OBJECTIVES: To detect prfA and hly toxin genes of Listeria monocytogenes using polymerase chain reaction (PCR) and colloidal gold technology. METHODS: L. monocytogenes DNA was extracted by boiling method. With prfA and hly of L. monocytogenes as the target genes, the 5' ends of upstream and downstream primers of prfA gene were labeled with 6-FAM and biotin, and the 5' ends of upstream and downstream primers of hly gene were labeled with digoxin and biotin, respectively, to establish the toxin gene detection method. Using cloning transformation, sequencing analysis, cloning of positive control products, the detection kid was developed and its specificity, sensitivity, reproducibility and stability were tested, followed by verification with sample testing. RESULTS: The concentration of L. monocytogenes DNA extracted by boiling method was 148.81±0.97 ng/μL, and the A₂₆₀/A₂₈₀ ratio ranged from 1.8 to 2.0. The PCR products showed a 100% homology with the gene sequences in GenBank database after cloning, transformation and sequencing. The colloidal gold strip yielded positive results only for L. monocytogenes samples without cross-reactions with Staphylococcus aureus, Escherichia coli or Bacillus cereus, and its minimum detection limit was 10^-2 ng/μL, demonstrating a 10-fold greater sensitivity of the test than agarose gel electrophoresis. The test also showed good reproducibility of the results when performed by different operators with good stability of the test strips after storage for 6 to 12 months. The test results showed that this kit could accurately and quickly detect L.monocytogenes in the test samples. CONCLUSIONS The detection kit developed in this study can simultaneously detect prfA and hly toxin genes of L. monocytogenes with good specificity, sensitivity, reproducibility and stability for use in food safety inspection.
Listeria monocytogenes/isolation & purification* ; Gold Colloid ; Bacterial Toxins/genetics* ; Polymerase Chain Reaction/methods* ; Hemolysin Proteins/genetics* ; Bacterial Proteins/genetics* ; DNA, Bacterial/genetics* ; Food Microbiology ; Heat-Shock Proteins

Objective:To establish a method of dual nueleic acid test strips for rapid detection of Bacillus cereus cytK and nhe toxin genes based on polymerase chain reaction(PCR)and colloidal gold technique,and to evaluate its specificity,sensitivity,repeatability and stability.Methods:Bacillus cereus DNA was extracted by boiling method.Specific primers were designed with Bacillus cereus cytK and nhe as the target genes.Clonal transformation was used to identify the PCR products.The optimal labeling amounts of colloidal gold-labeled streptavidin,quality control line(C line),cytK detection line(T1)and nhe detection line(T2)were determined.The nucleic acid test strips were assembled and its specificity,sensitivity,reproducibility and stability were evaluated.Results:The DNA concentration of Bacillus cereus was 248 mg·L-1,and the purities were 1.8-2.0.After cloning and plasmid sequencing,the similarities between the PCR products and the sequences of cytK and nhe registered in the GenBank database were 100％.Under the condition of pH 7.0,the optimal amount of streptavidin labeling per 200 μL of colloidal gold solution was 6.0 μL;the optimal marking amount was 2.00 g·L-1 for the quality control line(C line),0.550 g·L-1 for cytK gene detection line(T1)and 0.2 g·L-1 for nhe gene detection line(T2).In the specificity test,positive result on the test strips was seen only for Bacillus cereus,and no cross-reactivity was observed for Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and Bacillus subtilis,which were consistent with the electrophoresis results.Sensitivity assay showed that even when DNA concentration was reduced to 10-2 mg·L-1,three bands(C line,T1 line and T2 line)could be observed,and the detection limit of the test strip was one-tenth of agarose gel electrophoresis(10-1 mg·L-1).The nucleic acid test strips were verified by different operators in different laboratories,and the results were consistent.The stability of the test strips was verified at the 6th,9th and 12th months,and the results showed good stability.Conclusion:The dual nucleic acid test strip method established in this study can simutaneously detect the cytK and nhe toxin genes of Bacillus cereus with high sensitivity and specificity,achieving short-term visual detection.

