Rapid screening of 28 alkaloids in food poisoning samples by liquid chromatography-tandem mass spectrometry / 中国热带医学

@#Abstract: Objective　To investigate a poisoning incident caused by eating eight treasure congee, and establish liquid chromatography (LC)-mass spectrometry (MS)/MS screening method of 28 alkaloids to provide references for disposal of similar poisoning incidents. Methods　LC-MS/MS was used for screening 28 alkaloids in the urine, eight treasure congee and food raw material, and the detected alkaloids were quantified. Samples were extracted with 0.4% formic acid aqueous solution and separated by a Acquity UPLC BEH C18 column (1.7 μm, 100 × 2.1 mm). Acetonitrile-0.2% formic acid aqueous solution was used as the mobile phase and gradient elution was adopted. The ionization mode was electrospray positive ionization mode, and the detection method was multi-reaction monitoring (MRM). Analytes were quantified with the external standard method. Results　In the concentration range of 0-100 ng/mL, the linear correlation coefficient r were greater than 0.999 for 28 alkaloids. The recovery of 28 alkaloids in urine sample ranged from 63.0% to 105.0%, and the relative standard deviations (RSDs) were between 5.8% and 8.6%. The recovery of 28 alkaloids in eight treasure congee sample ranged from 72.0% to 109.0%, and the RSDs were between 6.3% and 9.7%. The recovery of 28 alkaloids in semen sesami nigrum sample ranged from 60.0% to 95.0%, and the RSDs were between 4.8% and 8.2%. Hyoscyamine (2 380.0 ng/mL), scopliamine (3.6 ng/mL) and rac-anisodamine (4.7 ng/mL) were detected in the patient's urine. Hyoscyamine (63.3 μg/g), scopliamine (5.7 μg/g) and rac-anisodamine (2.1 μg/g) were detected in eight treasure congee. Hyoscyamine (901.0 μg/g), scopliamine (80.0 μg/g) and rac-anisodamine (30.1 μg/g) were detected in the seed of Datura stramonium L. The ratio of scopliamine and hyoscyamine in the seed of D. stramonium was 1∶11, which complies with the characteristics of D. stramonium L. In urine sample, the proportion of scopliamine and rac-anisodamine was 0.15% and 0.20%, and hyoscyamine accounted for 99.65%. Conclusion　Seed morphology, the content range and proportion of three alkaloids are all in accord with the characteristics of D. stramonium. Combined with the clinical symptoms of atropine poisoning, it can be deduced that this incident is a family food poisoning caused by accidental consumption of seed of D. stramonium L. The method can provide technical support for the clinical diagnosis and treatment of alkaloid poisoning patients, and also provide a basis for emergency detection and disposal of alkaloid poisoning events.

Electroacupuncture inhibits dendritic spine remodeling through the srGAP3-Rac1 signaling pathway in rats with SNL

Previous studies have shown that peripheral nerve injury can lead to abnormal dendritic spine remodeling in spinal dorsal horn neurons. Inhibition of abnormal dendritic spine remodeling can relieve neuropathic pain. Electroacupuncture (EA) has a beneficial effect on the treatment of neuropathic pain, but the specific mechanism remains unclear. Evidence has shown that slit-robo GTPase activating protein 3 (srGAP3) and Rho GTPase (Rac1) play very important roles in dendritic spine remodeling. Here, we used srGAP3 siRNA and Rac1 activator CN04 to confirm the relationship between SrGAP3 and Rac1 and their roles in improving neuropathic pain with EA. Spinal nerve ligation (SNL) was used as the experimental model, and thermal withdrawal latency (TWL), mechanical withdrawal threshold (MWT), Western blotting, immunohistochemistry and Golgi-Cox staining were used to examine changes in behavioral performance, protein expression and dendritic spines. More dendritic spines and higher expression levels of srGAP3 were found in the initial phase of neuropathic pain. During the maintenance phase, dendritic spines were more mature, which was consistent with lower expression levels of srGAP3 and higher expression levels of Rac1-GTP. EA during the maintenance phase reduced the density and maturity of dendritic spines of rats with SNL, increased the levels of srGAP3 and reduced the levels of Rac1-GTP, while srGAP3 siRNA and CN04 reversed the therapeutic effects of EA. These results suggest that dendritic spines have different manifestations in different stages of neuropathic pain and that EA may inhibit the abnormal dendritic spine remodeling by regulating the srGAP3/Rac1 signaling pathway to alleviate neuropathic pain.

