BACKGROUND: Chronic kidney disease (CKD)-associated anemia (CKD-anemia) is associated with poor survival, and hemoglobin targets are often not achieved with current therapies. Phase 3 trials have demonstrated the treatment efficacy of roxadustat for CKD-anemia. This phase 4 study aims to evaluate the long-term (52-week) safety and effectiveness of roxadustat in a broad real-world patient population with CKD-anemia with and without dialysis in China. METHODS: This Phase 4 multicenter, open-label, prospective study, conducted from 24 November 2020 to 11 November 2022, evaluated the long-term safety and effectiveness of roxadustat for CKD-anemia in China. Patients aged ≥18 years with CKD-anemia with or without dialysis were included. The initial oral dose was 70-120 mg (weight-based followed by dose adjustment) over 52 weeks. The primary endpoint was safety based on adverse events (AEs). The secondary endpoints were hemoglobin changes from baseline and the proportion of patients who achieved mean hemoglobin ≥100 g/L. Effectiveness evaluable populations 1 (EE1) and EE2 included roxadustat-naïve and previously roxadustat-treated patients, respectively. The safety analysis set (SAF) included all patients who received ≥1 occasion. RESULTS: The EE1, EE2, and SAF populations included 1804, 193, and 2021 patients, respectively. In the SAF, the mean age was 50 ± 14 years, and 1087 patients (53.8%) were male. Mean baseline hemoglobin was 96.9 ± 14.0 g/L in EE1 and 100.3 ± 12.9 g/L in EE2. In EE1, the mean (95% confidence interval) hemoglobin changes from baseline over weeks 24-36 and 36-52 were 14.2 (13.5-14.9) g/L and 14.3 (13.5-15.0) g/L, respectively. Over weeks 24-36 and 36-52, 83.3% and 86.1% of patients in EE1 and 82.7% and 84.7% in EE2 achieved mean hemoglobin ≥100 g/L, respectively. In the SAF, 1643 (81.3%) patients experienced treatment-emergent AEs (TEAEs). Overall, 219 (10.8%) patients experienced drug-related TEAEs. Thirty-eight (1.9%) patients died of TEAEs (unrelated to the study drug). Vascular access thrombosis was uncommon. CONCLUSIONS: Roxadustat (52 weeks) increased hemoglobin and maintained the treatment target in Chinese patients with CKD-anemia with acceptable safety, supporting its use in real-world settings. REGISTRATION Chinese Clinical Trial Registry ( www.chictr.org.cn ) ChiCTR2100046322; CDE ( www.chinadrugtrials.org.cn ) CTR20201568.
Humans ; Male ; Female ; Anemia/etiology* ; Middle Aged ; Renal Insufficiency, Chronic/complications* ; Glycine/adverse effects* ; Isoquinolines/adverse effects* ; Aged ; Prospective Studies ; Adult ; Hemoglobins/metabolism* ; Treatment Outcome ; China ; Registries ; East Asian People

L-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand. Although microbial cell factories have been widely used for biosynthesis, there are still challenges such as genetic instability and low efficiency in the biosynthesis of L-citrulline. In this study, an efficient, plasmid-free, non-inducible L-citrulline-producing strain of Escherichia coli BL21(DE3) was engineered by combined strategies. Firstly, a chassis strain capable of synthesizing L-citrulline was constructed by block of L-citrulline degradation and removal of feedback inhibition, with the L-citrulline titer of 0.43 g/L. Secondly, a push-pull-restrain strategy was employed to enhance the L-citrulline biosynthesis, which realized the L-citrulline titer of 6.0 g/L. Thirdly, the NADPH synthesis and L-citrulline transport were strengthened to promote the synthesis efficiency, which achieved the L-citrulline titer of 11.6 g/L. Finally, fed-batch fermentation was performed with the engineered strain in a 3 L fermenter, in which the L-citrulline titer reached 44.9 g/L. This study lays the foundation for the industrial production of L-citrulline and provides insights for the modification of other amino acid metabolic networks.
Citrulline/biosynthesis* ; Escherichia coli/genetics* ; Metabolic Engineering/methods* ; Fermentation ; NADP/biosynthesis*