Objective:To discuss the effect of quorum sensing-related gene expression on biofilm formation and drug resistance in clinically multidrug-resistant Pseudomonas aeruginosa,and to clarify the mechanism of enhacing drug resistance.Methods:A total of 77 strains of Pseudomonas aeruginosa were collected.Based on drug resistance,the strains were divided into multidrug-resistant group and sensitive group.The optimal biofilm formation conditions were determined using the microtiter plate method;biofilm formations of the stains in both groups was observed under an optical microscope;crystal violet staining was used to semiquantitatively detect biofilm formation ability of P.aeruginosa in both groups;microbroth dilution method was used to determine the minimal inhibitory concentration(MIC)values of the quorum sensing inhibitor(C-30)against Pseudomonas aeruginosa in both groups;RNA was extracted from two groups using a commercial kit,while RNA from planktonic state and biofilm state of multidrug-resistant strains was extracted using modified TRIzol method;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of quorum sensing-related genes(lasR/I,RhlR/I,PqsR/A)of the stains in multidrug-resistant group and sensitive group,as well as before and after adding the quorum sensing inhibitor C-30.Results:Among 77 strains of Pseudomonas aeruginosa,56 were multidrug-resistant(multidrug-resistant group)and 21 were fully sensitive(sensitive group).Optimal biofilm formation occurred at a bacterial concentration of 1.5×108 CFU·mL-1 with 48 h incubation.The biofilm positivity rate was 91%,with strongly positive,moderately positive,weakly positive,and negative biofilms accounting for 16%,34%,41%,and 9%,respectively.The biofilm positivity rate in multidrug-resistant strains was 96%,and biofilm formation ability in multidrug-resistant group was higher than that in sensitive group(P<0.05).When the concentration of C-30 was 8 mg·L-1 the biofilm formation in most Pseudomonas aeruginosa was inhibited,with enhanced suppression at higher concentrations.The absorbtion(A)value of both planktonic-state and biofilm-state RNA ranged from 1.8 to 2.0.The RT-qPCR results showed that compared with planktonic state,the expression levels of lasR/I,RhlR/I,and PqsR/A mRNA of the stains in biofilm state were significantly increased(P<0.01).Compared with non-inhibitor group,the expression levels of lasR/I,RhlR/I,and PqsR/A mRNA in biofilm state of inhibitor-treated group were significantly decreased(P<0.01).Conclusion:High expression of quorum sensing-related genes in multidrug-resistant P.aeruginosa promotes biofilm formation,thereby enhancing drug resistance.

Objective: To understand the status and related knowledge requirements of health emergency literacy among college students in Shaanxi Province, so as to provide the basis for improving college students health emergency literacy. Methods: A total of 2 723 students from 18 colleges and universities in Shaanxi Province were selected by multi stage random sampling and simple random sampling methods in November 2023, and the survey of health literacy in emergency and knowledge requirements of health emergency literacy was conducted. Statistical analysis was carried out by using χ 2 test, Wilcoxon rank sum test, Kruskal-Wallis H test and Logistic regression analysis. Results: About 28.98% of the surveyed college students had a high level of health emergency literacy, which varied by students whether being only one child, whether having left behind experience, with different personality types, whether being student cadres, and with different frequencies of community or social activities ( χ 2=9.15, 7.90, 32.73, 16.29 , 120.25, P <0.05). The equivalence scores of the four dimensions of health emergency literacy from high to low were poisoning and nuclear and radiation (0.84), medical rescue (0.83), infectious disease (0.82), and basic knowledge and behavior ( 0.77 ). Logistic regression analysis found that college students with left-behind experience were negatively correlated with health emergency literacy and its four dimensions ( OR =0.74, 0.72, 0.80, 0.80, 0.83), while personality type (rational type), community or social activity frequency were positively correlated with the cognitive levels of health emergency literacy and its four dimensions among college students ( OR =1.57, 1.50, 1.33, 1.27, 1.38)( P <0.05). There was a higher level of basic knowledge and behavioral cognition among only child college students ( OR =3.73), and female students had a higher level of health emergency literacy, as well as awareness of infectious disease outbreaks and medical rescue ( OR =1.21, 1.28, 1.21)( P <0.05). The radar map showed that the level of health emergency literacy was positive development radar map. About 67.68 % of the students had a high willingness to acquire health emergency literacy knowledge, and the demand for basic health emergency knowledge and behavioral knowledge was the highest (52.37%). Conclusions College students have insufficient health emergency literacy, but they have the highest demand for health emergency. Publicity and education should be strengthened for students with left behind experience, irrational type, and low frequency of community or social activities.

