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 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.

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.

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*

Insufficient catalytic efficiency of flavonoid 6-hydroxylases in the fermentative production of scutellarin leads to the formation of at least about 18% of by-products. Here, the catalytic mechanisms of two flavonoid 6-hydroxylases, CYP82D4 and CYP706X, were investigated by molecular dynamics simulations and quantum chemical calculations. Our results show that CYP82D4 and CYP706X have almost identical energy barriers at the rate-determining step and thus similar reaction rates, while the relatively low substrate binding energy of CYP82D4 may facilitate product release, which is directly responsible for its higher catalytic efficiency. Based on the study of substrate entry and release processes, the catalytic efficiency of the L540A mutation of CYP82D4 increased by 1.37-fold, demonstrating the feasibility of theoretical calculations-guided engineering of flavonoid 6-hydroxylase. Overall, this study reveals the catalytic mechanism of flavonoid 6-hydroxylases, which may facilitate the modification and optimization of flavonoid 6-hydroxylases for efficient fermentative production of scutellarin.
Cytochrome P-450 Enzyme System/metabolism* ; Apigenin ; Glucuronates

OBJECTIVE: To summarize the clinical features, surgical methods, and prognosis of bucket-handle meniscal tears (BHMTs), and provide guidance for clinical treatment. METHODS: The clinical data of 91 BHMTs patients (91 knees), who met the selection criteria and were admitted between January 2015 and January 2021, was retrospectively analyzed. There were 68 males and 23 females. Age ranged from 16 to 58 years with an average of 34.4 years. The injury was caused by sports in 68 cases, traffic accident in 15 cases, and falls or sprains in 8 cases. There were 49 cases of left knee injury and 42 cases of right knee injury. The time from the onset of symptoms to the admission ranged from 1 day to 13 months (median, 18 days), including >1 month in 35 cases and ≤1 month in 56 cases. Medial BHMTs occurred in 52 cases and lateral BHMTs in 39 cases. There were 36 cases with ACL rupture and 12 cases with discoid meniscus. The knee extension was limited more than 10° in 55 cases. According to the condition of meniscus injury, the meniscus suture with Inside-out combined with All-inside techniques (54 cases) or meniscoplasty (37 cases) under arthroscopy were selected. ACL reconstruction was performed in all patients with ACL rupture with autogenous hamstring tendon. Postoperative complications were observed. International Knee Documentation Committee (IKDC) score, Lysholm score, and Tegner score were used to evaluate knee function, and clinical failure was recorded. RESULTS: Two patients developed intermuscular venous thrombosis, which improved after oral anticoagulant therapy. No vascular injury, postoperative infection, joint stiffness, or other complications occurred in all patients. All patients were followed up 24-95 months, with a median of 64 months. A total of 12 cases (13.19%) failed the operation and were re-operated or given oral anti-inflammatory analgesics and rehabilitation therapy. At last follow-up, IKDC score and Lysholm score of 91 patients significantly increased when compared with those before operation ( P<0.05), while Tegner score significantly decreased ( P<0.05). The above indexes of patients treated with meniscus suture and meniscoplasty were also significantly different from those before operation ( P<0.05). CONCLUSION BHMTs occurs mostly in young men and is one of the important reasons for the limitation of knee extension after trauma. Arthroscopic meniscus suture and meniscoplasty can obtain good effectiveness according to individual conditions of patients. But the latter can better preserve the shape and function of meniscus, and theoretically can obtain better long-term outcomes, which needs to be confirmed by further research with larger sample size.
Male ; Female ; Humans ; Adolescent ; Young Adult ; Adult ; Middle Aged ; Treatment Outcome ; Retrospective Studies ; Meniscus ; Knee Joint/surgery* ; Menisci, Tibial/surgery* ; Knee Injuries/diagnosis* ; Rupture ; Tibial Meniscus Injuries/surgery* ; Arthroscopy/methods* ; Anterior Cruciate Ligament Injuries/surgery*

