In recent years, the bacteriophage Φ29 (Phi29) DNA polymerase has garnered increasing attention due to its high-fidelity amplification capacity at constant temperatures. To advance the industrial application of this type of isothermal polymerases, this study mined and characterized new enzymes from the microbial metagenome based on the known Phi29 DNA polymerase sequence. The results revealed that a new enzyme, Php29 DNA polymerase, was identified in the microbial metagenome with plants as the hosts. This enzyme exhibited higher strand displacement activity, with a 59.5% similarity to bacteriophage Φ29. Experimental validation demonstrated that the enzyme had 3'→5' exonuclease activity, and its amplification products can serve as substrates for further catalytic reactions. The discovery and validation of Php29 DNA polymerase gives insights into the future industrial application of isothermal polymerases.
DNA-Directed DNA Polymerase/metabolism* ; Bacillus Phages/genetics* ; Metagenome

OBJECTIVE To understand the drug resistance genes,virulence genes and molecular epidemiological characteristics of extensively drug-resistant hypervirulent Klebsiella pneumoniae(XDR-hvKP)strains causing hospital-associated infection.METHODS The clinical isolates of XDR-hvKP were collected from Tongling People's Hospital from Jul.2020 to Dec.2022.The strains were identified by matrix-assisted laser desorption ionization-time-of-flight(MALDI-TOF)mass spectrometry,the common drug resistance genes and virulence genes were an-alyzed by Sanger sequencing,the capsular serotypes were determined by wzi gene sequencing;the drug resistance genes,virulence genes and ST subtypes were observed by means of whole-genome sequencing(WGS)technique.RESULTS Totally 18 strains of XDR-hvKP were collected,55.56％(10/18)of which were isolated from blood specimens,and 61.11％(11/18)were isolated from critical care medicine department.Sanger sequen-cing analysis showed that all of the strains carried blaKPC-2 drug resistance gene;rmpA2(100.00％)and rmpA,i-roN,iutA(94.44％,17/18)were the major virulence genes carried by the strains.WGS analysis indicated that all of the 18 XDR-hvKP isolates carried multiple drug resistance genes such as blaKPC-2 carbapenemase and the viru-lence genes like capsule(rmpA/rmpA2),aerobacterin(iucABCD-iutA),Salmonella(iroN)and yersinin(ybt).All of the ST subtypes were ST 11,and all of the capsular serotypes were KL 64.CONCLUSIONS The ST11-KL64 type XDR-hvKP strains carry blaKPC-2;rmpA,rmpA2,iroN and iutA are the major virulence genes.It is necessary to strengthen the monitoring of key population of the key departments and make joint efforts of multiple departments to contain the transmission of the strains.

The female reproductive system is essential for sustaining reproductive endocrine homeostasis,however,its vulnerability to various endogenous and exogenous insults,including pathological conditions,pharmacological agents,genetic predispositions,and environmental factors,often results in compromised fertility.The existing protective approaches(including surgical interventions,hormonal replacement therapies,and assisted reproductive techniques)are constrained by several limitations,such as adverse therapeutic effects,technical complexities,and their incapacity to reverse ovarian senescence.Artemisinin and its derivatives(ARTs),characterized by their unique endoperoxide bridge configuration,have exhibited outstanding therapeutic performance across multiple domains including malaria treatment,anticancer therapy,inflammation modulation,and parasitic infection control.Emerging research has identified their novel protective capabilities against various reproductive system pathologies.This comprehensive review systematically elucidates the molecular mechanisms underlying artemisinin-based interventions in reproductive pathologies and evaluates their clinical translation prospects,thereby proposing innovative strategies for the development of next-generation fertility-protective agents with enhanced safety and efficacy profiles.

