Lentiviral vectors(LVs)are key gene delivery tools for integrating target genes into the host genome,but they may also pose risks of insertional mutagenesis.The vector copy number(VCN)in cells is critical for determining the safety of gene modification.However,the reliability and accuracy of its quantification process are influenced by multiple factors.Developing cell reference materials with specific vector copy numbers represents a viable approach to enhance the reliability and consistency of measurement results,enabling quality control of the quantification process and traceability of outcomes.However,the preparation of such reference materials faces challenges in cell sample design,preparation protocols,and advanced quantification techniques.In this study,T lymphocyte cell line Jurkat-based reference materials with LV gene copy numbers of 1 and 2 copy/cell were developed.A high-precision duplex digital polymerase chain reaction(dPCR)method was established to quantify the LV gene and endogenous genes simultaneously.Additionally,the results of dPCR were cross-validated through next-generation sequencing and flow cytometric analysis.Ultimately,confocal microscopy characterization results showed that the developed cell reference materials had intact morphology.The quantification result of VCN-1 was(1.07±0.11)copy/cell,and that of VCN-2 was(2.09±0.21)copy/cell.These cell reference materials demonstrated compliance with stability and homogeneity requirements,and could be applied for quality control throughout the VCN measurement workflow and metrological traceability,improving the accuracy,comparability,and validity of copy number measurements.

Objective To evaluate platelet aggregation after 14 days of treatment with ticagrelor alone or aspirin plus ticagrelor in patients with stable coronary artery disease.Methods Patients with stable coronary artery disease receiving dual antiplatelet therapy with aspirin 100 mg/d plus clopidogrel 75 mg/d were randomly assigned to ticagrelor monotherapy group or ticagrelor plus aspirin group after washout of ticagrelor monotherapy.Platelet aggregation rate was measured after 7 days and 14 days.Results 57 patients(32 in Ticagrelor monotherapy group and 25 in Ticagrelor+Aspirin group)completed the follow-up.The mean age was(61.16±7.87)years.The platelet aggregation rates induced by arachidonic acid(AA),adenosine diphosphate(ADP),and collagen-induced were similar at baseline and randomization in both groups.After 14 days of treatment,the ADP-induced platelet aggregation rates in both groups were significantly lower than the baseline,but there was no statistical difference between the two groups[(21.50±7.86)％vs.(21.92±10.21)％,P=0.864].The ADP-induced platelet aggregation rates in both groups decreased compared to randomization,but the Ticagrelor monotherapy group was significantly higher than the Ticagrelor+Aspirin group[(48.22±24.01)％vs.(8.67±5.96)％,P＜0.001].In the monotherapy group,5 patients had a significant increase in the AA-induced platelet aggregation after the discontinuation of aspirin.After 14 days of continued Ticagrelor monotherapy,the AA-induced platelet aggregation rate decreased significantly again(＜30％).Conclusions Ticagrelor monotherapy exerts a strong inhibitory effect on ADP-induced platelet aggregation and a mild inhibitory effect on the AA-induced platelet aggregation.In addition to its strong inhibitory effect on ADP-induced platelet aggregation,the mild inhibitory effect of Ticagrelor on the AA-induced pathway may have some clinical significance for the early de-escalation of dual antiplatelet therapy to monotherapy in patients.

