Objective:To establish a liquid chromatography tandem mass spectrometry ( LC-MS/MS) method for the determination of 5-fluorouracil (5-Fu) in patient’s plasma and apply it in clinics patients validation. Methods:5-Fu was analyzed on an Agela Inno-val NH2 (2. 1 mm × 50 mm, 5 μm) column. Methanol:ultra pure water (2 ∶98) was used as the mobile phase with isocratic elution. The flow rate was 0. 3 ml ·min-1 and the column temperature was set at 40℃. The ion transitions with electrospray ionization negative model were m/z 128. 8→42. 1 and m/z 188. 6→42. 1 for 5-Fu and 5-bromouracil (the internal standard), respectively. The LC-MS/MS method was verified according to the guideline of quantitative analysis validation of biological samples ( Chinese Pharmacopoeia, 2015 edition, the fourth part) . Results:The calibration curve of 5-Fu was linear within the range of 10-1 000 ng · ml-1 . The lower limit of quantification was 10 ng · ml-1 . The precision, accuracy, matrix effect and stability within the linear range were all in line with the requirements of method validation. Conclusion:The LC-MS/MS method developed in the study for the determination of 5-Fu is simple, rapid, accurate and reproducible, which can be used for the plasma concentration detection of 5-Fu in patients.

Objective To analyze the correlation between miR-31 and ESCC in expression of miR-31 in the plasma of ESCC in Xinjiang Kazak and Han nationality patients. Methods The plasma samples were collected respectively from patients with ESCC in 20 cases and healthy subjects in 20 cases. The relatively expression of miR-31 was detected by real-time Q-PCR. Results The expression of miR-31 with ESCC were higher than those of the normal control group, which related to the degree of tumor differentiation in Kazak ESCC patients (P < 0.01); the levels of miR-31 relative expression in Kazak were higher than that of Han (P = 0.008, P = 0.027). Conclusion miR-31 may be involved in the occurrence of ESCC in Han and Kazak nationality. miR-31 might be another risk factor in high incidence of ESCC in Kazak than Han nationality.

Objective To evaluate the relationship between neuropeptide S (NPS) in the amygdala and 5-hydroxytryptamine (5-HT) and GABA in spinal dorsal horns of rats with neuropathic pain.Methods Eighty pathogen-free healthy male Sprague-Dawley rats,weighing 200-260 g,aged 2 months,were divided into 4 groups (n =20 each) using a random number table:sham operation group (group Sham),neuropathic pain group (group NP),low dose NPS group (group L-NPS) and high dose NPS group (group H-NPS).The neuropathic pain model was established by left L5,6 spinal nerve ligation (SNL) in anesthetized rats.NPS was injected into the bilateral amygdala at 3,6,9,12,15 and 18 days after SNL in LNPS group (10 pmol per side) and H-NPS group (100 pmol per side).The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 2 days before SNL and 1,4,7,11,14,17 and 21 days after SNL.Five rats were selected at 7,14 and 21 days after SNL and sacrificed,and the lumbar segment (L5) of the spinal cord was removed for detection of the expression of 5-HT and GABA in spinal dorsal horns by immunofluorescence histochemistry.Results Compared with group Sham,the MWT was significantly decreased,the TWL was shortened,and the expression of 5-HT and GABA in spinal dorsal horns was down-regulated in NP,L-NPS and H-NPS groups (P＜0.05).Compared with group NP,the MWT was significantly increased,the TWL was prolonged,and the expression of 5-HT and GABA in spinal dorsal horns was up-regulated in L-NPS and H-NPS groups (P＜0.05).Compared with group L-NPS,the MWT was significantly increased,the TWL was prolonged,and the expression of 5-HT and GABA in spinal dorsal horns was up-regulated in group H-NPS (P＜0.05).Conclusion The spinal mechanism of endogenous analgesia induced by NPS in the amygdala may be related to up-regulation of the expression of 5-HT and GABA in spinal dorsal horns of rats with neuropathic pain.

To prepare a lipid nanoparticle (LNP)-based subunit vaccine of Mycobacterium tuberculosis (Mtb) antigen EsxV and study its immunological characteristics, the LNP containing EsxV and c-di-AMP (EsxV: C: L) was prepared by thin film dispersion method, and its encapsulation rate, LNP morphology, particle size, surface charge and polyphase dispersion index were measured. BALB/c mice were immunized with EsxV: C: L by nasal drops. The levels of serum and mucosal antibodies, transcription and secretion of cytokines in lung and spleen, and the proportion of T cell subsets were detected after immunization. EsxV: C: L LNPs were obtained with uniform size and they were spherical and negatively charged. Compared with EsxV: C immunization, EsxV: C: L mucosal inoculation induced increased sIgA level in respiratory tract mucosa. Levels of IL-2 secreted from spleen and ratios of memory T cells and tissue-resident T cells in mice were also elevated. In conclusion, EsxV: C: L could induce stronger mucosal immunity and memory T cell immune responses, which may provide better protection against Mtb infection.
Animals ; Mice ; Mycobacterium tuberculosis ; Antigens, Bacterial ; Immunization ; Nanoparticles ; Vaccines, Subunit ; Mice, Inbred BALB C

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.
Humans ; Apolipoprotein E4/genetics* ; Cytosine ; Mutation ; Blastocyst ; Heterozygote ; Gene Editing ; CRISPR-Cas Systems