Proliferative diabetic retinopathy is a serious complication of diabetes in the eye that can lead to severe vision loss or even complete vision loss. In recent years, with the rapid development of surgical equipment and fundus examination technology, the indications for pars plana vitrectomy based surgical treatment have been expanded, surgical improvement and application, combined application of drugs, such as anti-vascular endothelial growth factor drugs, glucocorticoids, and surgical evaluation have made new progress. Surgical evaluation based on imaging can continuously monitor patients' eye conditions before, during and after surgery, and clinicians can choose different surgical plans and timing for different patients, which can help reduce patients' pain and achieve better visual outcomes.

Purpose: The causal relationship between the incidence and prognosis of chronic kidney disease (CKD) and serum testosterone levels in patients is not yet fully understood. This study aims to use the National Health and Nutrition Examination Survey (NHANES), a large-scale nationally representative sample, to investigate the relationship between CKD and testosterone. Materials and Methods: This study included six NHANES cycles for linear regression analysis, verified by multiple imputation methods. Stratified analysis and subgroup analysis were used to demonstrate the stability of CKD’s effect on testosterone. Furthermore, we used Kaplan-Meier plots and log-rank tests to evaluate differences in survival rates between CKD male patients with low and normal levels of testosterone. Results: From a total of 71,163 subjects, the cohort selected 28,663 eligible participants. Results showed that CKD patients had testosterone levels 28.423 ng/mL (24.762, 32.083) lower than non-CKD patients. The results of multiple imputations (β=27.700, 95% confidence interval: 23.427, 31.974) were consistent with those of linear regression analysis, and the numerical match was good. Stratified regression analysis, and subgroup analysis results showed that CKD had a significant impact on testosterone at different dimensions. Kaplan-Meier plots showed significantly reduced survival rates in low testosterone CKD male patients (p<0.0001). Conclusions The results of this big data analysis suggest that there may be a two-way risk between low levels of testosterone and CKD. The testosterone levels of CKD patients were significantly lower than those of the non-CKD population, and CKD patients with low testosterone levels had poorer prognoses. These results suggest that correcting testosterone levels in a timely manner can have preventive and therapeutic effects on the progression of CKD.

BACKGROUND: This study aimed to develop a comprehensive instrument for evaluating and ranking clinical practice guidelines, named Scientific, Transparent and Applicable Rankings tool (STAR), and test its reliability, validity, and usability. METHODS: This study set up a multidisciplinary working group including guideline methodologists, statisticians, journal editors, clinicians, and other experts. Scoping review, Delphi methods, and hierarchical analysis were used to develop the STAR tool. We evaluated the instrument's intrinsic and interrater reliability, content and criterion validity, and usability. RESULTS: STAR contained 39 items grouped into 11 domains. The mean intrinsic reliability of the domains, indicated by Cronbach's α coefficient, was 0.588 (95% confidence interval [CI]: 0.414, 0.762). Interrater reliability as assessed with Cohen's kappa coefficient was 0.774 (95% CI: 0.740, 0.807) for methodological evaluators and 0.618 (95% CI: 0.587, 0.648) for clinical evaluators. The overall content validity index was 0.905. Pearson's r correlation for criterion validity was 0.885 (95% CI: 0.804, 0.932). The mean usability score of the items was 4.6 and the median time spent to evaluate each guideline was 20 min. CONCLUSION The instrument performed well in terms of reliability, validity, and efficiency, and can be used for comprehensively evaluating and ranking guidelines.
Reproducibility of Results ; Surveys and Questionnaires ; Practice Guidelines as Topic ; Humans