Objective To evaluate platelet aggregation after 14 days of treatment with ticagrelor alone or aspirin plus ticagrelor in patients with stable coronary artery disease.Methods Patients with stable coronary artery disease receiving dual antiplatelet therapy with aspirin 100 mg/d plus clopidogrel 75 mg/d were randomly assigned to ticagrelor monotherapy group or ticagrelor plus aspirin group after washout of ticagrelor monotherapy.Platelet aggregation rate was measured after 7 days and 14 days.Results 57 patients(32 in Ticagrelor monotherapy group and 25 in Ticagrelor+Aspirin group)completed the follow-up.The mean age was(61.16±7.87)years.The platelet aggregation rates induced by arachidonic acid(AA),adenosine diphosphate(ADP),and collagen-induced were similar at baseline and randomization in both groups.After 14 days of treatment,the ADP-induced platelet aggregation rates in both groups were significantly lower than the baseline,but there was no statistical difference between the two groups[(21.50±7.86)％vs.(21.92±10.21)％,P=0.864].The ADP-induced platelet aggregation rates in both groups decreased compared to randomization,but the Ticagrelor monotherapy group was significantly higher than the Ticagrelor+Aspirin group[(48.22±24.01)％vs.(8.67±5.96)％,P＜0.001].In the monotherapy group,5 patients had a significant increase in the AA-induced platelet aggregation after the discontinuation of aspirin.After 14 days of continued Ticagrelor monotherapy,the AA-induced platelet aggregation rate decreased significantly again(＜30％).Conclusions Ticagrelor monotherapy exerts a strong inhibitory effect on ADP-induced platelet aggregation and a mild inhibitory effect on the AA-induced platelet aggregation.In addition to its strong inhibitory effect on ADP-induced platelet aggregation,the mild inhibitory effect of Ticagrelor on the AA-induced pathway may have some clinical significance for the early de-escalation of dual antiplatelet therapy to monotherapy in patients.

OBJECTIVE: To investigate the clinical characteristics, risk factors and prognosis of ulcerative keratitis (UK) in patients with rheumatoid arthritis (RA). METHODS: Sixteen patients with UK (the UK group) were screened among 4 773 RA patients hospitalized at Peking University People's Hospital from January 2003 to May 2021, and 72 patients with RA without UK in the control group were selected by propensity score matching (PSM). The clinical features, laboratory data and medications between the two groups were collected and analyzed. RESULTS: The average age of the patients with RA was 60 years at the time of the complication of UK. UK appeared on average in the 16th year of RA, with the majority of UK occurring in the 7th year of RA and beyond (14 cases), however, a minority of UK was the first symptom of RA (1 case). UK associated with RA manifested as simple marginal UK in 8 cases, marginal UK with perforation in 5 cases, marginal UK with uveitis in 2 cases and central UK with perforation in 1 case. The number of swollen joints was significantly higher in the UK group than in the control group [6.0(2.5, 23.0) vs. 3.0(1.0, 9.8), Z=-2.047, P=0.041], and the proportion of secondary Sjögren syndrome (SS, 37.5% vs. 6.9%, χ²=11.175, P=0.004) and interstitial lung disease (ILD, 37.5% vs. 8.3%, χ²=9.456, P=0.008) were significantly higher in the RA patients with UK than the patients without UK. The uses of sulfasalazine (12.5% vs. 48.6%, χ²=7.006, P=0.008), leflunomide (31.3% vs. 63.9%, χ²=5.723, P=0.017) and gold salt(6.3% vs. 33.8%, χ²=4.841, P=0.032)were significantly lower in the UK group than in the control group. However, there was no statistically significant comparison between the two groups for the 28-joint disease activity score-erythrocyte sedimentation rate (5.3±1.8 vs. 5.1±1.6, t=0.309, P=0.761). Logistic analysis showed the number of swollen joints (OR=1.148), secondary SS (OR=79.118), complicated with ILD (OR=6.596) and the use of sulfasalazine (OR=0.037) were independent relevant factors (P < 0.05) for UK in RA patients. CONCLUSION The number of swollen joints, secondary SS, complicated with ILD and the use of sulfasalazine are independent relevant factors for UK in patients with RA.
Arthritis, Rheumatoid/epidemiology* ; Corneal Ulcer/etiology* ; Humans ; Middle Aged ; Minority Groups ; Risk Factors

OBJECTIVETo determine whether erythropoietin (EPO) promotes rapid proliferation of glioma through Akt pathway.

METHODSWe detected the expression of EPO in human glioma tissues using immunohistochemistry. A nude mouse model bearing human glioma U87 cell xenograft was established and given intraperitoneal injection of EPO or saline every other day, and the tumor growth was observed. In the in vitro experiment, U87 cells were treated with PBS (control), EPO, or EPO with Akt inhibitor, and the expression of p-Akt and cyclin D1 was detected using Western blotting; the cell proliferation rate was determined using cell counting kit-8 and clone formation assay, and the cell cycle changes were analyzed with flow cytometry.

