Selection markers are essential tools in gene editing, the utility of such systems is inherently constrained by species-specific limitations, governed by divergent host genetic backgrounds and metabolic compatibility. To address this limitation, we leveraged the CRISPR/Cas9 system to develop a universal counter-selection tool. We designed and introduced an sgRNA expression cassettes as counter-selection markers, which directs the Cas9 protein to target and cleave genomic DNA, allowing for the selection of the strains where the sgRNA expression cassette has been replaced. Optimized to target multiple copy sites with sgRNA, this system significantly enhances cell lethality, boosting counter-selection efficiency to over 85.00%. This counter-selection tool is not limited to single strains and is suitable for various scenarios, including multi-copy plasmid assembly and plasmid editing, demonstrating broad application potential.
CRISPR-Cas Systems/genetics* ; Gene Editing/methods* ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Plasmids/genetics*

The examinations of tear quality and volume are important indicators for the diagnosis of dry eye disease. Tests commonly used in clinical practice to diagnose dry eye disease include testing of tear volume and tear film stability to assess the severity of the condition, and analyzing and labeling of tear components under laboratory conditions to diagnose clinical staging and also to guide clinical supplementation of relevant components to target treatment. Accurate assessment of tear production and compositional changes enables clinicians to effectively monitor the severity of dry eye disease and evaluate the effectiveness of therapeutic measures. With the development of optical imaging technology and artificial intelligence in recent years, the combination of clinical tear indicators and examination techniques has enabled the application of new testing methods that provide more convenient, rapid and accurate testing, greatly improving the diagnosis and treatment of dry eye disease in the clinic.

AIM: To investigate the effects of hypobaric hypoxia in plateau on tear indexes and related anatomical structures in rabbits.METHODS: A total of 18 healthy New Zealand rabbits were selected and randomly divided into plateau group and control group, with 9 rabbits(18 eyes)in each group. The plateau group was housed in the Simulated Climate Cabin for Special Environment of Northwest of China, simulating hypobaric hypoxia at an altitude of 6 000 m. The control group was housed in a clean animal room with atmospheric pressure and oxygen. Changes in the tear meniscus height and non-invasive tear break-up time were detected by using RHCT-1 corneal topographer dry eye comprehensive analysis system, changes in tear secretion was measured by Schirmer Ⅰ test, before intervention and on the 3, 7 d, 2 and 4 wk. Meanwhile, the changes in tear composition before and after intervention in the plateau environment were analyzed using Raman Spectroscopy. The histopathological changes of the lower lid conjunctiva, cornea, lacrimal gland, and Hardarian gland were observed by hematoxylin-eosin(HE)staining after 4 wk of intervention, and the expression of mucin 5AC(MUC5AC)in conjunctiva was detected by immunohistochemistry.RESULTS: Compared with the control group, Schirmer Ⅰ test, tear meniscus height, first and average non-invasive tear break-up time in the plateau group decreased significantly since 3 d, and the difference was significant with the extension of observation time(P<0.05). The above indexes increased from 2 wk. After 4 wk of intervention, the protein and lipid content of the tear composition of rabbits in the plateau group increased, and the nucleic acid content decreased compared with the pre-intervention period. Compared with the control group, rabbits in the plateau group showed thickening of corneal stromal edema, an increase in the number of conjunctival cup cells, increase in the level of expression of MUC5AC, an increase in the level of expression of MUC5AC, an atrophy and flattening of cytoplasm in lacrimal epithelial cells, an enlargement of glandular lumen, and no obvious destructive changes in the Hardarian glands.CONCLUSION: Acute plateau environment can destroy the homeostasis of rabbit ocular surface, so that the tear secretion and the tear film stability decreases, but within a certain period of time, rabbits undergo compensation with the habituation to the hypobaric hypoxia environment, which can increase the tear secretion to a certain extent and restore the tear film stability.

To understand the pathogenicity,virulence genes,drug resistance genes,and drug resistance of Staphylococcus aureus isolated from yaks in some areas of Lhasa and Nagqu City,55 yak nasal swab samples were analyzed for S.aureus in this study.The cocci were isolated and identified,and the carriage of virulence genes and drug resistance genes,as well as the pathogenicity and drug sensitivity of the isolated S.aureus strains,were detected with PCR,artificially infected mice,and the K-B drug susceptibility disk method.Seven strains of S.aureus were isolated from the 55 yak nasal swab samples,with an iso-lation rate of 12.73％.The isolated strains were all pathogenic to mice,with a fatality rate exceeding 60％.These seven strains of S.aureus carried three drug resistance genes(tetM,tet,and mecA)and six virulence genes(seb,sec,clfA,hla,hlb,and nuc).The detection rate of the three drug resistance genes was 100％,whereas the detection rate of the six virulence genes in the isolates ranged from 83.33％to 100％.The resistance rates of the isolated strains to penicillin,ampicillin,and cotrimox-azole reached 85.71％-100％,whereas the resistance rates to tetracycline and ceftazidime were both 28.57％.Thus,yaks in Lhasa and Nagqu cities were found to be infected by S.aureus strains carrying various drug-resistance genes and virulence genes.These strains were highly pathogenic and showed sensi-tivity to most antibacterial drugs.These findings may serve as a reference for treating S.aureus infection in yaks in the cities of Lhasa and Nagqu.

