Site-directed mutagenesis of bifunctional riboflavin kinase/FMN adenylyltransferase via CRISPR/Cas9 to enhance riboflavin production.

Vitamin B2 is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D, which respectively decreased by 29.90 % and 89.32 % compared to wild-type FAD synthase, were selected for CRISPR/Cas9 gene editing of the genome, generating engineered strains R203 and R210. pNEW-AZ was transformed into R203, generating R6. R6 produced 657.38 ± 47.48 mg/L riboflavin, a 21.69 % increase compared to R5. This study contributes to the high production of riboflavin in recombinant E. coli BL21.

Biosynthesis of the phycocyanin ß-subunit in Escherichia coli BL21 and its antioxidant activity and application in the preservation of fresh-cut apples.

In this study, the biosynthesis of phycocyanin ß-subunit (CpcB) in Escherichia coli BL21 was investigated, and its antioxidant activity and application in anti-browning of fresh-cut apples was explored. Four genes (cpcB, cpeS, hox1 and pcyA) involved in the biosynthesis of CpcB were cloned and transformed into E. coli BL21 by constructing recombinant plasmid pETDuet-5. The positive transformant was screened by ampicillin resistance. The analysis of SDS-PAGE and zinc fluorescence spectrum showed that CpcB was successfully expressed in E. coli BL21 with a molecular weight of 21 kDa. The purified CpcB had a maximum absorption peak at 615 nm, and its maximum florescence emission wavelength was 640 nm. It exhibited a stronger ability to scavenge four free radicals than Vc. The color change in fresh-cut apples was obviously delayed by the CpcB treatment. These results suggest that CpcB may be used as a potential anti-browning agent for food preservation.

Achieving a win-win situation by promoting internet recycling of waste electronics and preventing information leakage in a multi-party game.

With scientific and technological advancements and the diversification of residents' demands, the pace of electronic product replacement is accelerating, generating a rapidly increasing number of waste electronics. The rapid development of information technologies, such as the Internet, has brought significant opportunities for recycling waste electronics. However, this is hindered by information safety concerns from residents. To achieve a win-win situation of promoting Internet recycling of waste electronics and preventing information leakage, this study performed a game analysis using key stakeholders in the Internet recycling of waste electronics. The game analysis of recycling waste electronics revealed that the lower the personal information leakage, the more residents would participate in recycling. Strict government regulation would increase the credibility of Internet recycling companies in protecting information security. Further, if the government imposed high fines on companies that breach information security, Internet recycling companies would endeavor to protect information security. In conclusion, this study offers policy recommendations and a theoretical basis to achieve a win-win situation of promoting Internet recycling of waste electronics and preventing information leakage from the perspective of stakeholders.

Deep degradation of sulfamethoxazole by the Fe-Co/γ-Al2O3-catalysed photo-Fenton system.

The heterogeneous photo-Fenton system using Fe-Co/γ-Al2O3 as a catalyst was applied in the study of sulfamethoxazole(SMX) degradation. The morphology, structure, elemental composition and metal valence distribution of Fe-Co/γ-Al2O3 were found to be relatively stable before and after the reaction. The highest SMX degradation efficiency and mineralization (The ratio of organic matter being oxidized to carbon dioxide and water) were obtained under the conditions of 15% Fe-Co loading rate, 1:1 mass ratio of Fe and Co, 1 g/L catalyst dosage, 1.5 mL 30% H2O2 dosage, 18 W UV lamp power and 60 min reaction time, which were 98% and 66%, respectively. Radical quenching experiments and electronic paramagnetic resonance (EPR) characterization revealed that ·OH played an important role in the degradation and mineralization SMX in the Fe-Co/γ-Al2O3 heterogeneous photo-Fenton system. Combined with the analysis of N, S and intermediate products, there may be three degradation pathways of SMX in the heterogeneous photo-Fenton system. This work provides a technical reference for realizing the efficient degradation and mineralization of SMX in a heterogeneous photo-Fenton reaction system.

