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.

The incidence of male infertility has been progressively increasing, its pathogenesis still incompletely elucidated and associated with multifactorial etiologies. Among these, sexually transmitted infections (STIs) constitute a significant contributing factor to male infertility. STIs, caused by various pathogens including human papillomavirus (HPV), may impair male fertility through diverse pathophysiological mechanisms. Nevertheless, current investigations into the impact of HPV infection on male reproductive capacity remain limited. This review critically examines the effects of HPV infection on the anatomical and functional integrity of the male reproductive system, semen parameters, and sperm quality, while exploring the correlation between HPV-related clinical management and male fertility preservation. Through comprehensive elucidation of HPV's pathogenic mechanisms within the male reproductive system and identification of its potential etiological contributions to male infertility, this review aims to provide theoretical foundations for optimizing fertility guidance protocols and developing novel antiviral therapeutic interventions, thereby advancing clinical strategies for reproductive health management and inform future research directions in antiviral treatment development.

The incidence of male infertility has been progressively increasing, its pathogenesis still incompletely elucidated and associated with multifactorial etiologies. Among these, sexually transmitted infections (STIs) constitute a significant contributing factor to male infertility. STIs, caused by various pathogens including human papillomavirus (HPV), may impair male fertility through diverse pathophysiological mechanisms. Nevertheless, current investigations into the impact of HPV infection on male reproductive capacity remain limited. This review critically examines the effects of HPV infection on the anatomical and functional integrity of the male reproductive system, semen parameters, and sperm quality, while exploring the correlation between HPV-related clinical management and male fertility preservation. Through comprehensive elucidation of HPV's pathogenic mechanisms within the male reproductive system and identification of its potential etiological contributions to male infertility, this review aims to provide theoretical foundations for optimizing fertility guidance protocols and developing novel antiviral therapeutic interventions, thereby advancing clinical strategies for reproductive health management and inform future research directions in antiviral treatment development.

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 explore the alterations in functional connectivity density (FCD) resulted from mild cognitive impairment (MCI) and their correlations with cognitive scores in various cognitive domains in patients with Parkinson's disease (PD).Methods:Forty-three PD patients admitted to Department of Neurology, Sixth Affiliated Hospital of Nantong University from January 2022 to April 2024 were selected and divided into PD-MCI group (MoCA scores<26) and PD with normal cognition (PD-NC) group (MoCA scores≥26) according to Montreal Cognitive Assessment (MoCA). Another 23 middle-aged and elderly healthy volunteers (HC group) matched with PD patients in age, gender and education level were recruited at the same period. Resting-state functional MRI (rs-fMRI) data were collected and whole brain FCD was calculated. Differences of clinical data, whole brain FCD, and FCD in brain regions with significantly different FCD among the 3 groups were compared. Efficiency of FCD in brain regions with significantly different FCD between PD-MCI group and PD-NC group in differentially diagnosing PD-MCI and PD-NC was analyzed by receiver operating characteristic (ROC) curve. Pearson correlation was used to the analyze the correlations of FCD in brain regions with significantly different FCD with MoCA score and cognitive scores in various cognitive domains.Results:Among the 43 patients, 23 were into the PD-MCI group and 20 into the PD-NC group. PD-MCI group had significantly lower scores in the visuospatial and executive function, abstraction, and delayed memory cognitive domains than PD-NC group ( P<0.05). Brain regions with significantly different FCD among the 3 groups were the right parahippocampal gyrus, left gyrus rectus, right rolandic operculum, left middle occipital gyrus, right precentral gyrus, left middle frontal gyrus, and left medial superior frontal gyrus. Compared with the HC group, the PD-MCI group and PD-NC group had significantly increased FCD at the right parahippocampal gyrus, left gyrus rectus and right rolandic operculum, statistically decreased FCD at the right precentral gyrus, left middle frontal gyrus, and left medial superior frontal gyrus ( P<0.05). Compared with the HC group, the PD-MCI group had significantly increased FCD at the left middle occipital gyrus ( P<0.05). Compared with the PD-NC group, the PD-MCI group had significantly decreased FCD at the right parahippocampal gyrus, and statistically increased FCD at the left middle occipital gyrus and left middle frontal gyrus ( P<0.05). Area under ROC curve (AUC) of FCD in brain regions with significantly different FCD in discriminating PD-MCI and PD-NC was 0.878, with sensitivity of 90.0% and specificity of 91.3%. FCD at right parahippocampal gyrus, left middle occipital gyrus and left middle frontal gyrus was negatively correlated with MoCA score ( P<0.05); FCD at right parahippocampal gyrus was positively correlated with cognitive scores in the visuospatial and executive function, and delayed memory domains ( P<0.05); FCD at left middle occipital gyrus was negatively correlated with cognitive scores in the executive function and visual-spatial skills, and abstraction domains ( P<0.05); FCD at the left medial frontal gyrus was negatively correlated with cognitive scores in the visuospatial and executive function, abstraction and delayed memory domains ( P<0.05). Conclusions:Abnormal FCD can be noted in some brain regions of PD patients, enjoying differences between PD-MCI patients and PD-NC patients. Combined FCD in brain regions with significantly different FCD has high value in differentially diagnosing PD-MCI and PD-NC, and FCD in brain regions with significantly different FCD is correlated with cognitive function changes in PD patients.