Objective:To analyze the external quality control assessment results of urinary iodine, salt iodine and water iodine in iodine deficiency disorders laboratories in Qinghai Province, to evaluate the testing capacity of provincial, municipal and county-level laboratories and the operation of external quality control network, so as to provide quality assurance for consolidating and eliminating iodine deficiency disorders.Methods:In 2021, 1 provincial, 8 municipal, and 43 county-level laboratories in Qinghai Province had participated in the assessment of urinary iodine and salt iodine, while 1 provincial and 8 municipal-level laboratories had participated in the assessment of water iodine. The assessment results were evaluated using the method of reference value ± uncertainty of external quality control samples.Results:All laboratories that participated in the assessment had provided feedback. One provincial-level laboratory passed the assessment of urinary iodine, salt iodine, and water iodine. Among 8 municipal-level laboratories, 2 laboratories failed the urinary iodine assessment, with a pass rate of 6/8; the assessment of salt iodine and water iodine in 8 laboratories were all qualified. Among 43 county-level laboratories, 7 laboratories failed the urinary iodine assessment, with a pass rate of 83.7% (36/43); the assessment of salt iodine in 43 laboratories were all qualified.Conclusions:The external quality control network of iodine deficiency disorders laboratories in Qinghai Province has fully covered all municipal and county-level laboratories. The testing capability of provincial-level laboratory is stable and maintains a high level; the testing quality of some municipal and county-level laboratories is still unstable and needs to be further strengthened.

Objective:To verify the revised method of cerium sulfate catalytic spectrophotometry for iodide index of "Standard Examination Methods for Drinking Water-Nonmetal Parameters" (GB/T 5750.5-2006).Methods:From July to September 2019, the Laboratory of Department for Endemic Disease Prevention and Control of Qinghai Institute for Disease Prevention and Control verified the revised method (determination of iodide in drinking water by cerium sulfate catalytic spectrophotometry) of cerium sulfate catalytic spectrophotometry (hereinafter referred to as original method) in "Standard Examination Methods for Drinking Water-Nonmetal Parameters" (GB/T 5750.5-2006). The revised method was verified according to the requirements of "Standard Examination Methods for Drinking Water-Water Analysis Quality Control" (GB/T 5750.3-2006), including standard curve, detection limit, precision, accuracy and actual sample determination.Results:The linear range of the revised method was 0 - 20.0 μg/L, the correlation coefficient was - 0.999 4 - 0.999 8, and the detection limit was 0.231 μg/L. The relative standard deviation ( RSD) of low, medium and high iodine water samples of 6 times detection ranged from 1.4% to 9.6%, and the recoveries of low and medium water samples ranged from 89.0% to 108.0%. The detection results of national first-class reference materials for iodine composition analysis in water were within the range of standard value ± uncertainty. There was no significant difference in the test of results of 12 tap water samples between the revised method and the original standard method ( t = - 0.075, P > 0.05). Conclusion:The revised method has a good linear relationship of standard curve, high precision and accuracy, and good reproducibility, is simple and easy to operate, and is suitable for promotion and application.

OBJECTIVE：To establish the quality standard of the Tibetan medicine Ma lus Fructus （known as the fruit of Ese ）， and to provide scientific reference for quality control and utilization of resource. METHODS ：The qualitative analysis of 20 batches of Malus Fructus with different producing areas and different origins was carried out by morphological observation ，microscopic and TLC identification. According to the method stated in 2015 edition of Chinese Pharmacopoeia ，water content ，total ash content and extract content were determined. HPLC method was used to determine the contents of 6 kinds of flavones （chlorogenic acid ， hyperin，isoquercitrin，quercitin，phloridzin，quercetin）in Malus Fructus. RESULTS ：The properties of this medicinal material were obovate ，spheroidal or oval ，its surface was reddish brown to dark red ，with slight shrinkage. Its taste was sweet ，sour and slightly astringent. The microscopic characteristics of the powder were clear ，the stone cells were scattered individually or clustered，and the pulp parenchyma cells contained calcium oxalate cube crystal and cluster crystal. The results of TLC showed that there were fluorescent spots of the same color in the chromatogram of the tested product and the corresponding position of the reference substance （ursolic acid ）. The content ranges of moisture ，total ash and extracts were 9.02％-11.39％，2.29％-3.35％， 37.12％-58.41％ respectively. The linear ranges of chlorogenic acid ，hyperin，isoquercitrin，，quercetin phlorizin and quercetin were 3.12-50，1.25-20，1.25-20，3.12-50，18.7-300，0.5-8 μg/mL（all r were not less than 0.999 7）. RSDs of prescision ，stability and reproducibility tests were all lower than 3.0％（n＝6）. The average recoveries were 97.56％，98.28％，106.14％，103.74％， 98.37％，98.76％；RSDs were 1.57％，1.62％，1.38％，1.99％，2.15％，2.31％（n＝6）. The content ranges of the above six components in 20 baches of samples were 0.001 5％-0.028 8％，0.005 9％-0.022 0％，0.008 7％-0.030 4％，0.019 4％- 0.048 5％，0.068 5％-0.199 8％ and 0.005 1％-0.081 4％. CONCLUSIONS ：Established method is accurate and stable ，and can be used for quality control of Malus Fructus. It is preliminarily Δ 基金项目：四川省科技计划项目（No.2020YFS0495）；“杏林学 proposed that moisture and total ash content should not exceed 者”学科人才科研提升计划（No.CXTD2018016） 12％ and 3.5％，and extract （by dry product ）and phlorizin ＊硕士研究生 。研究方向 ：中药品种 、质量与资源 。E-mail： content should not be less than 40％ and 0.1％ respectively. 1159020313@qq.com # 通信作者：教授。研究方向：中药品种、质量与资源。E-mail：