Piperine suppresses growth and migration of human breast cancer cells through attenuation of Rac1 expression

Objective: To investigate the effect of piperine on human breast cancer cells. Methods: The effect of piperine on proliferation and migration of human breast cancer cells, MCF-7 and MDA-MB-231, was investigated using colony formation assays, wound healing assays, Matrigel migration assays, flow cytometry, RT-qPCR, and Western blotting assays. Results: Piperine inhibited the growth of MCF-7 and MDA-MB-231 cells and suppressed colony formation. Cell reduction at the G 0 / G 1 phase and cell arrest at the G 2 /M phase were observed in breast cancer cells. However, the significant effect was only demonstrated in MDA-MB-231 cells. Moreover, cancer cell migration was suppressed by piperine at low concentration. RT-qPCR and Western blotting assays showed that piperine downregulated Rac1 gene and protein expression. Conclusions: Piperine could inhibit growth and migration of breast cancer cells by reducing Rac1 gene and protein expression.

Relationship between long-term learning and memory impairment induced by sevoflurane anesthesia and PSD-95/Kalirin-7/Rac1 signaling pathway in neonatal rats / 中华麻醉学杂志

Objective:To evaluate the relationship between long-term learning and memory impairment induced by sevoflurane anesthesia and postsynaptic density protein-95 (PSD-95)/Kalirin-7/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling pathway in neonatal rats.Methods:Sixty SPF male Wistar rats, aged 7 days, weighing 12-18 g, were divided into 5 groups ( n=12 each) using a random number table method: control group (group C), 1% sevoflurane anesthesia for 2 h group (group S 1), 1% sevoflurane anesthesia for 4 h group (group S 2), 2% sevoflurane anesthesia for 2 h group (group S 3) and 2% sevoflurane anesthesia for 4 h group (group S 4). Morris water maze test was performed at 4, 8 and 12 weeks after anesthesia.The rats were sacrificed after the last Morris water maze test, and the hippocampal tissues were obtained for microscopic examination of the pathological changes (using HE staining), neuron apoptosis (by TUNEL staining), and expression of PSD-95, Kalirin-7 and Rac1 protein and mRNA (by Western blot and quantitative real-time polymerase chain reaction). The apoptosis rate was calculated. Results:Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the time of stay in the target quadrant was shortened, and the apoptosis rate of hippocampal neurons was increased at 4th, 8th and 12th weeks after anesthesia, phosphorylated Rac1/Rac1 ratio was decreased, and the expression of PSD-95 and Kalirin-7 protein and mRNA was down-regulated in S 1, S 2, S 3 and S 4 groups ( P<0.05). Compared with group S 4, the escape latency was significantly shortened, the number of crossing the original platform was increased, the time of stay in the target quadrant was prolonged, and the apoptosis rate of hippocampal neurons was decreased, phosphorylated Rac1/Rac1 ratio was increased, the expression of PSD-95 and Kalirin-7 protein and mRNA was up-regulated, and the histopathological changes of hippocampal tissues were attenuated in S 1, S 2 and S 3 groups ( P<0.05). Conclusions:The mechanism by which sevoflurane anesthesia induces long-term learning and memory impairment may be related to inhibition of activity of PSD-95/Kalirin-7/Rac1 signaling pathway in hippocampi of neonatal rats.

Rac1 Signaling in Amygdala Astrocytes Regulates Fear Memory Acquisition and Retrieval / 神经科学通报·英文版

The importance of astrocytes in behavior control is increasingly appreciated, but little is known about the effects of their dynamic activity in regulating learning and memory. In the present study, we constructed AAVs of photoactivatable and photoinactivatable Ras-related C3 botulinum toxin substrate 1 (Rac1) under the mGFAP promoter, which enabled the manipulation of Rac1 activity in astrocytes by optical stimulation in free-moving mice. We found that both up-regulation and down-regulation of astrocytic Rac1 activity in the basolateral amygdala (BLA) attenuated memory acquisition in a fear conditioning mouse model. Meanwhile, neuronal activation in the BLA induced by memory acquisition was inhibited under both the up- and down-regulation of astrocytic Rac1 activity during training. In terms of the impact on fear memory retrieval, we found both up- and down-regulation of BLA astrocytic Rac1 activity impaired memory retrieval of fear conditioning and memory retrieval-induced neuronal activation. Notably, the effect of astrocytic Rac1 on memory retrieval was reversible. Our results demonstrate that the normal activity of astrocytic Rac1 is necessary for the activation of neurons and memory formation. Both activation and inactivation of astrocytic Rac1 activity in the BLA reduced the excitability of neurons, and thereby impaired fear memory acquisition and retrieval.