Cucurbitaceae is an important family of flowering plants containing multiple species of important food plants, such as melons, cucumbers, squashes, and pumpkins. However, a highly efficient genetic transformation system has not been established for most of these species (Nanasato and Tabei, 2020). Watermelon (Citrullus lanatus), an economically important and globally cultivated fruit crop, is a model species for fruit quality research due to its rich diversity of fruit size, shape, flavor, aroma, texture, peel and flesh color, and nutritional composition (Guo et al., 2019). Through pan-genome sequencing, many candidate loci associated with fruit quality traits have been identified (Guo et al., 2019). However, few of these loci have been validated. The major barrier is the low transformation efficiency of the species, with only few successful cases of genetic transformation reported so far (Tian et al., 2017; Feng et al., 2021; Wang JF et al., 2021; Wang YP et al., 2021). For example, Tian et al. (2017) obtained only 16 transgenic lines from about 960 cotyledon fragments, yielding a transformation efficiency of 1.67%. Therefore, efficient genetic transformation could not only facilitate the functional genomic studies in watermelon as well as other horticultural species, but also speed up the transgenic and genome-editing breeding.
CRISPR-Cas Systems ; Citrullus/genetics* ; Cucurbitaceae/genetics* ; Gene Editing ; Plant Breeding ; Transformation, Genetic

Objective:To study the feasibility of using pelvic iterative cone beam CT images for dose calculation of radiation therapy planning, so as to provide support for adaptive radiotherapy.Methods:The CIRS 062 M phantom was scanned by Varian Halcyon v2.0 o-ring accelerator, and the average CT number under different scattering conditions was calculated, and then the ICBCT-ED conversion curve was established. CT images of CIRS 002PRA pelvic IMRT phantom and ICBCT images at different positions were collected. Treatment plan using VMAT technique based on CT image was designed and transplanted into ICBCT image with dose recalculated. The differences of gamma passing rate among target volume, organs at risk and 3-dimensional dose were compared. Based on the actual treatment plan of patients, the differences of 3-dimensional dose gamma passing rate in 10 pelvic patients were analyzed retrospectively.Results:There was a large CT value deviation at central position between the isolated no-scattering condition and the full-scattering condition, and the maximum deviation was 144 HU. The CT values of other positions in full-scattering condition were similar to those of the central position, and the maximum deviation was less than 50 HU. Based on the calculated result of ICBCT images at different positions of the pelvic phantom, the dose deviation of the target volume or organs at risk was less than 1 Gy. Compared with the plan based on CT images, the average 3-dimensional dose gamma passing rate under the criteria of 1% dose difference (DD)/1 mm distance-to-agreement (DTA) and 2% DD/2 mm DTA in plan based on ICBCT images were (88.86 ±1.18)% and (98.38±0.89)%, respectively. The ranges of average 3-dimensional dose gamma passing rate under the criteria of 2% DD/2 mm DTA and 3% DD/3 mm DTA in 10 patients with pelvic tumors were 90.03%-95.43% and 93.58%-97.78%, respectively, and the worst result was only 85.90% and 92.90%, respectively. The main reason of the worst result was the dose difference caused by large variation of bladder contour due to over-filling.Conclusions:Under comprehensive scattering conditions, the ICBCT-ED conversion curve is reconstructed and the treatment plan can be designed by using the ICBCT image of Halcyon v2.0 linear accelerator. The accuracy meets the standards of clinical application, which provides assurance for adaptive radiotherapy in the future.

This study proposes an image segmentation method based on bottleneck detection and watershed algorithm to solve the problem of overlapping cervical cell image. First, we use polygon approximation to get all feature points on the cell contour and then use bottleneck detection and ellipse fitting to obtain the correct split point pairs. Therefore, the approximate range of the overlapping region was determined. The watershed algorithm was used to obtain the internal boundary information for the gradient image of the region. Finally, the segmentation results of the overlapped cells were obtained by superimposing with the outer contour. The experimental results show that this algorithm can segment the contour of a single cell from the overlapping cervical cell images with good accuracy and integrity. The segmentation result is close to that of doctors' manual marking, and the segmentation result is better than other existing algorithms.
Algorithms ; Cervix Uteri/cytology* ; Female ; Humans ; Image Processing, Computer-Assisted