Case report: The MSI-L/p-MMR metastatic rectal cancer patient who failed systemic therapy responds to anti-PD-1 immunotherapy after stereotactic body radiation-therapy.

Background: Traditionally, patients with microsatellite stability (MSS)/microsatellite instability-Low (MSI-L)/proficient mismatch repair (p-MMR) metastatic colorectal cancer (mCRC) have had poor benefit from immunotherapy. Therefore, how to enhance the response of immunotherapy is still a challenge for MSS/MSI-L/p-MMR CRC patient. Case presentation: We report a special case of a rectal cancer patient with programmed death-ligand 1 (PD-L1) negative expression, MSI-L/p-MMR, tumor mutational burden-low (TMB-L) and liver metastases, who partial response (PR) to immunotherapy after systemic therapy failure including chemotherapy, anti-angiogenesis therapy and stereotactic body radiation-therapy (SBRT). The computed tomography (CT) results showed that among three liver metastases had been reduction or disappearance after Tislelizumab treatment for three times. Besides, the carcinoembryonic antigen (CEA) and carbohydrate antigen 199 (CA199) decrease and maintained at a low level for 3 months. The progression-free survival (PFS) of patient has exceeded 3 months. Conclusions: This case indicates that the patient with MSI-L/p-MMR mCRC can respond to anti-PD-1 immunotherapy after systemic therapy. And the SBRT (targeting liver metastases) may a method for increase-sensitivity of immunotherapy in CRC patients with MSI-L/p-MMR.

Comprehensive Analysis of Gut Microbiota and Fecal Bile Acid Profiles in Children With Biliary Atresia.

Background: Biliary atresia (BA) is the most common cholestatic liver disease in neonates. Herein, we aimed at characterizing the gut microbiota and fecal bile acid profiles of BA patients, defining the correlations between them, and evaluating the relationship between the clinical pathogenesis and changes in the gut microbiota and bile acid profiles. Methods: A total of 84 fecal samples from BA patients (n = 46) and matched healthy controls (HCs, n = 38) were subjected to sequencing by 16S rRNA gene amplification, and fecal bile acid were analyzed by targeted metabolomics. Findings: Compared with the controls, a structural separation of the intestinal flora of BA patients was uncovered, which was accompanied by changes in the composition of fecal bile acids. In the BA group, Actinobacillus, Monoglobus, and Agathobacter were enriched in patients without cholangitis (p < 0.05). Selenomonadaceae and Megamonas were more abundant in patients without recurrent cholangitis episodes (p < 0.05), while Lachnospiraceae and Ruminococcaceae were enriched in patients with multiple recurrences of cholangitis (p < 0.05). Postoperative jaundice clearance was associated with Campylobacter and Rikenellaceae (p < 0.05), and tauroursodeoxycholic acid was associated with jaundice clearance (p < 0.001). Conclusion: BA patients are characterized by different compositions of gut microbiota and bile acids, and their interaction is involved in the process of liver damage in BA, which may be closely related to the occurrence of postoperative cholangitis and jaundice clearance.

Growth assessments for children with recurrent tracheoesophageal fistulas.

PURPOSE: To assess the growth status of children with recurrent tracheoesophageal fistula (rTEF), and determine the possible risk factors of growth retardation (GR). METHODS: The medical records of 83 patients with rTEF who underwent surgical repair were retrospectively analyzed. The patients were retrospectively divided into two groups according to whether they had GR. The clinical variables were compared between the GR and non-GR groups. Univariate and multivariable logistic regression analysis were performed to identify the risk factors for GR. RESULTS: Eighty-three children diagnosed with rTEF were included in this study. After a median follow-up of 31.4 (19.8, 48.7) months, GR occurred in 28 patients (33.7%). Among them, six patients with only weight for age Z score (WAZ) < -2SD, five patients with only height for age Z score (HAZ) < -2SD, and six patients with only BMI for age Z score (BAZ) < -2SD, while seven patients with both WAZ and HAZ < -2SD and four patients with both WAZ, HAZ and BAZ < -2SD. Multivariate logistic regression analysis showed that birth weight, anastomotic stricture and dysphagia after rTEF repair were independent risk factors with OR of 0.325 (0.119, 0.891), 4.396 (1.451, 13.324) and 5.341 (1.153, 24.752) for GR, respectively. CONCLUSIONS: GR is a common complication after rTEF repair. Birth weight, anastomotic stricture and dysphagia after rTEF repair are independent risk factors affecting growth.