Objective:To investigate the effects of modified TROPIS surgery, incorporating both open and hanging stitches on the treatment of elevated anal fistula.Methods:For our study, 92 patients with high anal fistula, admitted between Jan. 2023 and Jun. 2024, were selected. They were divided into two categories: the observation group (45 modified TROPIS surgery) and the control group (47 cuts) ,to observe the clinical efficacy, postoperative recovery, quality of life and complications of the two groups.Results:Compared with the control group, the operation time, intraoperative blood loss and postoperative hospitalization time decreased in the observation group ( P<0.05) . Compared with the control group, the healing time of anal fistula in the observation group was (28.01±5.02) d, shorter than that in the control group (35.05±7.09) d, the healing rate was 97.78%, higher than that in the control group (80.85%) , and the recurrence rate was 4.44%, lower than that in the control group (19.14%) ( P<0.05) . The degree of postoperative pain and Wexner anal incontinence score in the observation group were lower than those in the control group ( P<0.05) . The life quality score of the observation group was higher than that of the control group ( P<0.05) . The complication rate of observation group was lower than that of control group ( P<0.05) . Conclusions:The modified TROPIS surgical method evidently provides advantages in anal fistula treatment. It can not only efficiently eliminate infections but also reduce anal movements and decrease the chances of post-surgical injuries and complications, presenting enhanced clinical options and suggesting a promising outlook for traditional anal fistula surgery.

Objective:To investigate the effects of modified TROPIS surgery, incorporating both open and hanging stitches on the treatment of elevated anal fistula.Methods:For our study, 92 patients with high anal fistula, admitted between Jan. 2023 and Jun. 2024, were selected. They were divided into two categories: the observation group (45 modified TROPIS surgery) and the control group (47 cuts) ,to observe the clinical efficacy, postoperative recovery, quality of life and complications of the two groups.Results:Compared with the control group, the operation time, intraoperative blood loss and postoperative hospitalization time decreased in the observation group ( P<0.05) . Compared with the control group, the healing time of anal fistula in the observation group was (28.01±5.02) d, shorter than that in the control group (35.05±7.09) d, the healing rate was 97.78%, higher than that in the control group (80.85%) , and the recurrence rate was 4.44%, lower than that in the control group (19.14%) ( P<0.05) . The degree of postoperative pain and Wexner anal incontinence score in the observation group were lower than those in the control group ( P<0.05) . The life quality score of the observation group was higher than that of the control group ( P<0.05) . The complication rate of observation group was lower than that of control group ( P<0.05) . Conclusions:The modified TROPIS surgical method evidently provides advantages in anal fistula treatment. It can not only efficiently eliminate infections but also reduce anal movements and decrease the chances of post-surgical injuries and complications, presenting enhanced clinical options and suggesting a promising outlook for traditional anal fistula surgery.

OBJECTIVE To understand the drug resistance genes,virulence genes and molecular epidemiological characteristics of extensively drug-resistant hypervirulent Klebsiella pneumoniae(XDR-hvKP)strains causing hospital-associated infection.METHODS The clinical isolates of XDR-hvKP were collected from Tongling People's Hospital from Jul.2020 to Dec.2022.The strains were identified by matrix-assisted laser desorption ionization-time-of-flight(MALDI-TOF)mass spectrometry,the common drug resistance genes and virulence genes were an-alyzed by Sanger sequencing,the capsular serotypes were determined by wzi gene sequencing;the drug resistance genes,virulence genes and ST subtypes were observed by means of whole-genome sequencing(WGS)technique.RESULTS Totally 18 strains of XDR-hvKP were collected,55.56％(10/18)of which were isolated from blood specimens,and 61.11％(11/18)were isolated from critical care medicine department.Sanger sequen-cing analysis showed that all of the strains carried blaKPC-2 drug resistance gene;rmpA2(100.00％)and rmpA,i-roN,iutA(94.44％,17/18)were the major virulence genes carried by the strains.WGS analysis indicated that all of the 18 XDR-hvKP isolates carried multiple drug resistance genes such as blaKPC-2 carbapenemase and the viru-lence genes like capsule(rmpA/rmpA2),aerobacterin(iucABCD-iutA),Salmonella(iroN)and yersinin(ybt).All of the ST subtypes were ST 11,and all of the capsular serotypes were KL 64.CONCLUSIONS The ST11-KL64 type XDR-hvKP strains carry blaKPC-2;rmpA,rmpA2,iroN and iutA are the major virulence genes.It is necessary to strengthen the monitoring of key population of the key departments and make joint efforts of multiple departments to contain the transmission of the strains.