BACKGROUND:Talus cartilage injury is a common motor system disease.This type of injury will affect the patient's daily life and work ability,and may worsen the condition if left untreated.Surgical treatment is commonly used,but the selection of surgical methods and the evaluation of medium-and long-term follow-up results have always been difficult clinical problems. OBJECTIVE:To explore the influence of T1ρ technique on the range of quantitative evaluation of talus osteochondral injury on the choice of surgical method and the results of medium-and long-term follow-up. METHODS:A total of 154 patients with osteochondral injury of talus admitted to The Second Hospital of Tangshan from January 2019 to August 2022 were retrospectively selected as the study subjects.The lesion site of talus was examined by MRI before operation,and the T1ρ and T2 values of different types were compared.Different surgical methods were selected according to the different T1ρ values.Group A(n=73)was treated with microfracture surgery with T1ρ<45 ms;group B(n=81)was treated with autogenous bone and cartilage transplantation with T1ρ≥45 ms.The general clinical characteristics and curative effects of patients under different surgical methods were compared;the important factors of postoperative recurrence were analyzed by multivariate Logistic regression,and the relationship between T1ρ value and postoperative recurrence was analyzed by restricted cubic spline graph,y=1-1/(1+e-z)regression equation to build a prediction model.The stability of the model was verified by cross-checking method. RESULTS AND CONCLUSION:(1)Classification of talus osteochondral injury in 154 patients(type Ⅰ:36 cases;type Ⅱ:37 cases;type Ⅲ:40 cases;type Ⅳ:41 cases),T1ρ and T2 values of the four groups were statistically significant(P<0.05);pairwise comparison was also statistically significant(all P<0.05).(2)After treatment of 154 patients,7 cases(4.6％)had local swelling,3 cases(2.0％)had pain aggravation,and 5 cases(3.3％)had wound infection.There were 2 cases(1.3％)with poor cartilage healing.(3)After treatment,there were statistically significant differences between groups A and B in terms of American Orthopaedic Foot&Ankle Society score,visual analog scale score,plantar flexor motion range,dorsoextension motion range,subchondral bone marrow edema volume,interleukin-6,interleukin-8,C-reactive protein,procalcitonin,platelet-derived growth factor,transforming growth factor-β1,and efficacy(P<0.05).The total effective rate of group B(90％)was higher than that of group A(85％)(P<0.05).(4)Age(OR=1.589,95％CI:0.305-1.252,P=0.036),interleukin-6(OR=1.737,95％CI:0.974-5.254,P=0.049),interleukin-8(OR=1.385,95％CI:1.066-4.355,P=0.034),C-reactive protein(OR=1.957,95％CI:1.323-2.178,P=0.035),transforming growth factor-β1(OR=1.459,95％CI:0.897-2.455,P=0.038),T1-ρ(OR=1.687,95％CI:0.854-3.321,P=0.026),T2(OR=1.843,95％CI:0.657-2.454,P=0.036),complications(OR=1.719,95％CI:0.654-3.464,P=0.019),and classification of osteochondral injury of talus(OR=3.789,95％CI:1.023-5.897,P=0.028)were independent risk factors for postoperative recurrence.Microfracture surgery(OR=0.751,95％CI:0.321-1.264,P=0.012)and autogenous bone and cartilage grafting(OR=0.649,95％CI:0.246-1.356,P=0.023)were independent protective factors for recurrence after medium-and long-term follow-up.(5)When T1ρ value≤35 ms,the risk of postoperative recurrence decreased rapidly,and when T1ρ value>35 ms,the risk of postoperative recurrence increased rapidly.(6)Further stepwise regression analysis showed that these nine risk factors were most closely associated with postoperative recurrence,and the formula for postoperative recurrence was obtained.The probability of postoperative recurrence was calculated using the regression equation.When P=0.75,the maximum value of Jorden index was 77.728,indicating that the model has a better prediction effect.(7)It is indicated that the quantitative evaluation of T1ρ before operation can effectively guide the selection of surgical methods,improve the success rate of surgery and the quality of life of patients.

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.
Humans ; Diglycerides ; Molecular Docking Simulation ; Binding Sites ; Catalysis ; Lipase/genetics*

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.
Bacillus subtilis/metabolism* ; Vitamin K 2/metabolism* ; Bioreactors/microbiology* ; Membrane Microdomains/metabolism*

Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.
N-Acetylneuraminic Acid/metabolism* ; Bacillus subtilis/metabolism* ; Promoter Regions, Genetic/genetics* ; Binding Sites ; Biosensing Techniques

Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serA^T410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serA^T410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA^* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.
Culture Media ; Ergothioneine/metabolism* ; Escherichia coli/metabolism* ; Fermentation ; Histidine/metabolism* ; Metabolic Engineering

Fermentation engineering is an industrial process that uses the transformation of microorganisms or other cells to produce a specific product in a specific bioreactor. Fermentation engineering has developed from an ancient food fermentation relying solely on experience accumulation to an important production mode of food, agriculture, medicine, chemical industry and other means of production and life. It has become a key technology to support the sustainable development of human beings, and is inseparable from the continuous progress of interdisciplinary technology. The interdisciplinary integration and the continuous upward movement of China's global industrial chain will inevitably put forward higher requirements for the cultivation of fermentation engineering composite talents in the new situation. In order to constantly improve the interdisciplinary fermentation engineering compound talent training system, in recent years, the research lab has been refining and improving the concept of talent training, and actively deepening the reform of talent training system. Systematic research and practice have been carried out around the aspects of training program, enrollment system, teacher background, subject setting, scientific research practice, evaluation system, etc., which has promoted the technological progress of fermentation engineering and related supporting industries, and contributed an important force to the transformation of China from a big fermentation country to a powerful fermentation country.
Agriculture ; China ; Fermentation ; Humans ; Industry

Genome-scale metabolic network model (GSMM) is an extremely important guiding tool in the targeted modification of industrial microbial strains, which helps researchers to quickly obtain industrial microbes with specific traits and has attracted increasing attention. Here we reviewe the development history of GSMM and summarized the construction method of GSMM. Furthermore, the development and application of GSMM in industrial microorganisms are elaborated by using four typical industrial microorganisms (Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae) as examples. In addition, prospects in the development trend of GSMM are proposed.
Corynebacterium glutamicum/genetics* ; Escherichia coli/genetics* ; Metabolic Engineering ; Metabolic Networks and Pathways/genetics*