The largest report on thoracoscopic surgery for recurrent tracheoesophageal fistula after esophageal atresia repair.

BACKGROUND: Although thoracoscopic surgery has become the routine surgical method for esophageal atresia/tracheoesophageal fistula (EA/TEF), thoracoscopic treatment for recurrent tracheoesophageal fistula (rTEF) is far from popularized. OBJECTIVES: We aimed to explore the safety and efficacy of thoracoscopic treatment for rTEF with a large-cohort study. METHODS: We retrospectively analyzed the clinical characteristics and outcomes of 103 consecutive patients who underwent thoracoscopic surgery for rTEF by one surgeon after EA/TEF repair at two different institutions in China from 2014 to 2021. RESULTS: One hundred and three pediatric patients (67 boys) were enrolled and the primary operations were performed via thoracoscopic (n = 75, 72.82%) or open surgery (n = 28, 27.18%). The median age at rTEF diagnosis was 5 (3, 10) months after the primary repair. Patients were diagnosed with recurrent fistula to the trachea (n = 97, 94.17%), bronchi (n = 4, 3.88%), and lung parenchyma (n = 2, 1.94%), and all of them underwent thoracoscopic surgery at a median age of 7 (5, 14) months with a median weight of 6200 (4870, 7650) g. After the repair of rTEF, the incidence of esophageal leakage, esophageal stricture, and TEF recurrence were 12.8%, 33.4%, and 10.8%, respectively. After the follow-up, 87 patients survived, 6 died, and 10 were lost to follow-up. CONCLUSIONS: The results of thoracoscopic surgery for rTEF were comparable with previously reported thoracotomy surgery. Owing to the clear field during the operation, rapid patient recovery and esthetic results, the thoracoscopic approach could be a better choice for experienced pediatric surgeons. LEVEL OF EVIDENCE: LEVEL IV.

Retrospective analysis of pneumothorax after repair of esophageal atresia/tracheoesophageal fistula.

BACKGROUND: To analyze the possible causes, treatment and outcomes of postoperative pneumothorax in patients with Gross type C esophageal atresia/tracheoesophageal fistula (EA/TEF). METHODS: Medical records of patients with Gross type C EA/TEF who were diagnosed and treated in Beijing Children's Hospital from January 2007 to January 2020 were retrospectively collected. They were divided into 2 groups according to whether postoperative pneumothorax occurred. Univariate and multivariate logistic regression analysis were performed to identify risk factors for pneumothorax. RESULTS: A total of 188 patients were included, including 85 (45 %) in the pneumothorax group and 103 (55 %) in the non-pneumothorax group. Multivariate logistic regression analysis showed that postoperative anastomotic leakage [P < 0.001, OR 3.516 (1.859, 6.648)] and mechanical ventilation [P = 0.012, OR 2.399 (1.210, 4.758)] were independent risk factors for pneumothorax after EA/TEF repair. Further analysis of main parameters of mechanical ventilation after surgery showed that none of them were clearly related to the occurrence of pneumothorax. Among the 85 patients with pneumothorax, 33 gave up after surgery and 52 received further treatment [conservative observation (n = 20), pleural puncture (n = 11), pleural closed drainage (n = 9), both pleural puncture and closed drainage (n = 12)]. All of the 52 patients were cured of pneumothorax at discharge. CONCLUSIONS: Anastomotic leakage and postoperative mechanical ventilation were risk factors for pneumothorax after repair of Gross type C EA/TEF, but the main parameters of mechanical ventilation had no clear correlation with pneumothorax. After symptomatic treatment, the prognosis of pneumothorax was good.