Development of small-molecule tropomyosin receptor kinase (TRK) inhibitors for

Tropomyosin receptor kinase A, B and C (TRKA, TRKB and TRKC), which are well-known members of the cell surface receptor tyrosine kinase (RTK) family, are encoded by the neurotrophic receptor tyrosine kinase 1, 2 and 3 (

Aspecto da artéria coronária circunflexa retroaórtica por ecocardiografia transesofágica / Retroaortic circumflex coronary artery appearance by transesophageal echocardiography

O sinal RAC (retroaortic anomalous coronary ou artéria coronária anômala retroaórtica) é um achado desconhecido, descrito ao ecocardiograma transtorácico como uma estrutura tubular ecogênica, localizada na face atrial do sulco atrioventricular. Apresentamos um caso onde o sinal RAC se apresenta na ecocardiografia transesofágica (ETE). O conhecimento do sinal RAC e a avaliação com ETE aumentam a sensibilidade e a especificidade e conferem a oportunidade de avaliar características anatômicas de alto risco, importantes na avaliação do risco de morte súbita.(AU)

Afzelin inhibits migration of MDA-MB-231 cells by suppressing FAK expression and Rac1 activation

Triple-negative breast cancer (TNBC) has the worst prognosis and the highest rate of metastasis among other types ofbreast cancer. These characteristics are supported by the dysregulation of focal adhesion kinase (FAK) and Rac1 whichare the key players of mesenchymal cell migration on TNBC. Afzelin is a secondary metabolite that is contained ina variety of plants. This study explored the anti-migration effect of afzelin and its interaction with FAK and Rac1 onthe highly invasive TNBC cell line, MDA-MB-231. Cell viability was assessed by 3-(4,5-dimethyl 2-thiazolyl)-2,5-diphenyltetrazolium bromide assay, and cell migration was evaluated using in vitro scratch assay. Rac1 activation wasanalyzed using the colorimetric assay, while vinculin and actin filaments were stained through immunofluorescence. Thequantity of total FAK and phosphorylated FAK tyr397 was detected by Western blotting. Afzelin decreased cell viabilityand inhibited two-dimensional cell migration in a dose-dependent manner. Under confocal laser scanning microscopy,vinculin localization at the cell edge demonstrated a reduction of focal adhesion formation by afzelin. Further explorationshowed that afzelin decreased FAK expression but did not affect FAK phosphorylation at tyr397. In addition, afzelindecreased Rac1-GTPase activation, which is a downstream effector of FAK. Taken together, these results suggest thatafzelin suppresses TNBC cell migration, through inhibition of FAK expression and Rac1-GTPase activation.

Inhibition of chemotherapy-related breast tumor EMT by application of redox-sensitive siRNA delivery system CSO-ss-SA/siRNA along with doxorubicin treatment / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) downregulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.

MicroRNA-34a promotes the senescence and apoptosis of human lens epithelial cells by up-regulating Cdc42 and Rac1 / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To observe the effect and mechanism of MicroRNA-34a on senescence and apoptosis of human lens epithelial cell line SRA01/04.<p>METHODS: MicroRNA-34a expression levels in ARC lens and transparent lens epithelial cells were detected by qRT-PCR. MicroRNA-34a mimics, MicroRNA-34a inhibitors and empty liposome(control group)were transfected into SRA01/04 cells by liposome transfection kit. Annexin V-FITC/PI double staining was used to detect the effect of MicroRNA-34a on the apoptosis of human lens cell line SRA01/04. The expression of Cdc42 and Rac1 protein was detected by western blot. <p>RESULTS: The expression level of MicroRNA-34a in anterior capsular tissue of transparent lens was significantly lower than that in ARC anterior capsular tissue(<i>P</i><0.05). The positive rates of SA-β-gal in the MicroRNA-34a mimics group, the control group and the MicroRNA-34a inhibitors group were(87.56±2.34)%,(12.22±2.74)% and(3.45±0.45)%, respectively. The positive rates of SA-β-gal in the MicroRNA-34a mimics group was significantly higher than the control group, while the SA-β-gal positive rate in the MicroRNA-34a inhibitors group was significantly lower than that in the control group(<i>P</i><0.05). The apoptosis rate of the MicroRNA-34a inhibitors group, control group and MicroRNA-34a mimics group were(5.87±1.22)%,(12.26±2.14)% and(29.45±3.12)%, respectively. The apoptosis rate of the MicroRNA-34a mimics group was significantly higher than that of the control group, while that of the MicroRNA-34a inhibitors group was significantly lower than that of the control group(<i>P</i><0.05). The expressions of Cdc42 and Rac1 in the MicroRNA-34a mimics group were significantly higher than those in the control group(<i>P</i><0.05), while the expressions of Cdc42 and Rac1 in the MicroRNA-34a inhibitors group were significantly lower than those in the control group(<i>P</i><0.05).<p>CONCLUSION: MicroRNA-34a may promote the senescence and apoptosis of human lens epithelial cells by up-regulating Cdc42 and Rac1.

