A sensitive and selective liquid chromatography–tandem mass spectrometric (LC ? MS/MS) method was established to determine 2-oxo-clopidogrel, a crucial intermediate metabolite in human plasma. A chromatographic separation was performed on a Sapphire C18 column following a liquid–liquid extraction sample preparation with methyl t-butyl ether. Detection was carried out on a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) with an electrospray ionization (ESI) mode. The method was validated in terms of specificity, accuracy, precision and limit of quantification. The calibration curves ranged from 0.50 to 50.0 ng/mL with good linearity. The stability was fully validated with addition of 1,4-dithio-DL-threitol (DTT) into the plasma sample prior to and in the preparation procedure. The validated method was proved to be suitable for use in pharmacokinetic study after single oral administration of 75 mg clopidogrel tablets in human subjects, which could make contribution to intensive study of the clinical drug–drug interactions of clopidogrel and individual treatment.

Objective:To study the masseter motor evoked potential(MEP)in patients with sleep bruxism(SB)and in healthy con-trols.Methods:30 subjects with SB and 30 healthy controls were included.MEPs were obtained by transcranial magnetic stimulation (TMS).Tests were done during daytime when the subjects were awake.The data were statistically analysed.Results:In the patients AMT was 55(52,55)%,latency of c-MEP (6.7 ±1.3)ms,the amplitude of c-MEP 0.19(0.15,0.29)mV,latency of r-MEP (2.3 ±0.4)ms,the central conduction time(CCT)4.4(3.3,5.2)ms.In the control subjects AMT was 52(52,55)%,latency of c-MEP (6.4 ±0.7)ms,the amplitude of c-MEP 0.23(0.17,0.28)mV,latency of r-MEP (2.4 ±0.4)ms,CCT 4.0 (3.4,4.4) ms.No significant difference was found between the 2 groups in the measurements evoked by TMS.Conclusion:The MEP after TMS in patients with SB is similar to that of healthy subjects,indicating that the excitability of the cortical motor system is not changed in bruxism subjects,at least when evaluated by TMS.

BACKGROUND: Hepatitis B virus (HBV) infection is one of the worldwide public health problem affecting about 300 million people. The envelope protein of HBV consists of three components known as preS1, preS2, and S antigen. According to the recent study, anti-HBs Ab showed effective neutralization ability against HBV from chronic hepatitis B and liver transplant patients, suggesting the possible development of therapeutic antibody. METHODS: Spleen cells immunized with S antigen of HBV were fused with myeloma cell line to obtain HBsAg specific monoclonal antibodies. High affinity antibodies against HBsAg (adr, ad and ay type) were selected by competitive ELISA method. Nucleotide sequence of the variable regions of monoclonal antibodies was analyzed by RT-PCR followed by conventional sequencing method. RESULTS: We produced 14 murine monoclonal antibodies which recognize S antigen of HBV. Two of them, A9-11 and C6-9 showed the highest affinity. The sequence analysis of A9-11 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain I (B) and light chain lambda 1, respectively. Likewise, the sequence analysis of C6-9 revealed that variable regions of the heavy chain and light chains are members of mouse heavy chain II (B) and light chain kappa 1, respectively. Neutralization assay showed that A9-11 and C6-9 effectively neutralize the HBV infection. CONCLUSION: These results suggest that A9-11 and C6-9 mouse monoclonal antibodies can be used for the development of therapeutic antibody for HBV infection.
Animals ; Antibodies ; Antibodies, Monoclonal* ; Base Sequence ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B Surface Antigens ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis B, Chronic ; Hepatitis* ; Humans ; Liver ; Mice ; Public Health ; Sequence Analysis ; Spleen