Objective:To explore the ultrasonic hemodynamic characteristics of early allograft dysfunction after left lateral lobe liver transplantation in children with biliary atresia.Methods:A total of 546 children with biliary atresia who underwent related left lateral lobe liver transplantation at Tianjin First Central Hospital from December 2012 to June 2021 were retrospectively selected, according to the early functional recovery of the transplanted liver, it was divided into a normal function recovery group (non-EAD group) and an early allograft dysfunction group (EAD group). The hepatic artery peak systolic flow velocity (PSV), end-diastolic flow velocity (EDV), resistance index (RI), portal vein diameter (PVD), portal vein flow velocity (PVV), portal vein flow (PVF), left hepatic vein diameter (LHVD) and left hepatic vein velocity (LHVV) were measured 1 to 7 days after surgery (a total of 3 703 ultrasound examination results), and the differences in ultrasound hemodynamic parameters between the two groups were compared. Binary logistic regression analysis was used to determine the correlation between hepatic artery RI=1.0, PSV<25 cm/s, PVV<15 cm/s, LHVV<15 cm/s and the occurrence of EAD within 7 days after surgery.Results:① Among the 546 children with biliary atresia, 262 children developed EAD after liver transplantation, and 284 children did not develop EAD. ②The portal vein flow of children in the EAD group was lower than that of the non-EAD group on 3 days, 4 days, 6 days and 7 days after surgery [3 days: 783 (560, 1 170) ml/(min·100 g) vs 942 (597, 1 381) ml/(min·100 g), P=0.006; 4 d: 862(594, 1 443) ml/(min·100 g) vs 1 068(748, 1 606) ml/(min·100 g), P=0.001; 6 d: 1 024 (631, 1 447) ml/(min·100 g) vs 1 141 (777, 1 709) ml/(min·100 g), P=0.005; 7 d: 937 (619, 1 408) ml/(min·100 g) vs 1 066 (670, 1 557) ml/(min·100 g), P=0.018]. The hepatic artery blood flow parameter RI was higher than that in the non-EAD group 7 days after surgery [0.72 (0.65, 0.79) vs 0.70 (0.63, 0.76), P=0.025]. There were no statistically significant differences in hepatic venous blood flow parameters between the two groups from 1 to 7 days (all P>0.05). ③Both the PVV and PVF in the EAD group and the non-EAD group showed an overall upward trend over time from 1 to 7 days after surgery, but compared with the non-EAD group, the portal vein flow in the EAD group increased more slowly from 3 to 7 days [PVF change rate: 0.01 (-0.25, 0.62)% vs 0.06 (-0.41, 0.41)%, P=0.003], while PSV, EDV and LHVV had no significant fluctuations. ④In the ultrasound hemodynamic abnormality index, the EAD group has a higher probability of transplanted hepatic artery RI=1.0 than the non-EAD group. Binary logistic regression analysis showed that hepatic artery RI=1.0 within 7 days after surgery was correlated with the occurrence of EAD [Exp(B)=2.413, P=0.005]. Conclusions:After left lateral lobe liver transplantation in children with biliary atresia, the portal vein of children with EAD showed a relatively low flow state.Abnormal ultrasound hemodynamic index hepatic artery RI=1.0 in children within 7 days after surgery can indicate the occurrence of EAD. Ultrasound examination can provide hemodynamic basis for early clinical detection of the presence of EAD.

