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.

BACKGROUND:Bone relies on the close connection between blood vessels and bone cells to maintain its integrity.Bones are in a physiologically hypoxic environment.Therefore,the study of angiogenesis and osteogenesis in hypoxic environment is closer to the microenvironment in vivo. OBJECTIVE:To explore the influence of Wenshen Tongluo Zhitong(WSTLZT)formula on H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell co-culture system in hypoxia environment and its related mechanism. METHODS:Enzyme digestion method and flow sorting technique were used to isolate and identify H-type bone microvascular endothelial cells.Mouse bone marrow mesenchymal stem cells were isolated and obtained by bone marrow adhesion method.H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell hypoxic co-culture system was established using Transwell chamber and anoxic culture workstation.WSTLZT formula powder was used to intervene in each group at a mass concentration of 50 and 100 μg/mL.The angiogenic function of H-type bone microvascular endothelial cells in the co-culture system was evaluated by scratch migration test and tube formation test.The osteogenic differentiation ability of bone marrow mesenchymal stem cells in the co-cultured system was evaluated by alkaline phosphatase staining and alizarin red staining.The protein and mRNA expression changes of PDGF/PI3K/AKT signal axis related molecules in H-type bone microvascular endothelial cells in the co-cultured system were detected by Western Blotting and q-PCR,respectively. RESULTS AND CONCLUSION:(1)Compared with the normal oxygen group,the scratch mobility and new blood vessel length of H-type bone microvascular endothelial cells were significantly higher(P<0.05);the osteogenic differentiation capacity of bone marrow mesenchymal stem cells was higher(P<0.05);the expression of PDGF/PI3K/AKT axis-related molecular protein and mRNA increased(P<0.05)in the hypoxia group.(2)Compared with the hypoxia group,scratch mobility and new blood vessel length were significantly increased in the H-type bone microvascular endothelial cells(P<0.05);bone marrow mesenchymal stem cells had stronger osteogenic function(P<0.05);the expression of PDGF/PI3K/AKT axis-related molecular proteins and mRNA further increased(P<0.05)after treatment with different dose concentrations of WSTLZT formula.These findings conclude that H-type angiogenesis and osteogenesis under hypoxia may be related to the PDGF/PI3K/AKT signaling axis,and WSTLZT formula may promote H-type vasculo-dependent bone formation by activating the PDGF/PI3K/AKT signaling axis,thereby preventing and treating osteoporosis.

Objective:In view of the incomplete evaluation system of actual combat injury treatment ability of grassroots officers and soldiers at present, this paper aims to construct a multi-dimensional evaluation system for combat injury treatment to provide theoretical guidance and evaluation means for warfighting-oriented training on combat injury treatment.Methods:Through literature review, expert consultation, and hierarchical analysis, we determined the factors to evaluate the comprehensive ability of field first aid for combat injuries of grassroots officers and soldiers from the perspective of actual combat, and then established a system for evaluating the actual combat injury treatment ability of grassroots officers and soldiers, in which the weights of factors in each dimension were determined.Results:An evaluation system of actual combat injury treatment ability of grassroots officers and soldiers was formed, which included 4 first-level indices and 16 second-level indices in tactics, strategies, treatment, and protection dimensions.Conclusions:The established evaluation system can provide a reference for actual combat injury treatment training at the grassroots level.

Objective:To investigate the role of evidence-based clinical guidelines combined with "rain classroom" smart teaching in standardized residency training in endocrinology department.Methods:The 100 trainees who underwent standardized residency training in the Department of Endocrinology at Xiamen Hospital from September 2022 to September 2023 were selected as the research subjects. They were divided into groups based on the parity of their admission batches, with even-numbered batches assigned to the control group and odd-numbered batches to the experimental group, with 50 trainees in each group. The control group received traditional teaching, while the experimental group was taught using evidence-based clinical guidelines combined with "rain classroom" smart teaching. Statistical comparisons were made between the two groups in terms of exit examination scores, teaching effectiveness, teaching satisfaction, and evaluation of trainees by teachers. SPSS 22.0 was used for t test and Chi-square test. Results:The experimental group demonstrated significantly higher scores in theoretical knowledge [(25.62±3.12) vs. (22.71±3.27)], case analysis [(25.18±3.24) vs. (22.24±3.35)], clinical operation [(25.01±3.19) vs. (22.12±3.29)], and daily performance [(8.12±1.01) vs. (7.21±1.13)] compared to the control group in exit examination ( P<0.001). Additionally, the experimental group showed superior performance in self-learning [(8.57±1.12) vs. (7.64±1.24)], clinical thinking [(8.61±1.16) vs. (7.78±1.28)], evidence-based thinking [(8.52±1.13) vs. (7.69±1.26)], clinical skills [(8.49±1.17) vs. (7.61±1.23)], communication skills [(8.53±1.18) vs. (7.67±1.29)], and literature review [(8.59±1.15) vs. (7.71±1.27)], with statistically significant differences ( P<0.05). Teaching satisfaction in the experimental group (96.00%) was significantly higher than that in the control group (82.00%) ( P=0.025). The pass rates rated by teachers were higher in the experimental group in medical history collection (94.00% vs. 80.00%), physical examination (96.00% vs. 82.00%), clinical thinking (92.00% vs. 76.00%), communication skills (94.00% vs. 80.00%), literature review (96.00% vs. 84.00%), specialized skills (94.00% vs. 78.00%), and diagnosis and treatment plan formulation (92.00% vs. 74.00%), with statistically significant differences ( P<0.05). Conclusions:The implementation of evidence-based clinical guidelines combined with "rain classroom" smart teaching in the standardized residency training in endocrinology department can significantly improve the exit examination scores and teaching satisfaction of trainees, teaching effectiveness, and evaluation of trainees by teachers. This teaching method has a positive role in improving the clinical comprehensive ability of trainees such as evidence-based medical thinking and standardized clinical diagnosis and treatment.