Clinical comparison between thoracoscopic and thoracotomy repair of Gross type C esophageal atresia.

BACKGROUND: To compare the clinical outcomes between thoracoscopic approach and thoracotomy surgery in patients with Gross type C Esophageal atresia (EA) and tracheoesophageal fistula (TEF). METHODS: Patients with Gross type C EA/TEF who underwent surgery from January 2007 to January 2020 at Beijing Children's Hospital were retrospectively analyzed. The patients were divided into two groups according to surgical approaches. The perioperative factors and postoperative complications were compared among the two groups. RESULTS: One hundred and ninety patients (132 boys and 58 girls) with a median birth weight of 2975 (2600, 3200) g were included. The primary operations were performed via thoracoscopic (n = 62) and thoracotomy (n = 128) approach. After comparison of clinical characteristics between the two groups, we found that there were statistically significant differences in associated anomalies, method of fistula closure, duration of mechanical ventilation after surgery, feeding option before discharge, management of pneumothorax, and prognosis (all P < 0.05). To a certain extent, thoracoscopic surgery reduced the incidence of anastomotic leakage and increased the incidence of anastomotic stricture in this study. However, there were no statistically significant differences between the two groups in terms of operative time, postoperative pneumothorax, anastomotic leakage, anastomotic stricture, and recurrent tracheoesophageal fistula (all P > 0.05). CONCLUSIONS: Thoracoscopy surgery for Gross type C EA/TEF is a safe and effective, minimally invasive technique with comparable operative time and incidence of postoperative complications.

Risk Factors and Reasons for Treatment Abandonment for Patients With Esophageal Atresia: A Study From a Tertiary Care Hospital in Beijing, China.

Background: This study aims to identify the risk factors and reasons for treatment abandonment for patients with esophageal atresia (EA) in a tertiary care hospital in China. Methods: A retrospective study was conducted on 360 patients with EA admitted to Beijing Children's Hospital between January 1, 2007 and June 1, 2020. Medical records for treatment abandonment and non-treatment abandonment patients were compared. Univariate and multivariate logistic regression analyses were conducted to identify potential risk factors for treatment abandonment. Results: After the diagnosis of EA, parents of 107 patients refused surgical repair and discharged against medical advice, and 253 patients underwent surgical repair. Among these 253 patients, parents of 59 patients abandoned treatment after surgery; 52 patients were discharged in an unstable condition, and parents of seven patients abandoned resuscitation leading to death in the hospital. By comparing clinical characteristics between treatment abandonment before surgery (n = 107) and non-treatment abandonment (n = 253) groups, we found that mother's parity >1, unplanned admission to intensive care unit before surgery, associated anomalies, and Gross type A/B were significant independent risk factors for treatment abandonment before surgery. Furthermore, birth weight <2,545 g, being discharged from neonatal center/intensive care unit and other departments, unplanned admission to intensive care unit after surgery, operative time >133 min, admission before 2016, pneumothorax, and anastomotic leakage were significant independent risk factors for treatment abandonment after surgery. The reasons for treatment abandonment included financial difficulties, multiple malformations with poor prognosis, belief of incurability and concerns about the prognosis of the diseases, postoperative complications, and extensive length of intensive care unit stay. Conclusions: Treatment abandonment of children with EA/TEF is still a common and serious problem in China. This study showed that EA/TEF patients in critical conditions, with associated anomalies, Gross type A/B, and who had occurrence of complications had high-risk for treatment abandonment.

Efficacy and safety of human urinary kallidinogenase for acute ischemic stroke: a meta-analysis.