Objective:To explore the relationship of negative life events,parent-child relationship,teacher-child relationship with the altruistic prosocial behaviors in migrant and unattended primary school children. Methods:Totally 1198 migrant children and 1631 unattended children were recruited. The dimension ofaltruistic prosocial behaviorof Child and Adolescent Prosocial Behavior Questionnaire (CAPBQ-A)was used to evaluate altruistic prosocial behaviors,Life Event Questionnaire (LEQ)was used to evaluate negative life events,parent trust and support dimensionof Social Psychological Relationship Questionnaire(SPRQ-P)to evaluate par-ent-child relationship, and teacher trust and support dimension (SPRQ-T ) to evaluate teacher-child relationship. Results:The CAPBQ-A scores were higher in migrant children than in the unattended children (P<0. 001). Being migrant or unattended children (β=-0. 07),LEQ score (β=-0. 04),SPRQ-P score (β=0. 13) and SPRQ-T score (β=0. 31 )predicted the CAPBQ-A score significantly. The SPRQ-P score had significantly stronger predictive effect on the migrant children compared to the unattended children (β=-0. 06 ). Yet the SPRQ-T score predicted the unattended children stronger (β=0. 06 ,Ps<0. 0 1 ). Conclusion:The unattended children's al-truistic prosocial behaviors are much worse than the migrant children. Personal relationships seem to play a more important role than negative life events in shaping children's altruistic prosocial behaviors. Moreover,the parent-child relationship seems to be more important to the migrant children,whereas the teacher-child relationship is more important to the unattended children.

AIM: To design a simple and effective auxiliary device for detection of the compound action poten-tial (CAP) and the force on the isolated sciatic nerve and gastrocnemius in toads, and to investigate its practicability. METHODS: A simple “L” shape device (L tube), which was composed of a nerve chamber and an organ bath, was made for fixing the isolated sciatic nerve and gastrocnemius.After fixing, the sciatic CAP and the gastrocnemius force were detected by BL-420S data acquisition and analysis system.The specimens were radomly divided into control group, and the lidocaine nerve and muscle groups.The sciatic nerve or gastrocnemius of each lidocaine group was firstly treated with the corresponding concentration of lidocaine, and then washed out with Ringer’s solution, and its reversible anesthetic action on nerve conduction and muscle force was analyzed to verify the practicability of the L tube device.RESULTS: The CAP and the force of the sciatic-gastrocnemius specimens were detected concurrently by fixing the specimens in the L tube, and the liquid in the nerve chamber and organ bath was changed easily.Compared with the control, lidocaine at 0.05 and 0.2 g/L significantly increased the sciatic threshold stimulus voltage and maximal stimulus voltage (P <0.05), prolonged the absolute refractory period (P <0.01), and slowed down the conduction velocity (P <0.01).Lidocaine at 1 g/L complete-
ly blocked the sciatic conduction.The rest tension of the gastrocnemius was increased, and the maximal twitch force was decreased significantly by 1 g/L lidocaine (P <0.01), but the threshold and maximal stimulus voltage did not show statis-tic difference.The parameters of the sicatic nerve and gastrocnemius completely or partially recovered to the control level after washing out.CONCLUSION: As an auxiliary device, L tube makes the detection of CAP and force on the isolated sciatic nerve and gastrocnemius in toads more conveniently.

Objective: To investigate the changes in the levels of interleukin-6 (IL-6) and let-7e in rats induced by coal mine dust, so as to provide the basis for the mechanism of coal worker's pneumoconiosis (CWP). Methods: Sixty-four clean and healthy male Sprague-Dawley rats were randomly divided into the control group, coal dust group, mixed dust group (mixed coal and silica dust) and quartz group. The rats in the control group were exposed to 1 mL physiological saline by non-exposure tracheal perfusion, and the rats in the dust-exposed groups were exposed to 1 mL dust suspension. Rats were sacrificed by anesthesia after 1 month and 6 months, lung tissue was observed using hematoxylin-eosin staining, the pathological change in the lungs was scored using the Szapiel scoring system, the levels of IL-6 in the bronchoalveolar lavage fluid were detected using enzyme-linked immunosorbent assay, and the expression of let-7e was determined by quantitative real-time PCR. Results: A month after exposure, a small amount of coal spots and inflammatory exudation were observed in the lung tissue of the coal dust group and the mixed dust group. The quartz group showed tissue structure destruction and mild fibrosis and thickening of alveolar septum. Six months after exposure, there were more coal spots and slightly thickened alveolar septum in the coal dust group, and hyperplasia of pulmonary interstitial fibers, destruction of alveolar structure and silica nodules were observed in the mixed dust group. In the quartz group, the alveolar structure was obviously destroyed, the interstitial fiber proliferation was significant and silica nodules were seen. Two-factor analysis of variance showed that the interaction between duration of exposure and dust type significantly influenced the pathological score of lung tissue, IL-6 levels, and let-7e expression levels (P<0.05). Under the same dust type, the pathological score of lung tissue and IL-6 levels were higher at 6 months after exposure than at 1 month, while the relative expression of let-7e was lower at 6 months after exposure than at 1 month (all P<0.05). Under the same duration of exposure, the pathological score of lung tissue and IL-6 levels were higher in the dust-exposed groups than in the control group, while the relative expression of let-7e was lower in the dust-exposed groups than in the control group (all P<0.05). Conclusions Coal dust can cause an increase in levels of IL-6 and a decrease in let-7e expression in rats. The type of dust and duration of exposure can interactively affect IL-6 and let-7e.