Objective:To investigate the role of evidence-based clinical guidelines combined with "rain classroom" smart teaching in standardized residency training in endocrinology department.Methods:The 100 trainees who underwent standardized residency training in the Department of Endocrinology at Xiamen Hospital from September 2022 to September 2023 were selected as the research subjects. They were divided into groups based on the parity of their admission batches, with even-numbered batches assigned to the control group and odd-numbered batches to the experimental group, with 50 trainees in each group. The control group received traditional teaching, while the experimental group was taught using evidence-based clinical guidelines combined with "rain classroom" smart teaching. Statistical comparisons were made between the two groups in terms of exit examination scores, teaching effectiveness, teaching satisfaction, and evaluation of trainees by teachers. SPSS 22.0 was used for t test and Chi-square test. Results:The experimental group demonstrated significantly higher scores in theoretical knowledge [(25.62±3.12) vs. (22.71±3.27)], case analysis [(25.18±3.24) vs. (22.24±3.35)], clinical operation [(25.01±3.19) vs. (22.12±3.29)], and daily performance [(8.12±1.01) vs. (7.21±1.13)] compared to the control group in exit examination ( P<0.001). Additionally, the experimental group showed superior performance in self-learning [(8.57±1.12) vs. (7.64±1.24)], clinical thinking [(8.61±1.16) vs. (7.78±1.28)], evidence-based thinking [(8.52±1.13) vs. (7.69±1.26)], clinical skills [(8.49±1.17) vs. (7.61±1.23)], communication skills [(8.53±1.18) vs. (7.67±1.29)], and literature review [(8.59±1.15) vs. (7.71±1.27)], with statistically significant differences ( P<0.05). Teaching satisfaction in the experimental group (96.00%) was significantly higher than that in the control group (82.00%) ( P=0.025). The pass rates rated by teachers were higher in the experimental group in medical history collection (94.00% vs. 80.00%), physical examination (96.00% vs. 82.00%), clinical thinking (92.00% vs. 76.00%), communication skills (94.00% vs. 80.00%), literature review (96.00% vs. 84.00%), specialized skills (94.00% vs. 78.00%), and diagnosis and treatment plan formulation (92.00% vs. 74.00%), with statistically significant differences ( P<0.05). Conclusions:The implementation of evidence-based clinical guidelines combined with "rain classroom" smart teaching in the standardized residency training in endocrinology department can significantly improve the exit examination scores and teaching satisfaction of trainees, teaching effectiveness, and evaluation of trainees by teachers. This teaching method has a positive role in improving the clinical comprehensive ability of trainees such as evidence-based medical thinking and standardized clinical diagnosis and treatment.

Objective:In view of the incomplete evaluation system of actual combat injury treatment ability of grassroots officers and soldiers at present, this paper aims to construct a multi-dimensional evaluation system for combat injury treatment to provide theoretical guidance and evaluation means for warfighting-oriented training on combat injury treatment.Methods:Through literature review, expert consultation, and hierarchical analysis, we determined the factors to evaluate the comprehensive ability of field first aid for combat injuries of grassroots officers and soldiers from the perspective of actual combat, and then established a system for evaluating the actual combat injury treatment ability of grassroots officers and soldiers, in which the weights of factors in each dimension were determined.Results:An evaluation system of actual combat injury treatment ability of grassroots officers and soldiers was formed, which included 4 first-level indices and 16 second-level indices in tactics, strategies, treatment, and protection dimensions.Conclusions:The established evaluation system can provide a reference for actual combat injury treatment training at the grassroots level.

