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

Objective:To explore the clinical and pathological characteristics and prognostic factors of gastric neuroendocrine carcinoma(G-NEC)and gastric mixed adenoendocrine carcinoma(G-MANEC).Methods:Retrospective analysis was conducted on the clinical data of 67 patients with G-NEC and G-MANEC who underwent surgical treatment at Heping Hospital Affiliated to Changzhi Medical College from May 2015 to May 2023.The study included an analysis of the pathological characteristics distinguishing G-NEC from G-MANEC.Results:Com-pared to gastric adenocarcinoma,patients with G-NEC and G-MANEC in the stomach showed a higher incidence of gastric cancer in the male gastric cardia and were diagnosed at a later age.Tumors with larger diameters increase susceptibility to anemia,low albumin levels,and in-vasion of nerves and vasculature.Deeper tumor infiltration is associated with increased local lymph node metastases,later TNM staging,and a higher likelihood of distant metastasis post-surgery.The prognosis of G-NEC and G-MANEC is worse than that of gastric adenocarcinoma(P=0.001).However,there is no statistically significant difference in the pathological characteristics(P>0.05)and prognosis analysis(P=0.212)between G-NEC and G-MANEC.Univariate survival analysis identified age,preoperative albumin,preoperative CEA,number of lymph node metastases,TNM staging,and postoperative distant metastasis as risk factors affecting patient's overall survival(OS).In the multivariate ana-lysis,age,preoperative albumin,TNM staging,and postoperative distant metastasiswere identified as independent risk factors for OS.Con-clusions:There is a significant difference in clinical characteristics between G-NEC,G-MANEC,and gastric adenocarcinoma,often diagnosed at an advanced stage,which is prone to distant metastasis post-surgery.Poor prognosis is observed in patients aged over 60 years,with pre-operative albumin<40g/L,TNM stage Ⅱ/Ⅲ,and postoperative distant metastasis.

To ensure the safety of the commercially available chenpi, a convenient and fast analytical method was developed for the determination of 133 pesticide residues in chenpi using gas chromatography-tandem mass spectrometry (GC-MS/MS). In this study, different extraction solvents, redissolution solvents and adsorbents were tested according to the recovery and purification effect to obtain a modified QuEChERS method. The samples were extracted with acetonitrile. During the clean-up step, octadecyl-modified silica (C18) and graphitized carbon black (GCB) were selected, and aminopropyl (NH2) was used instead of primary secondary amine (PSA) because of its weaker ion exchange capacity which had little effect on the recovery of ditalimfos. Samples were quantified by matrix-matched calibration with internal stan-dards. All pesticides showed good linearity in the respective range, both with values of r2 >0.99. The average recoveries of the pesticides spiked samples ranged from 70.0％ to 112.2％ with the RSDs of 0.2％–14.4％. The modified QuEChERS method was validated and applied to twenty real samples. Five pesticides were found in eight batches, but no pesticide exceeded the maximum residue limits (MRL, MRL reference to European commission).

Objective To explore the effect of betulinic acid on NAFLD and its mechanism. Methods We used the high-fat diet animal models, with or without feeding the standard chow diet containing betulinic acid for 2 months. During this period, the body weight was monitored regularly and metabolism cage was used to monitor the energy metabolism of the animals. After killing the mice, molecular biological analysis was performed on serum and tissue related to liver. Results In diet induced obese mice animal experiments, the mice body weight had been reduced and NAFLD had been improved significantly by betulinic acid. The various indexs of serum and liver tissue had also been significantly improved. The metabolic rate increased significantly. Fatty acid synthase gene and protein levels were significantly lower. Furthermore, FAS activity was significantly lower than the control mice. Liver FAS activity of the high fat mice and the high fat mice treated with betulinic acid were (1873.6 ± 85.7) and (1181.6 ± 85.7) pmol NADPH/ min/ mg protein, respectively. Conclusion Betulinic acid inhibited FAS at expression and activity level, and improved lipid deposition in liver.

Objective To evaluate the clinical value of 125I seeds implantation (RSI) for treatment of lymph nodes metastases (LNM) in patients with 131I refractory differentiated thyroid carcinoma (RAIR-DTC).Methods A total of 42 RAIR-DTC patients with LNM (14 males,28 females,median age 49 years) who underwent RSI guided by CT from January 2015 to June 2016 were retrospectively analyzed.All patients underwent CT and their serum thyroglobulin (Tg) levels were measured 2,4 and 6 months post-treatment.The LNM size and serum Tg levels before and after treatment were compared,and the curative effect of RSI for complications was observed.Paired t test,x2 test,repeated measurement analysis of variance,Spearman correlation analysis were used to analyze the data.Results Two patients (4.76％) achieved complete remission,9(21.43％) achieved partial remission,29(69.05％) had no change,2(4.76％) were with progressive disease in all 42 patients.Thetotal effective rate was 95.24％ (40/42).The LNM diameter post-treatment was (1.39±0.75) cm,significantly lower than that before treatment ((1.99±0.38) cm;t=5.557,P＜0.01).The Tg level 2,4 and 6 months posttreatment was (53.24± 14.05),(33.17±7.61) and (25.93± 10.91) μg/L,respectively,which was significantly lower than that before treatment ((57.72± 15.23) μg/L;F =23.612,all P＜0.05).The age,sex,metastatic site,and the number of seeds implanted in each lesion were not the influencing factors on RSI (x2 values:0.569-15.884,rs =0.277,all P＞0.05).The LNM size was the influencing factor on RSI (x2=4.524,P＜0.05).Conclusions 125I RSI can significantly alleviate the clinical symptoms of RAIR-DTC patients with LNM,and the LNM size has relevance for the treatment effect.The clinical follow-up time of serum Tg level can be extended to 6 months or even longer.