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta TSK114 was found to be of IgG1 isotype with kappa light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-alpha by surface plasmon resonance technique revealed a binding affinity (KD) of ~5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, respectively. TSK114 neutralized human TNF-alpha-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-alpha-neutralizing antibody with picomolar affinity.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal/chemistry/genetics/*immunology ; Antibody Affinity/*immunology ; Antibody Specificity ; Base Sequence ; Blotting, Western ; Cell Line ; Cytotoxicity, Immunologic ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin Variable Region/genetics ; Kinetics ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Neutralization Tests ; Sequence Analysis, Protein ; Tumor Necrosis Factor-alpha/*immunology

Objective To investigate whether cyclic GMP-AMP synthase ( cGAS ) , a cytosolic DNA sensor, could recognize the reverse transcription intermediate and induce the subsequent signaling path-way during the infection of human T cell leukemia virus type 1 ( HTLV-1 ) . Methods Biotin-labeled ssDNA90, a reverse transcription intermediate of HTLV-1, was transfected into HeLa cells and the interac-tion between it and cGAS was detected by co-immunoprecipitation experiments. HeLa cells were co-cultured with HTLV-1-positive MT2 cells and the interaction between cGAS and stimulator of interferon genes ( STING) was analyzed by co-immunoprecipitation experiments. The expression of STING in HeLa cells was silenced by siRNA. cGAS was transfected into the HeLa cells 24 h after the silencing and after 24 h, these cells were co-cultured with MT2 cells for another 24 h. Real-time PCR assay was used to measure the ex-pression of IFN-β, RANTES ( regulated upon activation, normal T-cell expressed, and secreted) , TNF-α, HTLV-1 protein Tax, p19 and HBZ. Immunoblot assay was performed to evaluate the phosphorylation of IRF3 and p65 in HeLa cells. Results cGAS interacted with ssDNA90. cGAS interacted with STING in the cytoplasm. In STING-silenced HeLa cells, cGAS transfection had no influence on the expression of IFN-β, RANTES , TNF-α, Tax , p19 or HBZ , nor did it affect the phosphorylation of IRF3 or p65 . Conclusions cGAS interacted with HTLV-1 RTI ssDNA90 and activated STING-dependent innate immune responses.