Inhibition of chemotherapy-related breast tumor EMT by application of redox-sensitive siRNA delivery system CSO-ss-SA/siRNA along with doxorubicin treatment / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) down-regulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.

Dose-and Time-Dependent Suppression of Rac1 and STIM1 in Acute Myeloid Leukaemia Cell Line Model

@#Introduction: Rac1 and STIM1 genes are emerging therapeutic targets for cancers. However, their roles in acute myeloid leukaemia (AML) are not well understood. The goal of this study was to evaluate the effects of dose and time on Rac1 and STIM1 knockdown in the AML cell line model (THP-1 cells). Methods: THP-1 cells were transfected with siRac1 at doses of 50, 100, and 200 nM or dsiSTIM1 at doses of 2, 5, and 10 nM. Expression level of Rac1 and STIM1 then were assessed at time points between 12 and 72 h post-transfection using real-time reverse transcription polymerase chain reaction. Results: Compared to the control, 87% Rac1 knockdown was attained with 50 nM siRac1 at 24 h post-transfection, and 70% STIM1 knockdown was achieved with 10 nM dsiSTIM1 at 48 h post-transfection. Conclusion: These results show that effective knockdown of Rac1 and STIM1 is possible, and therapy that includes Rac1 and STIM1 inhibitors eventually could provide a new and highly effective strategy for AML treatment.

miR-223-3p regulates proliferation and apoptosis of hepatocellular carcinoma SMMC-7721 cells by targeting RAC1 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects of miR-223-3p on the proliferation and apoptosis of hepatocellular carcinoma (HCC) cells by regulating Ras-related C3 botulinum toxin substrate 1 (RAC1) and its possible mechanism. Methods: Thirty pairs of HCC and corresponding para-cancer tissues resected in Jilin Central Hospital from August 2016 to August 2018 were collected for this study; in addition, human HCC cell lines SMMC-7721, BEL-7402, HepG2 and human normal hepatocyte QSG-7701 were also collected. The expression level of miR-223-3p in HCC tissue and cell lines was detected by qPCR. miR-223-3p mimics, miR-223-3p inhibitor and siRAC1 were transfected into SMMC-7221 cells, respectively. CCK-8 assay, Colony formation assay and Annexin V-FITC/PI staining Flow cytometry were used to detect the proliferation, clone formation and apoptosis of SMMC-7721 cells, respectively. The relationship between miR-223-3p and RAC1 was confirmed by Dual luciferase reporter gene assay. The protein level of RAC1 in SMMC-7721 cells was detected by Western blotting. Results: The expression of miR-223-3p in HCC tissues was significantly lower than that in paracaner tissues (P<0.01), and had significant correlation with pathological characteristics, such as tumor size, TNM stage, EdmondsonSteiner grade (all P<0.05 or P<0.01). miR-223-3p expression in HCC cell lines was significantly lower than that in QSG-7701 cells with the lowest expression in SMMC-7721 cells. Dual luciferase reporter gene assay confirmed that RAC1 was a target gene of miR-223-3p, and miR-223-3p negatively regulated RAC1 expression. Over-expression of miR-223-3p significantly inhibited the proliferation and colony formation (P<0.05 or P<0.01) of SMMC-7721 cells and promoted cell apoptosis (P<0.01). Contrarily, knockdown of miR-223-3p reversed the inhibitory effect of miR-223-3p mimics on cells. Conclusion: miR-223-3p over-expression inhibits proliferation and colony formation and promotes apoptosis of HCC cells, the mechanism of which may be related with its targeted down-regulation of RAC1.