The female reproductive system is essential for sustaining reproductive endocrine homeostasis,however,its vulnerability to various endogenous and exogenous insults,including pathological conditions,pharmacological agents,genetic predispositions,and environmental factors,often results in compromised fertility.The existing protective approaches(including surgical interventions,hormonal replacement therapies,and assisted reproductive techniques)are constrained by several limitations,such as adverse therapeutic effects,technical complexities,and their incapacity to reverse ovarian senescence.Artemisinin and its derivatives(ARTs),characterized by their unique endoperoxide bridge configuration,have exhibited outstanding therapeutic performance across multiple domains including malaria treatment,anticancer therapy,inflammation modulation,and parasitic infection control.Emerging research has identified their novel protective capabilities against various reproductive system pathologies.This comprehensive review systematically elucidates the molecular mechanisms underlying artemisinin-based interventions in reproductive pathologies and evaluates their clinical translation prospects,thereby proposing innovative strategies for the development of next-generation fertility-protective agents with enhanced safety and efficacy profiles.

The structures and activities of enzymes are influenced by pH of the environment. Understanding and distinguishing the adaptation mechanisms of enzymes to extreme pH values is of great significance for elucidating the molecular mechanisms and promoting the industrial applications of enzymes. In this study, the ESM-2 protein language model was used to encode the secreted microbial proteins with the optimal performance above pH 9 and below pH 5, which yielded 47 725 high-pH protein sequences and 66 079 low-pH protein sequences, respectively. A deep learning model was constructed to identify protein acid-base tolerance based on amino acid sequences. The model showcased significantly higher accuracy than other methods, with the overall accuracy of 94.8%, precision of 91.8%, and a recall rate of 93.4% on the test set. Furthermore, we built a website (https://enzymepred.biodesign.ac.cn), which enabled users to predict the acid-base tolerance by submitting the protein sequences of enzymes. This study has accelerated the application of enzymes in various fields, including biotechnology, pharmaceuticals, and chemicals. It provides a powerful tool for the rapid screening and optimization of industrial enzymes.
Deep Learning ; Hydrogen-Ion Concentration ; Amino Acid Sequence ; Enzymes/metabolism* ; Sequence Analysis, Protein ; Proteins/metabolism* ; Bacterial Proteins/metabolism*

Objective To investigate the relationship between serum interleukin-1β(IL-1β),C-reactive pro-tein to albumin ratio(CAR)and heparin-binding protein(HBP)levels and secondary pulmonary Candida albi-cans infection during chemotherapy in patients with lung cancer.Methods A total of 175 patients with lung cancer admitted to this hospital from January 2018 to April 2022 were selected,and all patients were treated with chemotherapy.According to whether they had secondary pulmonary Candida albicans infection,they were divided into infection group(37 cases)and non-infection group(138 cases).The clinical data,serum levels of IL-1β,HBP and CAR were compared between the two groups.Multivariate Logistic regression analysis was used to analyze the influencing factors of secondary pulmonary Candida albicans infection in patients with lung cancer during chemotherapy.The receiver operating characteristic(ROC)curve was used to analyze the pre-dictive value of serum IL-1β,CAR and HBP for secondary pulmonary Candida albicans infection during chemo-therapy,and the prognosis of lung cancer patients with different serum IL-1β,HBP levels and CAR were com-pared.Results There were significant differences in smoking,pathological stage,diabetes mellitus,chemo-therapy cycle,chronic obstructive pulmonary disease and anatomical location between the infection group and the non-infection group(P＜0.05).The serum levels of IL-1β,HBP and CAR in the infection group were higher than those in the non-infection group(P＜0.05).Multivariate Logistic regression analysis showed that smoking,diabetes,anatomical site,chronic obstructive pulmonary disease,pathological stage,chemotherapy cycle and serum IL-1β,HBP,CAR were the influencing factors of secondary pulmonary Candida albicans infec-tion in patients with lung cancer during chemotherapy(P＜0.05).The area under the curve(AUC)of serumIL-1β,CAR and HBP in predicting pulmonary Candida albicans infection was 0.779,0.732 and 0.796,respec-tively.The AUC of the combination of the three was 0.931,and the sensitivity and specificity were 86.49％and 90.58％,respectively.The survival rate of lung cancer patients with high levels of IL-1β,CAR and HBP was sig-nificantly lower than that of patients with low levels of Il-1β,CAR and HBP(P＜0.05).Conclusion The in-crease of serum IL-1β,CAR and HBP levels in patients with lung cancer is related to secondary pulmonary Candida albicans infection during chemotherapy.Detection of these serum Il-1β,CAR and HBP levels is helpful to predict the risk of pulmonary Candida albicans infection and death.

