OBJECTIVE: To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology. METHODS: We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated. RESULTS: This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively. CONCLUSION By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.
Humans ; COVID-19 ; CRISPR-Cas Systems ; Genotype ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics* ; RNA ; COVID-19 Testing

Objective: To explore the clinical application value of deep convoluti onal neural network for automatic detection and classification of benign and malignant thyroid nodules ultrasound images. Methods: A total of 1 012 ultrasound images of thyroid nodules were retrospectively selected and labeled. The YOLOv5 network model was constructed to accurately locate the location of thyroid nodules and automatically trim the area of the nodules. At the same time , a GoogLeNet network model was constructed to classify benign and malignant nodules after reduction. Results : In the collected data set , the mean accuracy of the target detection network for thyroid nodule location detection was 96. 2% . Meanwhile , the sensitivity, specificity, accuracy and AUC of the classification network for benign and malignant nodules were 0. 885 ,0. 822 ,0. 866 and 0. 92 respectively ,which were significantly higher than those of the AlexNet model (0. 81) , VGG model (0. 86) and MobileNet model (0. 76) . Conclusion The deep convo⁃ lutional neural network model has high localization and recognition ability for benign and malignant thyroid nodules in ultrasound images ,which is helpful to improve the accuracy of automatic image diagnosis.

Vascular damage is followed by vascular endothelial growth factor (VEGF) expression at high levels, which is an important mechanism for cerebral radiation necrosis (CRN) development. Antiangiogenic agents (Bevacizumab) alleviates brain edema symptoms caused by CRN through inhibiting VEGF and acting on vascular tissue around the brain necrosis area. Many studies have confirmed that Bevacizumab effectively relieves symptoms caused by brain necrosis, improves patients' performance status and brain necrosis imaging. Considering that the efficacy of antiangiogenic therapy is mainly related to the duration of drug action, low-dose antiangiogenic agents can achieve favorable efficacy. Prevention is the best treatment. The occurrence of CRN is associated with tumor-related factors and treatment-related factors. By controlling these factors, CRN can be effectively prevented.
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Angiogenesis Inhibitors/pharmacology* ; Bevacizumab/therapeutic use* ; Brain/metabolism* ; Consensus ; Humans ; Lung Neoplasms/drug therapy* ; Necrosis/etiology* ; Radiation Injuries/etiology* ; Vascular Endothelial Growth Factor A/metabolism*

Objective: To compare the clinical effects of endoscopic laminectomy with traditional hemilaminectomy for lumbar spinal stenosis. Methods: From January 2016 to April 2017, 61 patients with lumbar spinal stenosis were treated surgi-cally. Percutaneous endoscopic laminectomy was performed in 32 patients (minimally invasive group), including 13 males and 19 females, aged 38-76 years, with an average age of 58.47±7.51 years. Twenty-nine patients (open group) underwent hemilaminecto-my, including 11 males and 18 females, aged 38-75 years, with an average age of 57.17±9.99 years. The operation time, bleeding, incision length, bedridden time and hospitalization time were recorded. Visual analogue scale(VAS), Oswestry disability index (ODI), dural sac cross-sectional area (DSCA), ventral intervertebral space height (VH), dorsal intervertebral space height (DH) and lumbar mobility (range of motion, ROM) were compared between the two groups. Results: All of 61 patients were followed up for 14 to 27 months, with an average of 19.2±2.95 months. The operation time was 60.88±6.49 min in the minimally invasive group, and 52.07±9.45 min in the open group (t=4.277, P=0.000). The blood loss of minimally invasive group was 55.63±10.14 ml, and that of open group was 78.79±12.58 ml (t=7.952, P=0.000). The incision length of minimally invasive group was 23.31±4.56 mm, and open group 82.59±7.66 mm (t=12.047, P=0.000). Bed rest time was 21.97±6.42 h in minimally invasive group and 78.79±12.58 h in open group (t=12.047, P=0.000). The hospitalization time of the minimally invasive group was 8.53±2.75 d and the open group 11.34±3.12 d (t=3.745, P=0.000). All these parameters had statistical significance (P<0.05). At the last follow-up, the VAS score of minimally invasive group was 1.06±0.56, and the open group was 1.14±0.74 (t=0.469, P=0.634). ROM of open group was 5.66±1.12 degree, and ROM of minimally invasive group was 5.56±1.13 degree (t=0.140, P=0.710), VH of minimally invasive group was 14.75±2.81 mm, and open group was 14.44±2.89 mm (t=0.181, P=0.672). There was no significant difference between the two groups for these parameters. At the last follow-up, ODI score was 13.25%±1.08% in the minimally invasive group and 14.28%±2.10% in open group (t=5.911, P=0.018). DSCA score was 108.56±8.69 mm² in the minimally invasive group, and 117.28±11.09 mm² in open group (t=11.774, P=0.001). There were significant differences between the two groups for ODI and DS-CA. Conclusion Both endoscopic and open laminectomy have excellent clinical effects on lumbar spinal stenosis. Endoscop-ic laminectomy has the advantages of less local trauma, less damage to the stability of the lumbar spine and faster recovery. However, there's a higher technical requirement for endoscopic spine surgery.

