Objective:To develop a rapid, sensitive, and quantitative method for detecting Klebsiella pneumoniae using digital loop-mediated isothermal amplification(LAMP)-CRISPR/Cas12b. Methods:Five LAMP primers targeting the Kp-1 gene of Klebsiella pneumoniae and guide RNA (gRNA) for Cas12b were designed. The digital LAMP-CRISPR/Cas12b reaction mixture included 1×WarmStart ? LAMP master mix, 1×LAMP primers, 250 nmol/L Cas12b, 250 nmol/L gRNA, 3 μmol/L ssDNA reporter, 1 000 U/ml RNase inhibitor, 4 mmol/L Mg 2+, and DEPC water. After preparing the digital chip, it was incubated at 60℃ for one hour. Fluorescence distribution was then detected using a biochip analyzer to calculate the input DNA concentration. The specificity of the method was tested using genomic DNA from seven pathogenic microorganisms. The quantitative performance was assessed using serial dilutions of Klebsiella pneumoniae DNA ranging from 5-500 000 copies/μl. Clinical sputum samples were collected for comparison of quantitative performance with qPCR and qualitative performance with culture methods. Results:The digital LAMP-CRISPR/Cas12b method showed high specificity, yielding negative results for all six non-target pathogens. Quantitative performance tests indicated a sensitivity as low as 5 copies/μl, with a linear dynamic range of 5-50 000 copies/μl ( R2=0.927 4). Clinical sample quantitative testing showed that the correlation coefficient between digital LAMP-CRISPR/Cas12b and qPCR quantification was 0.917 0. Compared with the culture results of 72 samples, this method had a sensitivity of 100% and detected two additional samples with negative culture result, with a specificity of 91%; Compared with the culture method, qPCR had a sensitivity of 96% and a specificity of 83%. These results indicated that the digital LAMP-CRISPR/Cas12b method had good quantitative and qualitative detection performance for clinical sputum samples. Conclusions:This method offers advantages over qPCR, including rapidity, simplicity, and high precision. The digital LAMP-CRISPR/Cas12b method enables absolute quantification of Klebsiella pneumoniae in sputum samples, enhancing the accuracy of early screening for Klebsiella pneumoniae infections. These advantages make digital LAMP-CRISPR/Cas12b technology highly promising for the precise diagnosis of pathogenic microorganisms in field detection, primary healthcare, and resource-limited environments.

Objective:To develop a rapid, sensitive, and quantitative method for detecting Klebsiella pneumoniae using digital loop-mediated isothermal amplification(LAMP)-CRISPR/Cas12b. Methods:Five LAMP primers targeting the Kp-1 gene of Klebsiella pneumoniae and guide RNA (gRNA) for Cas12b were designed. The digital LAMP-CRISPR/Cas12b reaction mixture included 1×WarmStart ? LAMP master mix, 1×LAMP primers, 250 nmol/L Cas12b, 250 nmol/L gRNA, 3 μmol/L ssDNA reporter, 1 000 U/ml RNase inhibitor, 4 mmol/L Mg 2+, and DEPC water. After preparing the digital chip, it was incubated at 60℃ for one hour. Fluorescence distribution was then detected using a biochip analyzer to calculate the input DNA concentration. The specificity of the method was tested using genomic DNA from seven pathogenic microorganisms. The quantitative performance was assessed using serial dilutions of Klebsiella pneumoniae DNA ranging from 5-500 000 copies/μl. Clinical sputum samples were collected for comparison of quantitative performance with qPCR and qualitative performance with culture methods. Results:The digital LAMP-CRISPR/Cas12b method showed high specificity, yielding negative results for all six non-target pathogens. Quantitative performance tests indicated a sensitivity as low as 5 copies/μl, with a linear dynamic range of 5-50 000 copies/μl ( R2=0.927 4). Clinical sample quantitative testing showed that the correlation coefficient between digital LAMP-CRISPR/Cas12b and qPCR quantification was 0.917 0. Compared with the culture results of 72 samples, this method had a sensitivity of 100% and detected two additional samples with negative culture result, with a specificity of 91%; Compared with the culture method, qPCR had a sensitivity of 96% and a specificity of 83%. These results indicated that the digital LAMP-CRISPR/Cas12b method had good quantitative and qualitative detection performance for clinical sputum samples. Conclusions:This method offers advantages over qPCR, including rapidity, simplicity, and high precision. The digital LAMP-CRISPR/Cas12b method enables absolute quantification of Klebsiella pneumoniae in sputum samples, enhancing the accuracy of early screening for Klebsiella pneumoniae infections. These advantages make digital LAMP-CRISPR/Cas12b technology highly promising for the precise diagnosis of pathogenic microorganisms in field detection, primary healthcare, and resource-limited environments.