Objective:To investigate the efficacy of tibiofibular-based reconstruction technique with single femoral tunnel for Fanelli type C posterolateral complex (PLC) injury.Methods:A retrospective case series study was conducted to analyze the clinical data of 16 patients with Fanelli type C PLC injury admitted to Tianjin Hospital from July 2016 to July 2019, including 10 males and 6 females, aged 20-61 years [(36.5±13.9)years]. PLC reconstruction was performed by tibiofibular-based technique with single femoral tunnel using gracilis tendon and semi-tendinosus autografts. If the posterior and anterior cruciate ligaments (PCL/ACL) rupture were combined, arthroscopic single bundle reconstruction was performed simultaneously. If the posteromedial corner (PMC) injury was combined, PMC repair or reconstruction surgery was performed simultaneously. Operation time and intraoperative blood loss were recorded. When the bone needle and tunnel for PLC were drilled during the operation, the interference of the femoral tunnel through the cruciate ligament was observed under the arthroscope. Before and at 6 and 12 months after operation, the varus stability of the knee joint was evaluated with the difference of lateral joint space width of both knees and the International Knee Documentation Committee (IKDC) objective classification of varus stability of the knee joint; the external rotation stability was evaluated with the difference of external rotation angle of both knees and the IKDC objective classification of external rotation stability of the knee joint. Before, at 6 and 12 months after operation and at the last follow-up, IKDC 2000 subjective score and Lysholm score were compared. The occurrence of complications was observed.Results:All the patients were followed up for 12-36 months [24(15, 33)months]. The operation time was 100-220 minutes [175.0(111.3, 200.0)minutes], with intraoperative blood loss of 30-150 ml [(84.3±36.5)ml]. Intraoperative arthroscopy showed no interference of perforation between PLC and cruciate ligament femoral tunnel. The differences of lateral joint space width of both knees at 6 and 12 months after operation were 0.5(0.2, 1.4)mm and 0.6(0.2, 1.5)mm respectively, which were both significantly improved compared with 12.1(10.8, 12.6)mm before operation ( P<0.05), while there was no significant difference at 6 and 12 months after operation ( P>0.05). The IKDC objective classification of varus stability of the knee joint was grade A in 13 patients, grade B in two and grade C in one at 6 or 12 months after operation, and showed statistical difference from grade D in all the patients before operation ( P<0.01). At 6 and 12 months after operation, the difference of external rotation angle of both knees was -2.0(-3.2, 1.3)° and -1.4(-3.0, 1.7)° respectively, which were significantly improved compared with 16.8(13.9, 18.4)° before operation ( P<0.05), while there was no significant difference at 6 and 12 months after operation ( P>0.05). IKDC objective classification of external rotation stability of the knee joint was grade A in 14 patients, grade B in one and grade C in one at 6 or 12 months after operation, and showed statistical difference from grade C in 14 patients and grade D in 2 before operation ( P<0.01). At 6 and 12 months after operation and at the last follow-up, the IKDC 2000 subjective scores [(76.3±4.7)points, (80.3±4.4)points, (79.9±3.8)points respectively] and the Lysholm scores [(76.1±3.9)points, (81.1±4.3)points, (82.8±3.2)points respectively] were significantly improved compared with those before operation [(48.6±3.7)points and (52.6±2.4)points] ( P<0.05). The IKDC 2000 subjective scores and Lysholm scores were significantly improved at 12 months after operation and at the last follow-up than those at 6 months after operation ( P<0.05). There were no significant differences in the IKDC 2000 subjective scores and Lysholm scores at 12 months after operation and at the last follow-up ( P>0.05). There were no complications such as wound infection, vascular and nerve injury, joint stiffness or ectopic ossification. Conclusion:For Fanelli type C PLC injury, tibiofibular-based reconstruction technique with single femoral tunnel reduces the interference between the lateral femoral tunnels, significantly improves the varus and external rotation stability and the function of the knee joint, and has few complications and satisfactory short-term clinical efficacy.