Objective:To assess the value of amide proton transfer weighted (APTw) imaging in the evaluation of pH changes in infarct core (IC) and ischemic penumbra (IP) in subacute cerebral infarction.Methods:The data of twenty-three subacute cerebral infarction patients with unilateral steno-occlusive disease of the middle cerebral artery (subacute infarction group) from April to November 2019 in the First Affiliated Hospital of Dalian Medical University were prospectively analyzed. Fifteen healthy volunteers were enrolled in this study as the control group. All subjects underwent conventional MRI, DWI, 3D-pseudo continuous arterial spin labeling (3D-pCASL) and APTw sequences. Based on DWI images, relative cerebral blood flow (rCBF) and APTw images to determine the region of IC, blood flow penumbra [cerebral blood flow(CBF)-DWI mismatch area, IP CBF] and metabolic penumbra (APTw-DWI mismatched area, IP APT). 3D ROIs were used to semi-automatically measure the APTw signals and the volume of IC and IP CBF of the patients in subacute infarction group. The comparison of APTw signals between the infarct side and the contralateral side in the subacute infarction group, the comparison of bilateral APTw signals in the control group, and the comparison of APTw signals in the IC and IP CBF regions were performed by paired-sample t test or Wilcoxon signed-rank test. The paired-sample t test or Mann-Whitney U test was used to compare the APTw signals between the two groups. The Friedman test was applied to compare the difference of volumes among IP CBF1.5, IP CBF2.5 and IP APT . Results:There was no significant difference of the APTw signals among the IC, the contralateral side in the subacute infarction group and the control group ( P>0.05). The APTw signals of IP CBF and IC of the infarction group were statistically different ( P<0.05). Compared with the contralateral side of IP CBF1.5 (3.7±1.7, -1.84±1.48, 5.57±2.75), the APTwmax (3.07±1.41, t=-3.012, P=0.006), APTw min [-1.30 (-1.74, -0.57), Z=-2.099, P=0.036], and APTwmax-min(4.51±2.58, t=-3.273, P=0.003) signals in the IP CBF1.5 were decreased ( P<0.05). Compared with the contralateral side of IP CBF2.5 [-1.53 (-2.80, -0.91), 5.31±2.61], the APTw min [-1.08 (-1.60, -0.49), Z=-2.616, P=0.009] and APTwmax-min (4.41±2.72, t=-3.228, P=0.004) signals in the IP CBF2.5 were decreased. The volumes of IP CBF1.5 [107.51(50.08, 138.61)mm 3], IP APT [99.00 (53.27, 121.335) mm 3] and IP CBF2.5 [89.91 (51.53, 139.87) mm 3] were successively reduced (χ2=7.913, P=0.019), and the volume of IP CBF2.5 was significantly smaller than that of IP CBF1.5 ( P=0.037). Conclusion:The acid-base metabolism in the IC of subacute cerebral infarction is not obvious, but the blood flow penumbra has local acid-base metabolism imbalance, and the range of metabolic penumbra coincides with the blood flow penumbra.

Objective: To investigate the association between consumption of milk tea beverages and other sugary beverages with insomnia symptoms among college students, so as to the provide reference for promoting sleep quality among college students. Methods: From September to November 2023, a convenience sampling method was used to select 15 440 college students from seven universities in Shanghai, Jiangxi Province, Hubei Province, and Shanxi Province. A self developed questionnaire and the Insomnia Severity Index were used to assess milk tea beverage consumption, other sugary beverages and insomnia symptoms. Univariate analysis of insomnia symptoms among college students was performed using Chi square tests and Cochran-Armitage trend χ 2 tests. Multivariate Logistic regression analysis was conducted for the association between milk tea and other sugary beverage consumption with insomnia symptoms. Results: The detection rates of insomnia symptoms among college students was 40.83%. The frequency of consumption of different milk tea and various sugary drinks (juice, tea, milk, sodas,yogurt, functional drinks, coffee) per week was related to the detection trend rate of insomnia symptoms of college students ( χ 2 trend =38.38, 47.66, 74.16, 32.44, 65.78 , 38.71, 35.94, 91.59, P < 0.01 ). Multivariate analysis indicated that compared to individuals with no milk tea consumption, college students engaging in milk tea consumption had a significantly increased risk of insomnia symptoms ( OR =1.24, P <0.05). Students consuming milk tea at low to moderate frequencies (1-3, 4-5 times per week) showed a higher risk of insomnia symptoms compared to those with no consumption ( OR =1.20, 1.54, P <0.05). Furthermore, higher levels of milk tea consumption were significantly associated with insomnia symptoms (1 501-2 999 mL, OR =1.22; ≥3 000 mL, OR =1.36), and the consumption of other sugary beverages were also associated with insomnia symptoms of college students ( OR =1.10-1.55) ( P <0.05). Conclusions There is a relationship between consumption of milk tea beverage other sugary beverages with insomnia symptoms among college students. Colleges should regularly conduct dietary health education seminars to improve sleep quality among college students.