Objective:To explore the influencing factors of postoperative recurrence in patients with complex anal fistula, and to construct a nomogram model to predict the risk of postoperative recurrence and verify it.Methods:Clinical data of 310 patients with complex anal fistula who underwent fistulectomy in the hospital from Aug. 2019 to Mar. 2023 were retrospectively selected and divided into modeling group (93 cases) and validation group (217 cases) in a 3∶7 ratio according to system randomization method. Hospital electronic medical record system was used to collect patient baseline data and calculate the recurrence rate of patients 6 months after surgery. According to the data of the modeling group, multivariate Logistic regression was used to analyze the influencing factors of postoperative recurrence in patients with complex anal fistula. Based on the influencing factors, a nomogram model was established to predict the risk of postoperative recurrence, and external verification was performed based on the data of the validation group.Results:The recurrence rate at 6 months after operation was 20.43% (19/93) in the modeling group and 17.51% (38/217) in the validation group. There was no significant difference in recurrence rate between the two groups ( χ2=0.370, P=0.543) . The proportion of male, smoking history, diabetes mellitus, high anal fistula and unclear position of internal orifice in the recurrence group was higher than that in the non-recurrence group, and the body mass index and course of disease were higher than those in the non-recurrence group ( P<0.05) . Based on the above seven influencing factors, a nomogram model of the risk of recurrence of complex anal fistula after surgery was established. C index of the modeling group and the validation group was 0.984 and 0.798 respectively, the calibration curve was close to the ideal curve, and the Receiver operating characteristic AUC of the nomogram prediction model was>0.70, indicating that model consistency, prediction efficiency and differentiation were good. Conclusion:The nomogram prediction model based on gender, body mass index, smoking history, diabetes mellitus, course of disease, high anal fistula and internal orifice position can effectively predict the risk of postoperative recurrence in patients with complex anal fistula.

Objective:To observe the therapeutic timing and dosage of intravenous immunoglobulin (IVIG) in children with severe adenovirus pneumonia.Methods:Clinical data of children with severe adenovirus pneumonia treated with IVIG at the Department of Respiratory, Guangzhou Women and Children′s Medical Center, Guangzhou Medical University from January 2019 to January 2020 were retrospectively analyzed.Participants were classified as early presenters (5-10 days of illness course) and later presenters (11-15 days of illness course) according to the timing of IVIG treatment.They were further subdivided into plan 1 group[1 g/(kg·d) IVIG for 2 days] and plan 2 group [0.4-0.5 g/(kg·d) IVIG for 3-5 days]. Continuous variables and categorical variables between groups were analyzed by the nonparametric Mann- Whitney U test and the Fisher′ s exact test, respectively. Results:A total of 202 patients with the median age of 12 (12, 36) months were recruited, involving 128 early presenters (63.37%) and 74 later presen-ters (36.63%). Later presenters had a longer duration of fever [18.00(14.00, 23.25) days vs.11.00(9.00, 14.00) days], more demands for mechanical ventilation (33.78% vs.20.31%), and higher incidence of bronchiectasis (9.46% vs.1.56%) than those of early presenters (all P<0.05). For early presenters, no significant differences were detected in the demand for advanced life support, outcomes and sequelae between plan 1 group and plan 2 group (all P>0.05). For later presenters, a shorter duration of fever [18.00(14.00, 21.00) days vs.21.00(15.50, 30.75) days] and lower usage of extracorporeal membrane oxygenation (ECMO, 2.13% vs.18.52%) were observed in the plan 1 group than that of plan 2 group (all P<0.05). The incidence of post-infectious bronchiolitis obliterans and bronchiectasis as pulmonary sequelae was comparable between plan 1 group and plan 2 group ( P>0.05). The incidence of adverse events was 5.77% during IVIG infusion, showing no significant difference between plan 1 group and plan 2 group ( P>0.05). Conclusions:Early treatment of IVIG are very important to improve the prognosis of children with severe adenovirus pneumonia.For later presenters, a high dosage of IVIG is effective in reducing the ECMO use and shortening the duration of fever, thus providing clinical benefits.