Objective To study the changes of serum brain natriuretic peptide(BNP),uric acid(UA)and procalcitonin(PCT)levels in children patients with dilated cardiomyopathy(DCM).Methods 24 children pa-tients with DCM and 24 children undergoing healthy physical examination from March 2015 to September 2016 were selected as the observation group and control group.The observation group was divided into differ-ent Ross grade groups according to the improved Ross heart failure grade scoring.The levels of serum BNP, UA and PCT in each group were detected and their correlation with the main echocardiographic indicators was analyzed.Results The levels of BNP,UA and PCT had statistical difference between the observation group and control group(P<0.05);the BNP and UA levels had statistical difference among different Ross grades (P<0.05),moreover which were increased along with the severity increase,but the PCT level had no statisti-cally significant(P> 0.05);the BNP level was positively correlated with the DCM severity(r= 0.713,P=0.000),the UA level was positively correlated with the DCM severity(r=0.489,P=0.002),while the corre-lation between the PCT level and DCM severity had no statistical significance(r=0.288,P=0.076);the BNP had obvious correlation with main echocardiographic indicators(P<0.05).Conclusion The levels of serum BNP,UA and PCT in children patients with DCM are changed significantly,in which BNP and UA may be the good markers for reflecting the disease severity and progression or prognosis.

Objective To investigate the utilization rate of gold fiducial markers and reasons for abandonment of gold fiducial markers in the CyberKnife VSI System, and to provide reference data for implantation of gold fiducial markers and radiotherapy planning. Methods From March to August,2017,a total of 47 patients had gold fiducial markers implanted or pasted. In those patients, 42 patients had gold fiducial markers implanted,including 32 receiving computed tomography(CT)-guided 3D-printing coplanar template assisted implantation, 1 receiving CT-guided 3D-printing non-coplanar template assisted implantation,1 receiving CT-guided implantation,and 8 receiving ultrasound-guided implantation. A total of 44 patients received the CyberKnife treatment, including 2 patients who failed to use gold fiducial markers and were treated with spine tracking instead and 3 patients missing the treatment for other reasons. The numbers of utilized and abandoned gold fiducial markers were recorded for calculation of the utilization and abandonment rates. The reasons for abandonment of gold fiducial markers were analyzed and classified. Results A total of 134 gold fiducial markers were implanted into or pasted to the 44 patients.In all the gold fiducial markers, 111 were utilized and 23 abandoned, yielding a utilization rate of 82.8% and an abandonment rate of 17.2%.The reasons for abandonment of gold fiducial markers included large rigidity error(26.1%), unqualified implanted fold fiducial markers(17.4%), displacement of gold fiducial markers(26.1%), and others(30.4%). Conclusions Compared with the CT-guided or ultrasound-guided implantation of gold fiducial markers, the CT-guided 3D-printing coplanar or non-coplanar template assisted implantation of gold fiducial markers requires only two puncture needles for each implantation and implants two gold fiducial markers by a single needle,which reduces the number of puncture needles,risk of puncture-induced injury,and incidence of complications after implantation. Not all the gold fiducial markers implanted by a variety of ways will be utilized. Some gold fiducial markers will be abandoned for different reasons,which should be taken into account during implantation of gold fiducial markers.

Objective To establish a model for the calculation of biologically effective dose (BED) and EQD2 (Equivalent dose in 2 Gy fractions) in radioactive seed implantation brachytherapy.Methods The BED formula for EBRT(external beam radiotherapy) and for continuous low dose-rate irradiation established under the L-Q model were introduced.The EDQ2 formula for the continuous low dose-rate irradiation (radioactive seed implantation) was established according to the definition of EQD2 and the formula of BED.The α/β values of common tissues and the Tr 1/2 values reported in the literature were summarized.The EDQ2 formula were further simplified by using the actual values.The empirical formula of EDQ2 for early reaction tissues and late reaction tissues were proposed,named as Wang-Peng empirical formula.EDQ2≈ (10/12) D (Wang-Peng Formula 1) was fit for early response tissue,and EDQ2≈ D/2 (Wang-Peng Formula 2) for late reaction tissues.Further examples on the clinical applications of the proposed formula were given,including primary lung cancer,supraclavicular lymph node metastasis of esophageal cancer and celic lymph node metastasis of cervical carcinoma.Results According to the Wang-Peng empirical formula,the EDQ2 of the late reaction tissue adjacent to the tumor was only about half that of the tumor tissue,so the radioactive seed implantation brachytherapy naturally protected the late reaction tissue by the biological equivalent dose.The actual calculation,showed that the empirical formula of early reaction tissue was more accurate,but the empirical formula of late reaction orgtissue was less inaccurate and could only be roughly estimated.Conclusions The BED calculation formula introduced here and the set of EQD2 calculation formula and Wang-Peng empirical formula established here were theoretically feasible and could be used for the conversion and superposition between the physical dose of radioactive seed implantation brachytherapy and the external irradiation dose.But it should be careful to apply the formula,pay attention to the default conditions,and carefully interpret the calculated results.