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 investigate the relationship between insomnia,gastrointestinal symptoms,and glycosylated hemoglobin(HbA1c)levels in individuals diagnosed with type 2 diabetes mellitus(T2DM),as well as the related influencing factors.Methods A total of 910 T2DM patients treated in our multicenter from January 2022 to December 2022 were enrolled in this study.General information(gender,age,smoking and drinking history,exercise,course of disease,treatment and complications),HbA1c,Athens Insomnia Scale(AIS)scores and Gastrointestinal Symptoms Rating Scale(GSRS)scores of patients were collected.The differences of sleep and gastrointestinal symptoms between groups were analyzed,and the correlation between the differences and HbA1c was analyzed.Furthermore,the risk factors for non-standard HbA1c were analyzed.Results The AIS score and GSRS score in the HbA1c control group were less than those in the non-standard group(P＜0.01).Insomnia was reported by 37.0％of T2DM patients,and the HbA1c level in the insomnia group was significantly higher than that in the non-insomnia group(10.00％±2.38％vs.8.26％±1.73％,P＜0.01).Gastrointestinal symptoms were present in 57.5％of T2DM patients,and the HbA1c levels in the group with gastrointestinal symptoms were significantly higher than those in the group without gastrointestinal symptoms(9.26％±2.23％vs.8.43％±1.98％,P＜0.01).Furthermore,26.3％of T2DM patients experienced both insomnia and gastrointestinal symptoms.Remarkably,the HbA1c levels in the group with both insomnia and gastrointestinal symptoms were significantly higher than those in the group without either condition(10.18％±2.44％vs.8.45％±1.86％,P＜0.01).Correlation analysis demonstrated a significant association between sleep quality,gastrointestinal function,and HbA1c levels(P＜0.01).The logistic regression analysis result revealed that age,GSRS score,AIS score,and the presence of insomnia combined with gastrointestinal symptoms were independent risk factors for predicting HbA1c≥6.5％(P＜0.01).Having both insomnia and gastrointestinal symptoms concurrently was the strongest risk factor for substandard HbA1c control,and the risk of blood sugar control may increase about 5 times when both appear together.Conclusion Insomnia and gastrointestinal symptoms are common comorbidities in T2DM patients,showing a cross-interfering relationship,and they appear together with poor blood sugar control,interact causally,and amplify each other.

OBJECTIVE To study the intervention effects of Wenshen Tongluo Zhitong Recipe(WTZR)on macrophage senes-cence and senile osteoporosis.METHODS The senescent macrophage model was established using hydrogen peroxide(H2 O2)and subsequently divided into four groups:control,model,low-dose drug-treated serum,and high-dose drug-treated serum.β-galactosidase staining,Western blot and qPCR were employed to evaluate the mRNA expression of senescence markers p21 and p53.ROS staining and JC-1 staining were applied to assess mitochondrial function in macrophages.The mRNA levels of Interleukin(IL)-6,IL-10,CD206,and inducible nitric oxide synthase(iNOS)were determined by qPCR analysis.Immunofluorescence was used to evaluate argi-nase(ARG1)and iNOS protein expressions for assessing the impact of drug-containing serum on macrophage polarization.qPCR analy-sis was conducted to measure osteocalcin(OCN),collagen type Ⅰ alpha 1(Col1a1),runt-related transcription factor 2(Runx2)mRNA levels as osteoblast-related markers;ALP staining along with alizarin red staining were performed to evaluate the effect of macrophage conditioned medium treated with drug-containing serum on bone marrow mesenchymal stromal cell(BMSC)osteogenic differentiation.C57BL/6J mice were randomly allocated into four groups:control group,model group,WTZR low-dose group,and WTZR high-dose group.The senile osteoporosis(SOP)mouse model was established by D-galactose.Micro-CT scanning analyzed fe-mur microstructure while HE staining detected pathological changes in femur bone tissue samples collected from each experimental con-dition.Furthermore,Western blot was used to detect the senescence-related molecules p21 and p53 and the osteogenesis-related mark-ers OCN and Runx2,qPCR analysis measured tibial expression levels of senescence-related molecules(p21,p53)as well as macro-phage polarisation-related molecules(IL-6,iNOS,CD206,and IL-10)to assess the effect of this compound on a mouse model simula-ting SOP.RESULTS Following intervention with serum containing WTZR,there was a significant decrease in the number of senes-cent positive cells compared to the model group.Additionally,there was a notable decrease in p21 and p53 mRNA and proteins expres-sion(P＜0.05,P＜0.01,P＜0.001).Furthermore,drug-containing WTZR effectively inhibited ROS production induced by H2 O2 and mitigated mitochondrial membrane potential reduction in macrophages(P＜0.05,P＜0.001).Treatment with drug-containing WTZR re-sulted in down-regulated mRNA expression of M1-related gene iNOS(P＜0.05)while up-regulating mRNA expression of M2-related genes CD163 and CD206(P＜0.05).The drug-containing WTZR significantly reduced fluorescence intensity for iNOS(P＜0.01)while increasing ARG1(P＜0.05)fluorescence intensity.Moreover,conditioned medium from macrophages treated with drug-containing ser-um increased ALP positive cell count(P＜0.01,P＜0.001),alizarin red positive area(P＜0.05),as well as Col1a1,Runx2 and OCN mRNA expression levels(P＜0.05,P＜0.01).The Tb.N,BMD,and BV/TV were significantly higher in the WTZR group compared to the model group(P＜0.05,P＜0.01,P＜0.001);meanwhile,Tb.Sp was notably lower than that observed in the model group(P＜0.05,P＜0.01);bone trabeculae were significantly improved,increased in number and widened.Additionally,the compound could significantly inhibit the D-galactose induced up-regulation of tibial senescence-related genes and proteins p21 and p53(P＜0.05,P＜0.001,P＜0.0001),promote the expression of osteogenesis-related markers OCN and Runx2 protein(P＜0.01,P＜0.0001),promote the down-regulation of M1 related genes IL-6 and iNOS(P＜0.05),and promote the expression of M2 related genes IL-10 and CD206(P＜0.05,P＜0.01).CONCLUSION Wenshen Tongluo Zhitong Recipe may play an anti-osteoporosis effect by inhibiting macrophage senescence and promoting the osteogenic differentiation of BMSC.