Role of transient receptor potential cation channel subfamily M member 2 in hepatic ischemia-reperfusion injury in the mouse and the underlying mechanisms / 中南大学学报(医学版)

OBJECTIVES@#To investigate the role of transient receptor potential cation channel subfamily M member 2 (TRPM2) in hepatic ischemia-reperfusion injury of mouse (HIRI) and the possible mechanisms.@*METHODS@#Sixty adult male C57BL/6 mice were randomly divided into 4 groups: a sham group (S group), a HIRI model group (M group), a TRPM2 adenovirus interference vector group (T group), and a TRPM2 adenovirus control vector group (C group) (=15 in each group). The liver tissues of mice before perfusion were obtained. The efficiency of adenovirus infection was detected by fluorescence microscopy, and the silencing efficiency of adenovirus against TRPM2 was detected by real-time PCR.The abdominal aorta blood and liver tissues were collected from mice at 2, 4 and 8 h after reperfusion. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of mice were detected. Hepatic pathological changes were examined by hematoxylin-eosin (HE) staining. The protein expression of TRPM2 and Rac family small GTPase 1 (RAC1) in liver tissues was detected by Western blotting. Changes of malondialdehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO) activities in liver tissues were detected by enzyme-linked immunosorbent assay.@*RESULTS@#A strong signal of green fluorescence was observed in the liver tissues of mice in the T and C groups compared to the S or M group. Compared with the S, M or C group, the expression of TRPM2 mRNA in liver tissue in the T group was significantly down-regulated (all <0.05). The morphology of hepatocytes was normal in the S group under light microscope.Hepatic sinus dilatation, congestion, hepatocyte degeneration, central necrosis of lobule, and massive inflammatory granulocyte infiltration were observed in the M and C group, respectively. The degree of hepatocyte damage in the T group was significantly reduced compared with that in the M and C group, respectively. Compared with the S group, the serum ALT and AST activities in the M, T and C groups were significantly increased at 2, 4 and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum ALT and AST activities in the T group were significantly lower in serum of mice at 2, 4, and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum SOD activity in the T group was significantly increased at 2, 4, and 8 h after reperfusion (all <0.05), while the serum MDA and MPO activities were significantly decreased (all <0.05). The protein expression of TRPM2 and RAC1 in liver tissues in the T group were significantly lower than those in the M and C groups at 2, 4 and 8 h after reperfusion (all <0.05).@*CONCLUSIONS@#Pretreatment with TRPM2 adenovirus interference vector can effectively silence TRPM2 gene expression in liver tissues of mice and attenuate HIRI, which may be related to inhibiting oxidative stress and reducing the expression of RAC1 protein.

ARHGEF16 variants screening and mutation function analysis for children with total anomalous pulmonary venous connection / 上海交通大学学报（医学版）

Objective:To investigate the pathogenesis of total anomalous pulmonary venous connection (TAPVC), and to identify ARHGEF16 gene through full exon sequencing screening and analyze its mutation function. Methods:The blood, clinical data and auxiliary examination results of 78 children with TAPVC and 100 healthy controls were collected, and the genomic DNA was extracted for ARHGEF16 mutation screening. ARHGEF16 wild-type and mutant expression plasmids were constructed and transfected into 293T cells. mRNA and protein expression levels were detected by quantitative real-time PCR (RT-qPCR) and Western blotting, respectively. Protein-protein interaction exploration was performed by Cytoscape software. Results:Two novel variants c.C236>T (A79V) and c.G619>C (G207R) were found in TAPVC patients and were not found in healthy controls. Compared with the wild type, the mutants ARHGEF16 were up-regulated in both mRNA and protein expression levels. Protein interaction analysis showed that ARHGEF16 and RAC1 were directly associated; RAC1 expression was up-regulated in HEK293 cells with ARHGEF16 overexpression through RT-qPCR. Conclusion:The missense mutations of ARHGEF16 affect the mRNA and protein expression levels of ARHGEF16. Overexpression of ARHGEF16 up-regulates the expression of RAC1, suggesting that it may participate in the development and formation of TAPVC by regulating RAC1.

Novel ractopamine-protein carrier conjugation and its application to the lateral flow strip test for ractopamine detection in animal feed / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In this work, a novel conjugate of ractopamine and bovine serum albumin (RAC-BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC-BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC-BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection (LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075-0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%-103.7% with a relative standard deviation of 2.5%-3.6% for intra- and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC-BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.