OBJECTIVE: Human urinary kallidinogenase (HUK) is a glycoprotein extracted from human urine that is used to treat stroke by triggering positive regulation of the kallikrein-kinin system. Our aim was to evaluate the efficacy and safety of HUK treatment for acute ischemic stroke. METHODS: We searched the online databases PubMed, Embase, Cochrane Library, Google Scholar, and China National Knowledge Infrastructure (CNKI) for papers published between January 2015 and December 2019. The quality of each trial was assessed using the Cochrane Reviewers' Handbook. Randomized controlled trials of HUK in patients with acute ischemic stroke were included. RESULTS: Sixteen trials with 1326 participants were included. The HUK injection groups had more neurological improvement than the control groups in National Institutes of Health Stroke Scale scores (mean difference, -1.65; 95% confidence interval [CI], -2.12 to -1.71) and clinical efficacy (1.30; 95% CI, 1.21 to 1.41). Subgroup analysis indicated that age may influence heterogeneity. Eleven trials reported adverse effects and there were no significant differences between the control and HUK groups (risk difference, 0.01; 95% CI, -0.02 to 0.04). CONCLUSIONS: HUK ameliorates neurological symptoms in stroke patients with few adverse effects. Further high-quality, large-scale randomized trials are needed to confirm these results.

Catheter ablation of atrial fibrillation after pericardiectomy: multi-center experience in China.

BACKGROUND: To study the effectiveness and safety of atrial fibrillation (AF) catheter ablation after pericardiectomy. METHODS: Data of 24 consecutive AF patients after pericardiectomy underwent catheter ablation from five centers were collected and analyzed retrospectively. All patients were followed up at 1, 3, and 12 months after catheter ablation. Results of a repeated blood test, electrocardiogram, and echocardiography during follow-up were also collected. Adverse events such as recurrence of AF, heart failure, stroke/transient ischemic attack (TIA) and minor, and major bleeding were recorded. All patients underwent brain magnetic resonance imaging (MRI) at the end of 12 months follow-up. RESULTS: Patients were young (20-73 years old, 48.1±11.0). Fifteen (62.5%) patients were male. CHA2DS2-VASc score (0-3, 0.21±0.41) was low in these 24 patients. Among these patients, 11 (45.8%) were paroxysmal AF, 8 (33.3%) were persistent AF, and 5 (20.8%) were long-lasting persistent AF. Left atrium diameter over 45 mm was detected in 17 (70.8%) patients. All patients underwent catheter ablation successfully. No peri-ablation procedure-related complication happened. Oral anticoagulant therapy was stopped 3 months after the final ablation. Anti-arrhythmia drugs were continued for all patients after ablation. For 12 months follow-up, AF recurred in 10 (41.7%) patients 3-7 months after the first ablation. MRI detected silent cerebral infarction (SCI) in 2 (8.3%) patients. No other adverse events occurred during follow-up. CONCLUSIONS: It is safe for AF patients to undergo catheter ablation after pericardiectomy, but the rate of recurrence of AF is high.

Chronic heart failure in patients with nonalcoholic fatty liver disease: prevalence, clinical features, and relevance.

Objective This study was performed to assess the prevalence of nonalcoholic fatty liver (NAFL) in patients with symptomatic congestive heart failure (CHF) and compare the clinical features with those of patients without NAFL. Methods In total, 102 patients with CHF were divided into NAFL and non-NAFL groups according to their hepatic ultrasonography findings. All patients underwent transthoracic echocardiography and cardiac magnetic resonance examination. Follow-up was performed for major cardiovascular events (MACE) and readmission due to heart failure at 1, 3, 6, and 12 months after the index hospitalization. Results NAFL was detected in 37 of 102 patients (36.27%). Compared with the non-NAFL group, patients with NAFL were younger, had a higher body mass index and left ventricular (LV) mass index, and had more severe fibrosis. MACE and readmission occurred in 15 patients in the NAFL group and 29 patients in the non-NAFL group, without a significant difference. Linear regression analysis revealed that after adjusting for confounders, NAFL was independently associated with the LV fibrosis size and the ratio of the LV fibrosis size to the LV mass index. Conclusions NAFL is present in more than one-third of patients with CHF and is associated with the severity of LV fibrosis.