Objective:To investigate the value of brain functional connectivity density (FCD) derived from resting-state functional magnetic resonance imaging (rs-fMRI) in differentially diagnosing 2 subtypes of Parkinson's disease (PD): tremor dominant (TD) and non-tremor dominant (nTD), as well as its correlation with clinical symptom scores.Methods:A retrospective analysis was performed; 45 PD patients (25 with TD and 20 with nTD), admitted to Department of Neurology, Drum Tower Hospital Affiliated to Medical School of Nanjing University were chosen; and 24 middle-aged and elderly volunteers matched in age, gender and years of education at the same time were chosen as normal controls (NCs). The rs-fMRI data were collected and FCD map of the whole brain was computed. The FCD differences in whole brain and brain regions enjoying different FCD were compared among the 3 groups. ROC curve was used to analyze the differential diagnostic efficacy of FCD in brain regions enjoying different FCD in TD and nTD. Correlations of FCD in brain regions enjoying different FCD with disease course, Hoehn-Yahr (H-Y) grading, unified Parkinson's disease rating scale (UPDRS) III scores, tremor scores and non-tremor scores were analyzed by Pearson or Spearman correlation analyses.Results:Brain regions enjoying different FCD among the TD, nTD, and NC groups were the right lingual gyrus, right orbital medial frontal gyrus, right insula, left superior temporal gyrus, left insula, right anterior central gyrus, left posterior central gyrus, right medial and paracingulate gyrus, and left angular gyrus. FCD in the right orbital medial frontal gyrus and left angular gyrus of TD group was significantly higher than that in the nTD group ( P<0.05); while FCD in the right insula, right anterior central gyrus, left posterior central gyrus and right medial and paracingulate gyrus of TD group was significantly lower than that in the nTD group ( P<0.05). Area under the curve of combined application of FCD in brain regions enjoying different FCD in differentially diagnosing TD and nTD was 0.842, with sensitivity and specificity of 80.0%. FCD in the left superior temporal gyrus and left posterior central gyrus was positively correlated with H-Y grading ( P<0.05), while FCD in the left angular gyrus was negatively correlated with H-Y grading ( P<0.05). FCD in the left superior temporal gyrus was positively correlated with UPDRS III scores ( P<0.05). FCD in the right lingual gyrus, right insula, left insula, right anterior central gyrus, left posterior central gyrus, right medial and paracingulate gyrus was negatively correlated with tremor scores ( P<0.05). FCD in the left superior temporal gyrus, right medial and paracingulate gyrus was positively correlated with non-tremor scores ( P<0.05), whereas that in the left angular gyrus was negatively correlated with non-tremor scores ( P<0.05). Conclusion:Abnormalities in FCD exist in certain brain regions of PD patients, with disparities between TD and nTD. Combined application of FCD in brain regions enjoying different FCD can effectively differentiate TD and nTD, and this FCD is also partially correlated with the symptom scores of PD patients.