OBJECTIVE: The barks, leaves, and branches of Cinnamomum cassia have been historically used as a traditional Chinese medicine, spice, and food preservative, in which phenylpropanoids are responsible compounds. However phenylpropanoid biosynthesis pathways are not clear in C. cassia. We elucidated the pathways by descriptive analyses of differentially expressed genes related to phenylpropanoid biosynthesis as well as to identify various phenylpropanoid metabolites. METHODS: Chemical analysis, metabolome sequencing, and transcriptome sequencing were performed to investigate the molecular mechanisms underlying the difference of active components content in the barks, branches and leaves of C. cassia. RESULTS: Metabolomic analysis revealed that small amounts of flavonoids, coumarine, and cinnamaldehyde accumulated in both leaves and branches. Transcriptome analysis showed that genes associated with phenylpropanoid and flavonoid biosynthesis were downregulated in the leaves and branches relative to the barks. The observed differences in essential oil content among the three tissues may be attributable to the differential expression of genes involved in the phenylpropanoid and flavonoid metabolic pathways. CONCLUSION This study identified the key genes in the phenylpropanoid pathway controling the flavonoid, coumarine, and cinnamaldehyde contents in the barks, branches and leaves by comparing the transcriptome and metabolome. These findings may be valuable in assessing phenylpropanoid and flavonoid metabolites and identifying specific candidate genes that are related to the synthesis of phenylpropanoids and flavonoids in C. cassia.

Objective:To investigate epidemiological characteristics and macrolide-resistance of hospitalized children with Mycoplasma pneumoniae (MP) infections in Beijing from 2016 to 2019, so as to provide basis for the prevention and treatment of pediatric Mycoplasma pneumoniae pneumonia (MPP).Methods:The clinical data were analyzed retrospectively from 8 691 children hospitalized with community acquired pneumonia in Beijing Children′s Hospital between January 2016 and September 2019.MP RNA was detected by simultaneous amplification and testing (SAT), and macrolide resistance of MP was examined by MP and macrolide-resistant isolate diagnostic kit (PCR with fluorescence probes). Chi- square test was used for categorical analysis. Results:Among 8 691 cases detected by SAT, the overall detection rate of MP was 28.10% (2 442/8 691 cases). The detection rates of MP from 2016 to 2019 were 26.23%, 31.36%, 27.84 % and 26.57%, respectively.The detection rate of MP in 2017 was significantly higher than that in other years ( χ2=16.11, P<0.05). The detection rate of MP in females was 29.65%(1 107/3 733 cases), which was evidently higher than that in males 26.93%(1 335/4 958 cases) ( χ2=7.85, P<0.05). The positive rates of MP in summer[32.21% (726/2 254 cases)] and autumn[39.76%(852/2 143 cases)] were significantly higher than those in spring[17.00% (327/1 924 cases)] and winter[22.66%(537/2 370 cases)] ( χ2=315.15, P<0.001). The percentages of MP were 35.06%(732/2 088 cases) in preschoolers and 37.71%(1 160/3 076 cases) in school-age children, which were significantly higher than 11.20%(232/2 072 cases) in infants and 22.01% (318/1 445 cases) in toddlers ( χ2=509.89, P<0.001). Macrolide resistance detection was conducted in 1 524 patients by fluorescent PCR.Among them, 1 386 patients were positive for drug resistance, and the positive rate was 90.94%.The prevalence of macrolide-resistant MP from 2016 to 2019 were 88.19%, 90.93%, 90.56% and 92.90%, respectively.Macrolide-resistant rates were not related with gender, age and season. Conclusions:MP can be detected in all seasons, but most prevalently in summer and autumn.Girls are more prone to MP infections than boys.The detection rate of MP increases with age, and the positive rate is higher in preschoolers and school-age children.During the 4-year study period, the drug resistant rate of MP remain high.

Polymeric micelles have exhibited attractive properties as drug carriers, such as high stability in vivo and good biocompatibility, and been successfully used to dissolve various drugs of poor aqueous solubilities. In this study, we developed a new type of polymeric micelles with mannose-mediated targeting and pH-responsive drug release properties for anticancer drug delivery. The polymeric micelles were prepared from an amphiphilic polymer, poly (glycidyl methacrylate)-g-mannose (PGMA-Mannose). An anticancer drug, doxorubicin (DOX), was encapsulated into the micelles during the micellization, and could be released rapidly under acidic condition. The specificity of cellular uptake of the micelles by two different cell lines was studied using confocal laser scanning microscopy and the MTT assay. DOX-loaded micelles were efficiently trapped by mannose-receptor-overexpressing cancer cells MDA-MB-231, whereas mannose- receptor-poor cells HEK293 showed much lower endocytosis towards the micelles under the same conditions. Thus, DOX-loaded micelles displayed higher cytotoxicity to MDA-MB-231 cancer cells as compared with free DOX. The present study demonstrates that PGMA-Mannose micelles are a promising targeted drug delivery system for cancer therapy.
Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Delivery Systems ; HEK293 Cells ; Humans ; Lectins, C-Type ; metabolism ; Mannose ; chemistry ; Mannose-Binding Lectins ; metabolism ; Micelles ; Receptors, Cell Surface ; metabolism