Objective The objective of this study was to evaluate the correlation between maximum standard uptake values( SUVmax ) of 18 F-FDG PET/CT and BRAF mutation in patients with papillary thyroid car-cinoma(PTC).Methods Atotalof51patients(meanageof49.3±12.9years)whounderwent18F-FDGPET/CT imaging and biopsy before thyroidectomy were retrospectively analyzed. Based on the pathological results, there were 48 patients with PTC and 3 patients with follicular thyroid carcinoma(FTC). The SUVmax of thyroid nodule was measured by semi-quantitative analysis. The correlation between clinical data( gender,age,tumor size and thyroglobulin concentration) and SUVmax was also analyzed. The difference of SUVmax between the two groups was analyzed by comparing the BRAF V600E mutation group and the non-mutation group. Results In patients with PTC,27 patients had BRAF V600E mutations and 11 patients had no tumor mutation. The SUVmax of BRAF V600E mutation group was significantly higher than that in the non-mutated group(5. 5 ± 3. 9 vs. 2. 2 ± 1. 2,P=0. 002). The SUVmax of patients with tumor diameter≥1 cm was significantly higher than that in patients with tumor <1 cm(P<0. 05). The SUVmax of patients with elevated thyroglobulin concentration was higher than that in normal patients(P<0. 05). No BRAF V600E mutation was observed in the FTC group. Conclusion The BRAF V600E mutant gene has a high SUVmax value in patients with PTC. There were significant difference in SUVmax among different tumor size and serum thyroglobulin concentration.

Objective To evaluate the value of multi-modality imaging (PET/CT+CAG+CMRI) in post myocardial infract (MI) patients followed coronary artery bypass graft (CABG) and autologous bone marrow stem cell (BMSC) therapy.Methods A total of 43 patients with MI (27 males, 16 females, age range: 47-72 years) were prospectively enrolled in the year 2012 between January and December.All patients underwent CABG+BMSC transplantation and were divided into 3 groups according to the time interval between two treatments (group 1: 0-3 d;group 2: 4-14 d;group 3: 15-30 d).All patients were orderly scanned with CMRI, PET/CT (13N-NH3·H2O/18F-FDG) and CAG at different time-points pre-/post-treatment.The quantitative parameters included vascular stenosis degree(VSD), LVEF, percentage size of infarction (PSI), the number of segments in mismatched myocardial perfusion/metabolic and the K value for radioactive distribution grading.One-way analysis of variance and the least significant difference t test were used to compare parameters before and after treatment in the same group and among three different groups.Results Regarding PET/CT diagnostic efficacy of abnormal myocardial segments, the sensitivity, specificity, positive predictive value and negative predictive value were 95.4%(540/566), 87.3%(144/165), 96.3%(540/561) and 84.7%(144/170), respectively.After CABG and BMSC transplantation treatments for 12 months, VSD decreased significantly((69.1±9.5)%;F=12.854, P<0.05), comparing with the baseline ((74.8±7.9)%;t=3.074, P<0.05).Comparing to the baseline, LVEF in 3 groups increased slightly(F values: 0.906,0.298,0.059, all P>0.05).PSI of patients in group 2 decreased greatly after 12 months treatment ((35.70±12.59)%;F=3.792, t values:-2.916-4.059, all P<0.05).K values for radioactive distribution grade decreased obviously after 1 month and 12 months treatment comparing to the baseline (11.79±1.87,12.39±2.35,14.05±2.15;F=4.212, t values:-4.619,-0.989, all P<0.05).Number of myocardial perfusion/metabolic abnormal segments in group 2 after 1 month treatment was lower comparing to the baseline and 24 months treatment (10.17±0.66, 12.92±0.99, 14.17±1.21;F=3.543, t values:-2.146,-2.898, all P<0.05).The PSI, mismatched segments and K values post-treatment were not significantly different between group 1 and 3 (F values: 0.093-1.364, all P>0.05).Conclusions Multi-modality imaging may be used for accurately detecting abnormal myocardium and predicting prognosis.CABG+BMSC therapy during day 4-14 post-MI may temporarily improve perfusion and metabolism in viable myocardium, but the long term prognosis seemed not be improved.