Objective To analyze the sequence of the novel conjugative plasmid pO157_Sal detected in outbreak isolates of Escherichia coli O157∶H7 .Methods The traE genes of the outbreak isolates in China were amplified by polymerase chain reaction (PCR) and the products were sequenced .The TraE sequences of Escherichia coli O157 ∶ H7 strains from other sources were retrieved from GenBank . Phylogenetic tree based on the TraE sequences was constructed by Neibhor-joining analysis .The whole plasmid sequences of pO157_Sal and pEC4115 were compared .Results The sequences of traE gene were identical among the Chinese isolates . There were homologous sequences of TraE in Escherichia coli O157∶H7 isolates from different sources .Twenty-one out of the 52 pO157_Sal genes were homologous to genes of pEC4115 with amino acid level identity ranging from 28% to 51% .Conclusions Although similar TraE sequences and similar plasmid are found in Escherichia coli O157∶H7 isolates from different sources ,pO157_Sal is only observed in Chinese outbreak isolates .The TraE sequences are conservative among the outbreak isolates ,indicating they are from the same specific source .

To explore and compare the response of the protease inhibitors or non-nucleoside reverse transcriptase inhibitors-based therapeutic impact on metabolic indices in hepatitis C virus (HCV)/human immunodeficiency virus(HIV) co-infected patients.A randomized,open,and control approach was performed to enroll 273 cases of HCV/HIV co-infected patients on their initial visits and to choose protease inhibitors(PIs group) or non-nucleoside reverse transcriptase inhibitors (NNRTIs group) based therapy treatments for one year.Laboratory results of metabolic indices before and after the treatment were collected.After treatment,the levels of triglyceride in NNRTIs and Pls groups were (1.93 ± 0.99) mmol/L and (1.62 ± 0.93) mmol/L respectively,high density lipoprotein-cholesterol were(1.28 ± 0.55) mmol/L and (1.08 ± 0.53) mmol/L,low density lipoprotein-cholesterol were (2.60 ± 1.44) mmol/L and (2.22 ± 1.16) mmol/L,fasting plasma glucose were (5.92 ± 1.21) mmol/L and (4.79 ± 0.47) mmol/L,serum creatinine were (70.5 ± 14.6) μmol/L and (56.6 ± 8.3) μmol/L,and serum amylase were(66.9 ± 27.5) U/L and(62.7 ± 33.8) U/L respectively.The difference between the two groups was statistically significant(all P＜0.01).There is a therapeutic impact on metabolic indices in patients wtih HCV / HIV co-infection after non-nucleoside reverse transcriptase inhibitors-based regimen.

Objective To investigate the role of epidermal growth factor receptor and cyclooxygenase-2 pathways in the erlotinib and celecoxib enhanced radiation sensitivity in A549 human lung cancer cell line. Methods IC20 of erlotinib and celecoxibon in A549 human lung cancer cells was measured by MTT assay,Clonogenic assays were used to evaluate the antitumor effects of the drugs and Xirradiation.Flow cytometry was used to assess the apoptosis and cell cycle alteration,and Western blot was used for the detection of Akt and phospho-Akt.Results Both erlotinib and celecoxib could inhibit the proliferation of A549 cells in vitro in a dose-dependent manner and their values of IC20 were (5.15 ± 0.14)and (40.32 ± 1.26) μmol/L,respectively. For radiation survival,the values of Dq,Do,SF2 of the combination of two drugs were lower than those of either drug ( t =6.62,P ＜ 0.05).The SER of celecoxib,erlotinib and their combination were 1.299,1.503 and 2.217,respectively.Flow cytometry assay showed that both celecoxib and erlotinib could enhance radiation-induced G0/G1 arrest,reduce the cell numbmer in S phase,and enhance radiation-induced apoptosis,especially for the combination of drugs.Western blot assay showed that the expressions of Akt protein were similar in all groups.However,pAkt expression was suppresssed by erlotinib and celecoxib,but promoted by radiation.pAkt had the lowest expression in the radiated cells with the treatment of two drugs ( t =4.89,P ＜ 0.05 ).Conclusions The edotinib and/or celecoxib could enhance radiosensitivity probably by increasing cell apoptosis and reducing the number of Sphase cells with low radiosensitivity.