The CRISPR/Cas systems comprising the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an acquired immune system unique to archaea or bacteria. Since its development as a gene editing tool, it has rapidly become a popular research direction in the field of synthetic biology due to its advantages of high efficiency, precision, and versatility. This technique has since revolutionized the research of many fields including life sciences, bioengineering technology, food science, and crop breeding. Currently, the single gene editing and regulation techniques based on CRISPR/Cas systems have been increasingly improved, but challenges still exist in the multiplex gene editing and regulation. This review focuses on the development and application of multiplex gene editing and regulation techniques based on the CRISPR/Cas systems, and summarizes the techniques for multiplex gene editing or regulation within a single cell or within a cell population. This includes the multiplex gene editing techniques developed based on the CRISPR/Cas systems with double-strand breaks; or with single-strand breaks; or with multiple gene regulation techniques, etc. These works have enriched the tools for the multiplex gene editing and regulation and contributed to the application of CRISPR/Cas systems in the multiple fields.
Gene Editing ; CRISPR-Cas Systems/genetics* ; Bacteria/genetics* ; Archaea ; Bioengineering

Purpose To assess the feasibility of nuclear score combined with ultrasound features on indeterminate thyroid nodules in fine needle aspiration cytological diagnosis.Methods A consecutive cohort of 119 thyroid fine-needle aspiration(FNA)specimens with indeterminate diagnosis(TBSRTC cate-gory Ⅲ-Ⅴ)and available histopathologic follow-up was evalua-ted.Original sonographic images and cytological slides of each nodule were reviewed by two experienced sonographers and two experienced cytopathologists respectively,and evaluated jointly to obtain consensus.The identification of the optimal cut-off points of simplified nuclear score and ultrasound features score for the diagnosis of malignancy or low-risk neoplasm were evalu-ated using the receiver operating characteristic(ROC)curves and the assessment of the area under the ROC curve(AUC).The specificity,sensitivity,positive predictive value(PPV)and negative predictive value(NPV)of nuclear score and US score were evaluated from crosstabs based on Cut-off points and signif-icance were calculated.The diagnostic accuracy of simplified nuclear score combined with ultrasound features was estimated by Logistic regression and ROC curve analysis.Results Nucle-ar grooves,intra-nuclear inclusions and chromatin clearing were more common in malignancy/low-risk neoplasms(P = 0.001,0.013 and 0.001 respectively).A Cut-off point of≥2 for the simplified nuclear score was sensitive for defining malignancy/low-risk neoplasm,and the sensitivity was 99.1%.Nodule height greater than width,absent halo sign,irregular margin and absent intranodular vascularization were more common in malig-nancy/low-risk neoplasms(P = 0.003,0.007,0.003 and 0.004 respectively).When the Cut-off point was set as≥5,the PPV and specificity for defining malignancy/low-risk neoplasm were 95.9%and 78.6%respectively.ROC curve analysis showed that the diagnostic accuracy of nuclear score combined with US score(0.873,95%CI = 0.777-0.970)was higher than that of nuclear score alone(0.817,95%CI = 0.687-0.948,P =0.092)or US score alone(0.758,95%CI 0.637-0.879,P =0.047).But no significant difference was detec-ted between the combined score and the nuclear score.Conclu-sion The addition of simplified nuclear score and US score to FNA cytology can increase the diagnostic accuracy of cytology in indeterminate categories.

Objective: To explore the role of serum microRNA (miRNAs) levels in the detection of pneumoconiosis, and to establish a combined application model of multiple serums miRNAs for pneumoconiosis diagnosis. Methods: 152 cases were selected in the case group and the control group respectively. The TaqMan Low Density Array method was used to screen out the candidate miRNAs for early screening of pneumoconiosis, and RT-qPCR was used to verify. According to the area under the curve (AUC) , the sensitivity and specificity of the candidate indicators were investigated. The logistic regression model was established by the two-class logistic regression model. Results: The expression of 7 candidate miRNAs in the serum of pneumoconiosis patients was significantly different (P<0.05) . The receiver operating curve (ROC) of the above 7 miRNAs was analyzed, miRNA-21, miRNA-200c, miRNA-16, miRNA-206, miRNA-155, miRNA-29a had statistical significance, and their ROC-AUC is 0.629~0.932. Logistic regression model was: logitP=13.769+0.536×miRNA-21-0.878×miRNA-200C-0.012×miRNA-16-0.111×miRNA-206+0.117×miRNA-155-1.192×miRNA-29a. Conclusion Multiple serum miRNAs combined application models may be used for the diagnosis of pneumoconiosis patients.