Novel ractopamine–protein carrier conjugation and its application to the lateral flow strip test for ractopamine detection in animal feed / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In this work, a novel conjugate of ractopamine and bovine serum albumin (RAC–BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC–BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC–BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection (LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075–0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%–103.7% with a relative standard deviation of 2.5%–3.6% for intra- and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC–BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.

Construction of luciferase reporter gene cell model and its application in screening rhein derivatives targeting RAC1 / 中国药理学通报

Aim To establish luciferase reporter gene expression cell models of CNEi-RACI-Luc2 based on the target of RAC1 promoter, and explore the application of screening anti-tumor active of rhein derivatives targeted regulating RAC1 at transcriptional level. Methods The lentiviral carrying luciferase reporter vector was designed and synthesized using RAC1 promoter se-quence, and CNE1 cells were infected with recombinant plasmid lentiviral to obtain cell lines that stably expressed firefly luciferase. Luciferase reporter assay was used to detect the luciferase luminescence value after stimulating with RAC1 activator PMA and inhibitor NSC23766 that targeted regulating the RAC1 promoter activities in cells, and RAC 1 expression was verified by Western blot. The effect of series of rhein derivatives on the lu-cifcrase activity of RAC1 promoter was observed, and RAC1 expression was determined by Western blot. Results The identification result of double enzyme digestion showed that a lentiviral expression vector carrying luciferase reporter vector rc-combined with RAC1 promoter was successfully constructed. Lcntivirus-infcctcd CNE1 cells were screened by puromycin, the CNE1-RAC1-Luc2 cells stably expressing firefly luciferase were obtained, and the Iransfection efficiency was over 90%. The RAC1 luciferase reporter assay system was sensitive to FMA and NSC23766 and consistent with the result of RAC1 protein expression by Western blot. The regulation of series of rhein derivatives to RAC1 luciferase activity of CNE1-RAC1-Luc2 cells was consistent with the results of Western blot. Conclusions The cell model of luciferase reporting system containing RAC1 promoter can be successfully constructed, which provides a practical platform for high throughput screening of RAC1-targeted drugs.

Inhibition of Rac1-dependent forgetting alleviates memory deficits in animal models of Alzheimer's disease

Accelerated forgetting has been identified as a feature of Alzheimer's disease (AD), but the therapeutic efficacy of the manipulation of biological mechanisms of forgetting has not been assessed in AD animal models. Ras-related C3 botulinum toxin substrate 1 (Rac1), a small GTPase, has been shown to regulate active forgetting in Drosophila and mice. Here, we showed that Rac1 activity is aberrantly elevated in the hippocampal tissues of AD patients and AD animal models. Moreover, amyloid-beta 42 could induce Rac1 activation in cultured cells. The elevation of Rac1 activity not only accelerated 6-hour spatial memory decay in 3-month-old APP/PS1 mice, but also significantly contributed to severe memory loss in aged APP/PS1 mice. A similar age-dependent Rac1 activity-based memory loss was also observed in an AD fly model. Moreover, inhibition of Rac1 activity could ameliorate cognitive defects and synaptic plasticity in AD animal models. Finally, two novel compounds, identified through behavioral screening of a randomly selected pool of brain permeable small molecules for their positive effect in rescuing memory loss in both fly and mouse models, were found to be capable of inhibiting Rac1 activity. Thus, multiple lines of evidence corroborate in supporting the idea that inhibition of Rac1 activity is effective for treating AD-related memory loss.

Rac1 Modulates Excitatory Synaptic Transmission in Mouse Retinal Ganglion Cells / 神经科学通报·英文版

Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of the Rho GTPase family which plays important roles in dendritic spine morphology and plasticity, is a key regulator of cytoskeletal reorganization in dendrites and spines. Here, we investigated whether and how Rac1 modulates synaptic transmission in mouse retinal ganglion cells (RGCs) using selective conditional knockout of Rac1 (Rac1-cKO). Rac1-cKO significantly reduced the frequency of AMPA receptor-mediated miniature excitatory postsynaptic currents, while glycine/GABA receptor-mediated miniature inhibitory postsynaptic currents were not affected. Although the total GluA1 protein level was increased in Rac1-cKO mice, its expression in the membrane component was unchanged. Rac1-cKO did not affect spine-like branch density in single dendrites, but significantly reduced the dendritic complexity, which resulted in a decrease in the total number of dendritic spine-like branches. These results suggest that Rac1 selectively affects excitatory synaptic transmission in RGCs by modulating dendritic complexity.