Objective To investigate the effect of gradient heat stress on phagocytosis of hepatic Kupffer cells (KCs) in vitro in rats. Methods Rat Kupffer cells were isolated in vitro and the temperature for gradient heat stress was set at 37, 39, 41 and 43℃. After thermal stimulation, cell injury was detected by PI and Hochest33342 staining. CCK-8 assay was used to investigate difference in cellular proliferation rate over 24h between the groups. Flow cytometry was used to investigate the influence of heat stress on the phagocytosis of KCs. Results Compared to the normal control group, cells in each heat stress group exhibited varying degrees of damage, especially cells in 43℃ group. The ratio of damage cells increased with the increase of heat stress severity (P<0.05). Proliferation assay indicated that the proliferation rate of cells in each heat stress group was significantly decreased in comparison with normal control group 6h after heat stress (P<0.05). After 12h recovery, decrease in proliferation rate was observed only in 43℃ group (P<0.001), and no difference in the rate of proliferation could be observed between the heat stress groups and normal control group after 24h recovery. Flow cytometry showed, that the phagocytosis of KCs decreased in heat stress groups compared with control group, especially in 43℃ group (P<0.05). This phenomenon disappeared after 24h recovery. Conclusion Heat stress can inhibit the phagocytosis of rat liver KCs through its cytotoxic effect on KCs, and subsequently inhibits its proliferative ability. Further investigation of the effect of heat stress on KCs may help understand the pathogenesis of heat stress.

Objective To investigate the protective effect of heat shock protein (HSP) 70 on the acute lung injury (ALI) of rats with heat stroke.Methods Sixty four rats were randomly (by employing a random number table) assigned into a sham-heated group (Sham group),heat stress group (HS group),and HS plus gluttamine treatment group (HS+GLN group) and HS plus quercet in treatment group (HS+QU group),16 each.All rats were housed in a artificial climate chamber,with the rats in the sham groups exposed to a temperature of 23 ℃ and humidity of 55％ ± 5％,while the rats of HS,HS+GLN and HS+QU groups to an ambient temperature of 39 ℃ and humidity of 65％.During heat stress or sham heating,rectal temperature (Tr),systolic blood pressure (SBP) and pulse rate (PR) were monitored to observe the difference in heat stress response among the groups.The time point at which the SBP started to drop from the peak level was taken as the point of HS onset.At the onset of HS,heat exposure was terminated,then the rats were immediately removed from the chamber,and returned to room temperature.The rats were scarified 0h and 6h after HS onset respectively.After bronchoalveolar lavage fluid (BALF) was collected,the lungs of all animals were harvested for pathological examination of lung injury.The concentrations of IL-1β,TNF-α and IL-6 in BALF and HSP70 in lung homogenate were measured by using an enzyme linked immunosorbent assay kit.Results Compared with HS and HS+QU groups,the rats in HS+GLN group required significantly greater heat load to induce HS (P＜0.001),and had longer survival time span after HS onset.Compared with Sham group,the concentration of HSP70 in lung homogenate in HS group increased in a time-dependent manner (P＜0.001).In comparison with HS group,the concentration of HSP70 in lung homogenate from HS+GLN group was significantly elevated at each time point (P＜0.001),while the treatment with QU significantly inhibited the expression of HSP70 (P＜0.001).The concentration of IL-1β,TNF-α and IL-6 in BALF significantly decreased in HS+GLN group compared with those in HS group and HS+QU group (P＜0.001).The pathological results showed that the lung injury was milder in HS+GLN group,while the opposite in HS+QU group.Conclusion HSP70 could protect HS rats against ALI by enhancing their thermo-tolerance and inhibiting inflammatory response.

Objective To investigate the role of oxidative stress in acute liver injury in a heat stroke model of conscious rats,and to explore its underlying mechanism.Methods Thirty-two rats were randomly (by using a random number table) assigned into a sham-heated control group (Sham group,n=8),a sham-heated group treated with NAC (Sham-NAC group,n=8),a heat stroke group (HS group,n=8) and a heat stoke group treated with NAC (HS-NAC,n=8).Rats were prepared with pre-warm chamber to initiate heat stoke.The change of rectum temperature (Tr),heart rate (HR) and systolic blood pressure (SBP) were monitored,and the time point of HS onset was recorded.Rats were sacrificed 12h after HS onset.ALT,serum TBIL,IL-6,IL-1β,TNF-α,MDA,T-SOD and GSH in the liver homogenates were measured.Liver tissues were harvested for determining the concentration of reactive oxygen species (ROS),neutrophil infiltration and the histological changes.Results During HS onset,no significant differences were observed in Tr,HR,SBP and heat exposure time between HS group and HS-NAC group (P＞0.05).However,the survival time was significantly longer in HS-NAC group than in HS group (P=0.039).12 hours after HS onset,the concentrations of ROS and MDA in the liver homogenates were significantly higher in HS group than in the other groups (P=0.000),while the concentrations of T-SOD and GSH were much lower than in the other groups (P=0.000).The serum concentrations of ALT and TBIL were significantly higher in HS group than in the other groups (P=0.000).Compare with HS group,the pathological injury was alleviated in HS-NAC group (P=0.000).The neutrophil infiltration level and the concentrations of IL-6,IL-1 β and TNF-α in liver tissue were significantly higher in HS group than in HS-NAC group (P=0.000).Conclusion Oxidative stress may play an important role in the pathogenesis of HS liver injury through its cytotoxic effect and by inducing inflammatory responses.