Objective: To explore the therapeutic effects of dendritic cells (DC) modified by the dust-mite-allergen(Der p1) gene on mouse model of allergic rhinitis (AR). Methods: DC modified by the Der p1 gene (Der p1-DC) were prepared.Using random number table, 24 Balb\c mice were divided into four groups: immature DC (imDC)/AR group, dexamethasone/AR group, Der p1-DC/AR group and control group, with 6 mice in each group.AR mouse model was built with Der p1 and the mouse model of AR was established.The AR mice were respectively given by abdominal injection of Der p1-DC, imDC and dexamethasone.Normal control mice were treated with physiologic saline.ELISA method was used for determining the content of IgE, IgG1and histamine in blood.The relative expression of mRNA of IL-4 and IL-13 on nasal mucosa with protein was analyzed by RT-PCR and Westen blot methods.All the data were statistically analyzed by SPSS 19.0 statistical software, and the variance analysis was used in multiple groups of average samples. Results: The contents of IgE, IgG1 and histamine in the mice of Der p1-DC/AR group were lower than those in imDC/AR group ((0.560±0.110) OD 450 nm vs (1.150±0.280) OD 450 nm, (0.690±0.054) OD 450 nm vs (0.920±0.125) OD 450 nm, (4 145±670) pg/ml vs (7 685±669) pg/ml, t value was 4.80, 4.14, 9.16, respectively, all P<0.05), and the expression of IL-4 and IL-13 on nasal mucosa in Der p1-DC/AR group was remarkedly lower than those in imDC/AR group (0.41±0.25 vs 1.59±1.02, 0.26±0.01 vs 1.10±0.09, t value was 2.75, 22.72, respectively, all P<0.05). There was no statistically significant difference between the mice treated with Der p1-DC and dexamethasone group. Conclusions The results showed that Der p1-DC could reduce inflammation in AR mice and decrease the expression of IL-4 and IL-13. It suggested that Der p1-DC can be used in the immunotherapy of AR mouse.

Objective: To study the effects p-phenylenediamine (PPD) on lung function and health-related quality of life of occupational exposed workers. Methods: This study was based on data from a company that produce hair dye containing PPD in China. Workers who exposed to PPD were selected as the study group, and workers un-exposed to PPD were selected as the control group. Questionnaires on health-related quality of life of workers using the 36-item Short Form Health Survey (SF-36) . Occupational health examination assessment results were tested in Taizhou Cancer Hospital. The lung function test includes forced vital capacity (FVC) , forced expiratory volume in one second (FEV_1.0) , and ratio of FEV_1.0 to FVC (FEV_1.0/FVC) . Results: The difference in systolic blood pressure between the PPD exposed group and the control group was statistically significant (P<0.05) . FVC, FEV_1.0, and FEV_1.0/FVC of the lung function indexes in the exposed group were lower than those in the control group (P<0.05) . In the health-related quality of life, body pain (P=0.002) , general health (P=0.029) , vitality (P=0.038) , and mental health (P=0.003) were lower in the exposed group than in the control group. Conclusion Occupational exposed to PPD may induce hazard to the workers’lung function and may cause detrimental effect on workers’ health-related quality of life.