RESULTSCompared with low-grade glioma tissues, high-grade glioma tissues exhibited a significantly increased EPO expression (P=0.0002). In the tumor-bearing mice, EPO treatment significantly increased the expression of EPO (P=0.0006) and p-Akt (P=0.0003) in the tumor and obviously increased the tumor volume (P<0.0001) and weight (P=0.0003). In U87 cells cultured in vitro, EPO treatment obviously accelerated the cell proliferation (P=0.020 on day 3 and 0.028 on day 5), promoted clone formation (P=0.0010), and increased proliferation index (P=0.0028); EPO significantly enhanced the protein expression of p-Akt (P=0.0020) and cyclin D1 (P=0.0022). The application of Akt inhibitor significantly suppressed the effect of EPO in enhancing cyclin D1 and p-Akt expression (both P<0.0001) and promoting cell proliferation.

CONCLUSIONEPO can significantly accelerate the proliferation of glioma through Akt pathway.

Objective To observe the effects of aerobic training and aerobic combined with resistance training on motor function, like muscle strength,cardiopulmonary endurance and so on,in patients with chronic kidney disease(CKD). Methods From July,2015 to August,2016,60 patients with CKD were randomly divided into control group(A,n=20), aerobic training group(B,n=20)and aerobic combined with resistance training group(C,n=20).Group B per-formed cycle ergometer at 50% peak oxygen uptake(VO2peak)for 30 minutes a time,and group C performed one section of Thera-Band resistance training based on group B, three times a week for twelve weeks.All patients were evaluated with one repetition maximum-upper limb (1 RM-U), one repetition maximum-lower limb (1 RM-L),Cardiopulmonary Exercise Test(CPET),Arm Curl Test(ACT),30-second Chair Stand(CS-30),Six-Min-ute Walk Test(6MWT),and estimated gomerular filtration rate(eGFR)and serum creatinine(sCr)were calculat-ed and recorded before and after training. Results There was no significant difference in all indexes among three groups before training(F<1.841,P>0.05).After training,all indexes improved in groups B and C(t>2.162,P<0.05),and were better in groups B and C than in group A(t>2.132, P<0.05).After training, 1 RM-U, 1 RM-L, VO2peak,ACT, CS-30 and 6MWT were better in group C than in group B(t>2.081,P<0.05). Conclusion Aerobic training could improve the motor function of patients with CKD,and it is more effective combined with resistance training.

Objective To investigate the differences in dosimetry between conformal radiotherapy (CRT),field-in-field intensity-modulated radiotherapy (FIF-IMRT),and intensity-modulated radiotherapy (IMRT) after breast-conserving surgery for left-sided breast cancer.Methods A total of 31 patients who underwent breast-conserving surgery for left-sided breast cancer were randomly selected,and the plans for CRT,FIF-IMRT,and IMRT were developed.The dose-volume histogram (DVH) was used for self-control study,and the non-parametric test was used to compare the differences in target volume and doses to organs at risk (OARs).Results All the three methods met the requirements of the prescribed doses.The CRT group had a higher V105 of the target volume and higher heart V30 and Dmax (P=0.000,0.000,0.000).The IMRT group had higher V5 and Dmean (P=0.000,0.000),as well as a higher lung V5 and a lower lung V40 (P=0.000,0.000).The FIF-IMRT group had the lowest Dmean (P=0.000),and the IMRT group had significantly higher Dmean and Dmax of the right lung and the spinal cord than the other two groups (P=0.000,0.000,0.000,0.000).The FIF-IMRT group had a significantly lower single hop count than the other two groups (P=0.000).Conclusions CRT has a good dose distribution in the target volume,but greatly injures the surrounding tissues.FIF-IMRT can well protect OARs and cause less damage to the device.

OBJECTIVETo observe the clinical effect and safety of circumcision stapler in the treatment of phimosis and redundant prepuce.

METHODSWe treated 120 patients with redundant prepuce or phimosis using circumcision stapler and another 60 by conventional dorsal-incision circumcision. We observed intraoperative blood loss, operation time, postoperative pain, wound healing time, cosmetic appearance of the penis, and postoperative complications and compared them between the two groups of patients.

