneys in a time-dependent manner. Such cytotoxicity was correlated with activation of ER stress, evidenced by upregulation of IRE-1α and its target protein spliced X-box binding protein-1 (XBP-1 s). Interestingly, inhibition of IRE-1α kinase activity by KIRA6 was more protective against Cd-induced apoptosis than inhibition of its RNase activity by STF-083010. Mechanistically, Cd promoted the binding of IRE-1α with signal transducer and activator of transcription-3 (STAT3) leading to elevated phosphorylation of STAT3 at Ser727 and thus inactivation of STAT3 signaling, which resulted in aggravation of Cd-induced apoptosis in HK-2 cells. Collectively, our findings indicate that IRE-1α coordinate ER stress and STAT3 signaling in mediating Cd-induced renal toxicity, suggesting that targeting IRE-1α might be a potential therapeutic approach for Cd-induced renal dysfunction and disease.

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography-electrospray tandem mass spectrometry.

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra(®) MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r(2) = 0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.

Expression of Serum PTTG1 in Laryngeal Carcinoma and Its Correlation to Prognosis.

OBJECTIVES: The purpose of this study was to investigate serum pituitary tumor transforming gene (PTTG1) expression in laryngeal carcinoma and its relationship with the clinical pathological characteristics and prognosis. METHODS: Expression of serum PTTG1 was measured by enzyme-linked immunosorbent assay in 110 patients with laryngeal carcinoma and 60 patients with vocal cord polyps. Expression of the serum PTTG1 levels relationship with the clinicopathological characteristics and prognosis were analyzed. RESULTS: In laryngeal carcinoma patients' serum, the PTTG1 median concentration was 141.43 pg/mL (interquartile range [IQR], 111.387 to 160.837 pg/mL), significantly higher than that of the vocal cord polyp group of 94.01 pg/mL (IQR, 81.26 to 108.59 pg/mL), and the difference was statistically significant (z=-6.715, P<0.001). PTTG1 expression with lymph node metastasis, clinical stage, and patients with laryngeal carcinoma was significantly correlated with the tumor differentiation degree (P<0.05). The total survival rate of the PTTG1 high expression group was significantly lower than the low expression group, and the difference of total survival time of the two groups was statistically significant (P<0.001). CONCLUSION: The PTTG1 expression level can be used as an index for evaluating prognosis of laryngeal cancer. High PTTG1 expression is one of the factors of poor prognosis of laryngeal carcinoma patients.

Synapse and Active Zone Assembly in the Absence of Presynaptic Ca2+ Channels and Ca2+ Entry.

Presynaptic CaV2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold CaV2s to presynaptic release sites, and synapse structure is CaV2 independent. Here, we ablated all three CaV2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although CaV2 knockout impaired the localization of ß subunits, α2δ-1 localized normally. Rescue with CaV2 restored exocytosis, and CaV2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of CaV2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor CaV2s via CaV2 C-termini.

Effect Of Chinese Herb Danzhi Xiaoyao Pills On Pharmacokinetics Of Venlafaxine In Beagles.

OBJECTIVE: To investigate the effects of Chinese herb Danzhi Xiaoyao pills on the pharmacokinetics of venlafaxine and its metabolites O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) in beagles by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: Six beagles (half male, half female) were chosen to test, being fasted before the experiment but having free access to drinking water 1 day before being fed drugs. After oral administration of venlafaxine hydrochloride tablets (10.28 mg/kg), the blood samples were collected in succession at different points in time. After 1-week washout period, Danzhi Xiaoyao pills (0.6g/kg) were given through oral administration to the six beagles every morning until the 7th day, venlafaxine hydrochloride tablets (10.28 mg/kg) were given after feeding Danzhi Xiaoyao pills (0.6g/kg) half an hour and blood samples were collected continuously at different points. All samples were analyzed by UPLC-MS/MS, and the main pharmacokinetic parameters of venlafaxine, ODV and NDV were computed by DAS 2.0. RESULTS: The Cmax of the venlafaxine group (control group) and the combination group (experimental group) were (2267.26±252.89) ng/mL and (1542.64±190.73) ng/mL, respectively. The AUC(0-∞) of the two groups were (13,934.79±3609.23) ng·h/mL and (8001.91±2167.58) ng·h/mL, respectively. The ODV Cmax of the two groups were (2253.80±215.81) ng/mL and (2721.37±118.20) ng/mL, and AUC(0-∞) were (13,974.99±2784.04) ng·h/mL and (17,539.44±1894.29) ng·h/mL, respectively. The NDV Cmax of the two groups were (50.98±5.76) ng/mL and (58.74±12.33) ng/mL, and AUC(0-∞) were (179.26±34.94) ng·h/mL and (220.68±51.41) ng·h/mL, respectively. After administration of Danzhi Xiaoyao pills, the Cmax and AUC(0-∞) of venlafaxine decreased significantly, indicating that the plasma exposure of venlafaxine decreased. The increase of Cmax and AUC(0-∞) of ODV and NDV indicated a rise in plasma exposure. CONCLUSION: Danzhi Xiaoyao pills can accelerate the metabolism of venlafaxine in beagles. In clinical, when venlafaxine was co-administrated with Danzhi Xiaoyao pills, dose adjustment of venlafaxine should be taken into account.