Objective To explore the relationship between serum Visfatin,pentraxin 3(PTX3),and carbo-hydrate antigen 19-9(CA19-9)levels and prognosis in patients with acute cholecystitis.Methods A total of 228 patients with acute cholecystitis admitted to a hospital from September 2020 to September 2023 were se-lected as the study objects.According to the severity of the patients,228 patients were divided into mild group(n=81),moderate group(n=102)and severe group(n=45).The patients were divided into good prognosis group(n=186)and bad prognosis group(n=42).General data of patients were collected,serum Visfatin and PTX3 levels were detected by enzyme linked immunosorbent assay,and serum CA19-9 levels were determined by chemiluminescence immunoassay.Univariate and multivariate analysis of prognostic factors in patients with acute cholecystitis was performed,and the diagnostic value of serum Visfatin,PTX3 and CA19-9 levels in patients with acute cholecystitis was analyzed by drawing receiver operating characteristic curve.Results Ser-um Visfatin,PTX3 and CA19-9 in moderate and severe groups were significantly different from those in mild group(P＜0.05).There were significant differences in serum Visfatin,PTX3 and CA19-9 between severe group and moderate group(P＜0.05).Gallbladder thickness,long diameter and short diameter of gallbladder,serum Visfatin,PTX3 and CA19-9 levels in the poor prognosis group were significantly different from those in the good prognosis group(P＜0.05).Multivariate Logistic regression analysis showed that serum Visfatin,PTX3 and CA19-9 levels were the influential factors for poor prognosis of patients(P＜0.05).The area under the curve of serum Visfatin,PTX3 and CA19-9 combined diagnosis was significantly larger than that of Visfa-tin alone diagnosis(Z=4.577,P＜0.001)and PTX3 alone diagnosis(Z=3.132,P=0.002)and the area un-der the curve of CA19-9 diagnosis alone(Z=2.766,P=0.006).Conclusion Serum Visfatin,PTX3 and CA19-9 are elevated in patients with acute cholecystitis,which is related to the patient's condition.The combi-nation of the three is of better value in the diagnosis of poor prognosis.