Objective: To study the effect of p-phenylenediamine (PPD) on liver and kidney function in occupational exposed workers. Methods: Workers in a hair dye production enterprise which used p-phenylenediamine as a raw material for production were selected as the main research population. Then we conducted a questionnaire survey on the basic conditions of workers and conducted occupational health checkups on general health status, liver and kidney function. Occupational health examination assessment results were tested in Taizhou Cancer Hospital. All data was built using EpiData 3.1 software, and statistical analysis was performed using software SPSS 20.0. Results: The liver function indicators including direct bilirubin, prealbumin, total protein, and white protein, globulin, aspartate aminotransferase, glutamyl transpeptidase, and total bilirubin in the workers exposed to high concentration of PPD were at high normal values, and these indicators were significantly different from low PPD concentration group (P<0.05) . The serum creatinine and serum uric acid in the renal function index were significantly higher in workers exposed to PPD than in workers exposed to low concentrations and in the control group (P<0.05) . Conclusion Occupational exposed to PPD may have a hazard to the workers’ liver and kidney function. Long-term occupational exposure to PPD may lead to increased cumulative exposure of workers, which may cause potential chronic liver and kidney damage in occupationally exposed populations.

OBJECTIVETo evaluate the biomechanical effects of the anterior cervical transpedicular-screw system (ACTPS), compared to the anterior cervical screw plate system (ACLP), in the subaxial cervical spine after 2-level corpectomy.

METHODSA verified intact finite element subaxial cervical (C3-C7) model was established and analyzed by Mimics 10.0, Rapidform XOR3, Hypermesh 10.0, CATIA5V19, ANSYS 14.0 softwares based on the CT data (C1-T1) was collected from a 28 years old male volunteer. The axial force of 75 N and moment couple of 1N·m was loaded on the upper surface of C3, which made the model movement in flexion extension, lateral bending, rotating direction, respectively. Then, recorded the range of motion, and compared the results with the in vitro biomechanical experimental data to verify the effectiveness of the model. The ACTPS model and the ACLP model were analyzed using the finite element method. The range of motion at the operation segments (C4-C7), the range of motion at the adjacent segment (C3-C4) and stress distribution under flexion, extension, lateral bending, and axial rotation were calculated, and compared the range of motion with intact model.

RESULTSThere were 85,832 elements and 23,612 nodes in the intact model of subaxial cervical spine (C3-C7) in this experiment,and the range of motion of intact model validated with the reported cadaveric experimental data. In ACTPS group the stress was been well-distributed, but the stress concentrated on the interface between screw and the titanium plate in ACLP group. There were obvious differences of the maximum stress value between the two groups. The range of motion of fixed segments in ACTPS group was smaller than ACLP group, however adjacent segment range of motion showed no significant difference. Compared with the intact group, the range of motion in flexion extension, lateral flexion, rotation direction was decreased respectively about 25°, 20° and 8°, the range of motion at adjacent segment (C3-C4) was correspondingly compensated about 0.3°, 3° and 0.1°.

CONCLUSIONSACTPS is better than ACLP in terms of biomechanical properties. It offers rigid stability, and may be more suitable for reconstruction stability of 2-level and more than 2 levels corpectomy in the subaxial cervical spine. Meanwhile, the risk of fracture of ACTPS system is lower than that of the ACLP system.

Adult ; Biomechanical Phenomena ; Bone Screws ; Cervical Vertebrae ; surgery ; Finite Element Analysis ; Humans ; Male ; Models, Anatomic ; Range of Motion, Articular ; Rotation

OBJECTIVETo establish a method to determine total bromine in urine.

METHODDiluted urine samples were directly introduced into ICP-MS then quantized by standard curve.

RESULTTotal bromine in urine was linear within 1.0~50 mg/L with r > 0.999, When spiked at a concentration of 0.020 mg/L, 0.050 mg/L, 0.150 mg/L, the recovery was 95%~98%, intra-assay precision was 1.4% 3.2%, inter-assay precision was 3.4% to 5.0%. Urine could store in -20 °C refrigerator 3 months without any bromine loss.

CONCLUSIONUsing ICP-MS to determine the urinary total bromine, the method is fast, accurate, wide linear range of features, could meet with the requirement of Part 5 of occupational health standards guide: Method determination of chemical substances in biological materials (GBZ/T 210.5-2008), a strong competitive advantage in a wide range of survey, suitable for promotion.

Bromine ; urine ; Humans ; Mass Spectrometry

Acetylcysteine ; urine ; Humans ; Hydrocarbons, Brominated ; metabolism ; urine

Blood Glucose ; Humans ; Hydrocarbons, Brominated ; toxicity ; Occupational Exposure ; adverse effects