RESULTSStapler circumcision showed obvious advantages over the conventional method in intraoperative blood loss ([2. 3 ± 1. 3] vs [15.6 ± 2.9] ml), operation time ([7.1 ± 1.4] vs [22.6 ± 4.6] min), wound healing time ([12.0 ± 2.9] as [16.3 ± 3. 1] d), postoperative pain score (1. 9 ± 1. 3 vs 5. 2 ± 1. 7), incision edema, and cosmetic appearance of the penis (all P <0. 05). Besides, stapler circumcision exempted the patients from stitch-removal pain. However, the incidence rate of postoperative local ecchymosis was significantly higher in the circumcision stapler group than in the conventional circumcision group (20. 8% vs 8. 3% , P <0. 05).

CONCLUSIONCircumcision stapler, with its advantages of easier manipulation, shorter operation time, better cosmetic penile appearance, less pain, and fewer complications, is superior to conventional circumcision in the treatment of phimosis and redundant prepuce.

Blood Loss, Surgical ; Circumcision, Male ; instrumentation ; methods ; Ecchymosis ; etiology ; Humans ; Male ; Pain, Postoperative ; Penis ; abnormalities ; Phimosis ; therapy ; Postoperative Complications ; Postoperative Period ; Surgical Staplers ; adverse effects ; Wound Healing

OBJECTIVETo investigate the expression and distribution of mast cell tryptase (MCT) in scar, and to discuss the different MCT gene expression in keloid, hypertrophic scar and normal skin.

METHODS20 samples of keloid, 20 samples of hypertrophic scar and 20 samples of normal skin were collected. The distribution of MCT was investigated by immunofluorescence histochemistry, and the MCT mRNA expression was detected by Relative Quantification real-time fluorescent PCR.

RESULTSMCT gene was mainly located in the collagen fiber bundles of the scar, especially in the superficial layer of scar. MCT mRNA expression was significantly higher in keloid than that in hypertrophic scar and normal skin (P < 0.01). Averagely, the MCT gene expression in keloid was 2.5 times and 5.4 times of that in hypertrophic scar and normal skin.

CONCLUSIONSMCT gene may play a role in the pathogenesis of scar.

Adolescent ; Adult ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Humans ; Keloid ; metabolism ; pathology ; RNA, Messenger ; genetics ; Skin ; metabolism ; pathology ; Tryptases ; genetics ; metabolism ; Young Adult

OBJECTIVETo investigate the relationship between thrombolysis in myocardial infarction (TIMI) risk score and efficacy of different treatment strategies in patients with non-ST-segment elevation acute coronary syndromes (NSTE-ACS).

METHODSFrom Oct. 2001 to Oct. 2003, 545 consecutive patients with NSTE-ACS were randomly assigned to early conservative strategy (n = 284) or early invasive strategy group (n = 261). The combined cardiovascular events (a combination of cardiac death, nonfatal myocardial infarction, nonfatal heart failure and re-hospital admission due to recurrent ischemia angina) within 30 days and 6 months were analyzed and related to the TIMI risk score at admission.

RESULTSRehospitalization due to recurrent ischemia angina of 30 days and the combined cardiovascular events of 30 days and 6 months were significantly lower in early invasive strategy group (3.5%, 10.0%, 21.1%) compared with early conservative strategy group (8.1%, 16.9%, 28.2%, all P < 0.05). Subgroup analysis indicated early invasive strategy could significantly decrease the 30 d incidence of the combined end point events in patients with high TIMI risk score and the 6 months incidence of the combined end point events in patients with moderate and high TIMI risk score (all P < 0.01), but the incidence was similar between the two different strategies in patients with low TIMI risk score.

CONCLUSIONSEarly invasive strategy may significantly reduce combined cardiovascular events in NSTE-ACS patients with moderate and high TIMI risk score compared with early conservative strategy.

Aged ; Angina, Unstable ; drug therapy ; physiopathology ; Coronary Disease ; drug therapy ; physiopathology ; Electrocardiography ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; drug therapy ; Risk Assessment ; Thrombolytic Therapy