Objective To explore the application value of ultrasound radiomics technology based on grayscale ultrasound images in the differential diagnosis of histological classification of glomerulonephritis.Methods A total of 204 patients with renal biopsy were selected from our hospital,and according to pathological results,they were divided into the membranous nephropathy group(n=133)and the mesangial proliferative glomerulonephritis group(n=71).The ultrasound images were sketched and the image omics features were extracted by two physicians.The pathological results and ultrasound data of renal biopsy were collected from the two groups,and the ultrasound radiomics features were preliminarily screened by the maximum correlation and minimum redundancy algorithm(mRMR)algorithm for all the obtained omics feature data.Then the optimal effective features were selected from the screened features by minimum absolute shrinkage and selection operator(LASSO)algorithm,and random forest(RF),support vector machine(SVM),logistic regression(LR),four kinds of classifiers of K-nearest neighbor(KNN)method were used to establish a prediction model.All cases were randomly divided into the training set and the validation set according to the ratio of 7∶3,and the four models were trained by the training set,and then validated in the validation set,and the best prediction model was selected by comparing the receiver operating characteristic(ROC)curve,Delong test and GiViTI calibration curve.Decision curve analysis(DCA)was used to evaluate the clinical utility of the model.Results The radiomics method was used to extract 837 radiomics features per image,and 16 meaningful features were finally screened out by using the mRMR+LASSO algorithm.Among the four prediction models of RF,SVM,LR and KNN,the best performing model was LR model,with the AUC of 0.944,the specificity of 0.867 and the sensitivity of 0.878.The GiViTI calibration curve showed that the model had good accuracy(P＞0.05),and the decision curve showed that the prediction model had good clinical practical value.Conclusion Ultrasound radiomics has a good ability to distinguish the more common histological types of glomerulonephritis,and is a non-invasive method with good application prospects.

Background In recent years, a growing number of studies have indicated that perfluorooctanoic acid (PFOA) exposure can impact heart development, though the specific mechanisms remain elusive. The aryl hydrocarbon receptor (AHR) is a critical environmental sensor capable of inducing oxidative stress and cell apoptosis. Objective To explore the role of AHR in the cardiac developmental toxicity of PFOA by using zebrafish embryo as an in vivo model. Methods Zebrafish embryos at 2 h post-fertilization (2 hpf) were exposed to dimethyl sulfoxide (DMSO) control, 1, 10, 100, and 1000 μg·L⁻¹ of PFOA. The AHR inhibitor CH223191 (CH) was added to the high-concentration group (1000 μg·L⁻¹) to form an intervention group. Under a dissecting microscope, the survival rate, mortality rate, heart rate, and heart malformation rate of 72 hpf zebrafish larvae were assessed. The activity of AHR was measured using 7-ethoxyresorufin-O-dealkylation (EROD) staining. The levels of intracellular and mitochondrial ROS in the heart of zebrafish larvae were assessed using dichloro-dihydro-fluorescein diacetate and MitoSOX™ Red, respectively. Apoptosis was examined using acridine orange (AO) staining and Immunofluorescence method. Total RNA was extracted from dissected hearts, and mRNA expression levels of oxidative stress-related genes (sod2, cat) and apoptosis-related gene (p53) were analyzed using quantitative PCR. Results Compared to the DMSO control group, PFOA at even the lowest concentration (1 μg·L⁻¹) increased heart malformation rate (P＜0.05) and reduced heart rate (P＜0.01) in the zebrafish larvae at 72 hpf, and the results showed a clear concentration-response relationship. Adding the AHR inhibitor CH significantly decreased heart malformation rate and restored heart rate to the control group level. The EROD results showed that PFOA at 1000 μg·L⁻¹ increased AHR activity (P＜0.001). Further studies revealed that PFOA caused a dose-dependent increase in intracellular ROS (P<0.001) and an increase in mitochondrial ROS (P<0.001) in the heart region of zebrafish larvae. A high dose of PFOA (1000 μg·L⁻¹) also induced elevated mRNA expression levels of oxidative stress-related genes sod2 and cat (P<0.05). Addition of the AHR inhibitor CH effectively antagonized PFOA-induced (1000 μg·L⁻¹) oxidative stress in the heart of zebrafish larvae (P<0.001). In addition, PFOA exposure induced a concentration-dependent increase in apoptotic bodies in the heart of zebrafish larvae (P<0.05). Moreover, PFOA at 1000 μg·L⁻¹ caused an increase in the cleaved-caspase 3 immunofluorescence signal (P<0.05) as well as overexpression of the apoptosis-associated gene p53 (P<0.001).which were attenuated by the CH supplementation. Conclusion PFOA triggers oxidative stress and apoptosis via AHR activation in the heart of zebrafish larvae, resulting in cardiac defects.