Objective: To evaluate the effect of tumor shape and location on pulmonary dose-volume parameters by intensity-modulated radiation therapy (IMRT) in patients with non-small cell lung cancer (NSCLC), aiming to provide a reference basis for establishing limits of the pulmonary dose-volume parameters during IMRT. Methods: Clinical data of 208 NSCLC patients undergoing radical IMRT from June 2009 to June 2016 were retrospectively analyzed. According to the tumor shape and location, 208 cases were divided into the vertical bar group (n=127) and the horizontal bar group (n=81), the superior lung group (n=103) and the inferior lung group (n=105). Regression model curve was used to evaluate the effect of tumor shape and location upon the common pulmonary dose-volume parameters(V₅, V₂₀, MLD, AVS₅ and AVS₂₀). Results: In all groups, the fitting curves of V₅, V₂₀ and MLD were manifested in the quadratic equation pattern, and AVS₅ and AVS₂₀ in the logarithmic equation manner. In the vertical bar group, the V₅(P=0.015), V₂₀(P=0.047) and MLD (P=0.012) were significantly higher, whereas the AVS₅(P=0.044) was significantly lower compared with those in the horizontal bar group. No statistical significance was observed in AVS₂₀ between two groups (P=0.490). The tumor location exerted significant effect upon V₅ alone (P=0.009). Conclusions When the tumors presents in the vertical bar shape, the limits of the common lung dose-volume parameters are likely to exceed those of tumors in the vertical bar shape. Lung tumors located in the inferior lobe exerts a more significant effect upon the low-dose region volume compared with the tumors in the superior lobe.

Objective To discuss the pharmaceutical care protocol and methods for clinical pharmacists taking part in cancer pain management. Methods A patient developed delirium and drowsiness after using oxycodone hydrochloride prolonged-release tablet ( OXYCONTIN) and morphine hydrochloride tablet for pain titration treatment. The clinical pharmacist analyzed the reasons of delirium and drowsiness,and suggested a dose reduction of OXYCONTIN or converting OXYCONTIN to another opioid analgesic. Results The suggestion of the clinical pharmacist was partly accepted. After adjusting the treatment, the delirium and drowsiness disappeared gradually, and the pain was well controlled. Conclusion In clinical practice, especially when faced with a rare adverse drug reaction, clinical pharmacists are helpful for ensuring the safety and effectiveness in pain management, as well as improving the level of the treatment, by the implementation of individualized drug therapy.

Objective To investigate the effect of statin on trabecular bone microstructure by using trabecular bone score (TBS), a new type of bone microstructure evaluation index. Methods A total of 253 middle and aged patients hospitalized in the First Affiliated Hospital of Nanjing Medical University between January 2014 and March 2016 were retrospectively analyzed. According to whether statin was used or not, patients were divided into two groups: 90 patients in the statin use group (statin was use for more than 1 year) and 163 in the control group (not taken any statin). Serum biochemical indicators, such as triacylglycerol, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, alkaline phosphatase, fasting blood glucose and 25 hydroxy vitamin D, were compared between the two groups. Dual energy X-ray absorptiometry (DXA) was used to measure the bone mineral density (BMD) of lumbar spine and femoral neck. TBS was calculated with TBS iNsight? software, and the DXA image of lumbar spine were analyzed. Results Values of total cholesterol and low density lipoprotein cholesterol were significantly lower in statin group compared with those of control group (P<0.001), while no significant differences in other biochemical indicators between two groups (P>0.05). There was higher lumbar spine BMD statin group compared to that of control group (g/cm2:1.04 ± 0.19 vs. 0.96 ± 0.14, P<0.01). There was higher lumbar spine BMD and higher TBS (1.31±0.09 vs. 1.26±0.09, P<0.01) in statin group than that of control group. There was no difference in the femoral neck BMD between the two groups (P>0.05). Conclusion Statin increases lumbar spine BMD and improves trabecular bone microstructure in middle and aged people.