Objective:To investigate the effect of medial open wedge high tibial osteotomy (HTO) on moderate and severe medial compartmental knee osteoarthritis.Methods:This study retrospectively reviewed patients treated with medial open wedge HTO between January 2017 and January 2019. All cases were followed up for more than 2 years. There were 28 patients with severe osteoarthritis, including 11 males and 17 females, aged 56.36±5.06 years. There were 32 patients with moderate osteoarthritis, including 12 males and 20 females, aged 54.16±6.3 years. Kellgren-Lawrence (K-L) grading, hip-knee-ankle angle (HKA), weight bearing line (WBL) ratio, joint line convergence angle (JLCA), medial joint space (MJS), posterior tibial slope (PTS), medial meniscus extrusion (MME) and medial meniscus extrusion ratio (MMER) were evaluated on radiographs before and 2 years after surgery. The International Cartilage Repair Society (ICRS) of the cartilage of medial femoral condyle (MFC) and medial tibial condyle (MTC) were evaluated under arthroscopy. The clinical outcomes were assessed by the Western Ontario and McMaster University (WOMAC) score, visual analogue scale (VAS) and the degree of medial collateral ligament (MCL) pseudo-relaxation. Predictors of MCL pseudo-relaxation were screened by binary logistic regression analysis.Results:K-L grading, HKA, WBL ratio and MJS improved in both groups after surgery, with no statistical significance between groups ( P>0.05). The postoperative JLCA was improved, whereas MME and MMER were not. And the severe group was higher than the moderate group before and after surgery ( P<0.05). Cartilage of MFC: in the severe group, 2 cases of ICRS 3 grade, 26 of 4 grade improved to 11 of 2 grade, 12 of 3 grade, 5 of 4 grade; in the moderate group, 12 cases of ICRS 2 grade, 18 of 3 grade, 2 of 4 grade improved to 30 of 2 grade, 2 of 3 grade. Cartilage of MTC: in the severe group, 2 cases of ICRS 3 grade, 26 of 4 grade improved to 17 of 2 grade, 8 of 3 grade, 3 of 4 grade; in the moderate group, 11 of 2 grade, 8 of 3 grade, 3 of 4 grade improved to 27 of 2 grade, 5 of 3 grade. The postoperative WOMAC score of the severe group improved from 50.71±8.07 to 3.86±1.84, while in the moderate group it improved from 44.09±6.63 to 3.34±2.24. The postoperative VAS score of the severe group decreased from 7.14±1.21 to 3.34±2.24, whereas it decreased from 6.38±1.24 to 0.44±0.62 in the moderate group ( P<0.05). There was no significant difference between the postoperative groups ( P>0.05). In the severe group, the degree of pseudo-relaxation of the MCL improved from preoperative 25 of I degree, 3 of II degree to postoperative 28 of 0 degree at the 0° position, and from 25 of I degree, 3 of II degree to 24 of 0 degree, 4 of I degree at 30° position ( P<0.05). In the moderate group, the degree of pseudo-relaxation of the MCL improved from preoperative 31 of 0 degree, 1 of I degree to postoperative 32 of 0 degree at the 0° position, and from 28 of 0 degree, 4 of I degree to 32 of 0 degree at the 30° position ( P<0.05). JLCA ( OR=0.08, 95% CI: 0.007, 0.948, P=0.045), MME ( OR=0.11, 95% CI: 0.100, 1.32, P=0.082) and MMER ( OR=0.66, 95% CI: 0.422, 1.030, P=0.067) were independent predictors of MCL pseudo-relaxation (the difference was statistically significant at P<0.10). Conclusion:Medial open wedge HTO has significant short-term clinical effect on the treatment of moderate and severe medial compartmental knee osteoarthritis, which can effectively relieve pain symptoms, improve joint function, and restore medial knee stability. Preoperative large JLCA, MME and MMER indicate pseudo-relaxation of the MCL.

Objective:To investigate the factors affecting phenotypes in the patients of methylmalonic acidemia combined with homocysteinemia cblC type with MMACHC c. 609G>A homologous variant. Methods:A retrospective study on the clinical manifestations, complications, treatment, and outcome in 164patients of cblC type with MMACHC c. 609G>A homologous variant was conducted.The patients were diagnosed by biochemical and genetic analysisfrom January 1998 to December 2020. Results:Among the 164 patients, 2 cases were prenatally diagnosed and began treatment after birth. They are 3 and 12 years old with normal physical and mental development. Twenty-one cases were diagnosed by newborn screening. Among them, 15 cases had with normal development. They were treated fromthe age of two weeks at the asymptomatic period. Six cases began treatment aged 1 to 3 months after onset. Their development was delayed. One hundred and forty-one cases were clinically diagnosed. Their onset age ranges from a few minutes after birth to 6 years old. 110 cases had early-onset (78.0%). 31 cases had late-onset (22.0%). Five of them died. 24 patients lost to follow-up. Of the 141 clinically diagnosed patients, 130 (92.2%) with psychomotor retardation, 69 (48.9%) with epilepsy, 39 (27.7%) with anemia, 30 (21.3%) had visual impairment, 27 (19.1%) had hydrocephalus, 26 (18.4%) had feeding difficulties, 7 (5.0%) with liver damage, and 5 (3.5%) with metabolic syndrome. The frequency of hydrocephalus and seizures was significantly higher in the early-onset group. The urinary methylmalonic acid increased significantly in the patients with epilepsy. During the long-term follow-up, the level of plasma total homocysteine in the seizure-uncontrolled group was significantly higher than that in the seizure-controlled group, the difference had a statistical significance ( P<0.05). Conclusion:Most of the patients with MMACHC c. 609G>A homozygous variant had early-onset disease, with a high mortality and disability rate. If not treated in time, it will lead to neurological damage, resulting in epilepsy, mental retardation, hydrocephalus, and multiple organ damage. Pre-symptomatic diagnosis and treatment are crucial to prevent irreversible neurological damage. Neonatal screening and prenatal diagnosis are important to improve the outcome of the patients.