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*

Objective: To establish a progressive research strategy for “colonic components analysis - efficacy verification and mechanism exploration - gut microbiota”, screen pharmacodynamic substances, and investigate their mechanism via gut microbiota. Methods: The pharmacodynamics of Gegen Qinlian decoction (GQD) were assessed using a mouse model of dextran sulfate sodium-induced ulcerative colitis (UC). Ultra-performance liquid chromatography-quadrupole-orbitrap mass spectrometer was used to identify the prototype and metabolic components of GQD in the colon during UC. To analyze the structure and function of characteristic genera of GQD and its active components, 16S rRNA sequencing was performed. Results: We identified 67 prototypic and 14 metabolic components of GQD in the UC colon. The primary prototype components are flavonoids and alkaloids, including puerarin (PUE), baicalin (BAI), and berberine (BER). The metabolism was predominantly sulfonation. Efficacy verification showed that the main active components, puerarin, baicalin, and berberine, had good therapeutic effects on UC. The results of 16S rRNA gene sequencing showed that GQD improved UC by regulating the structure and function of the gut microbiota. The abundance of gut microbiota involved in the metabolism of the prototype components was influenced by the corresponding components. The function prediction results showed that PUE was the most comparable to GQD, with 24 consistent pathways. BAI and BER showed comparable gut microbiota regulation pathways. Characteristic pathways of BER include glucometabolic processes. Conclusion This study focused on the key issues in the gut microbiota pathway and developed a progressive research strategy to understand the transformation mechanisms of colonic components. This research systematically analyzed the active components and metabolic transformation of GQD in the colon during the pathological state of UC, as well as changes in the structure and function of the gut microbiota, clarified the mechanism of GQD and its active components in improving UC via the gut microbiota pathway.

Objective:To compare the differences of long-term survival rates between hemodialysis (HD) and peritoneal dialysis (PD) in the oldest old end-stage renal disease (ESRD) patients, and analyze the influencing factors of mortality.Methods:It was a retrospective cohort study. The clinical data from the oldest old patients (≥80 years old) who underwent HD or PD for the first time and maintained dialysis treatment for ≥3 months in the Zhejiang Dialysis Registration System from January 1, 2008 to December 31, 2021 were collected. The follow-up endpoint was until the patients' death or December 31, 2022. The propensity score matching method was used to match groups. Kaplan-Meier method and log-rank test were used to compare the differences of long-term survival rates between the two groups. Cox regression analysis was used to analyze the risk factors of mortality.Results:A total of 5 880 the oldest old dialysis patients were included in this study, with 5 363 patients in the initial HD group and 517 patients in the initial PD group. After matching, there were 517 patients in the HD group and 517 patients in the PD group. The median survival time of HD group before matching was 39.9 months, with 1-year, 3-year, and 5-year survival rates of 85.4%, 54.9%, and 30.0%, respectively. The median survival time of PD group was 32.9 months, with 1-year, 3-year, and 5-year survival rates of 82.5%, 47.1%, and 22.3%, respectively. After matching, the median survival time of HD group was 40.3 months, and the 1-year, 3-year, and 5-year survival rates were 86.1%, 57.8%, and 29.1%, respectively. The survival rate of PD group remained unchanged. The difference of 1-year survival rate between the two groups was not statistically significant, but the 2-year, 3-year, 4-year, 5-year, and overall survival rates in HD group were higher than those in PD group (Log‐rank test, χ2=4.897, P=0.027; χ2=9.693, P=0.002; χ2=10.194, P=0.001; χ2=7.868, P=0.005; χ2=12.510, P<0.001). Multivariate Cox regression analysis showed that HD (HD/PD, HR=0.794, 95% CI 0.669-0.943, P=0.009), increasing age ( HR=1.069, 95% CI 1.038-1.110, P<0.001), comorbidity with chronic obstructive pulmonary disease ( HR=1.510, 95% CI 1.065-2.139, P=0.021) and serum albumin <35 g/L ( HR=1.393, 95% CI 1.165-1.665, P<0.001) were independent correlated factors of mortality. Conclusions:There is no significant difference in the 1-year survival rate between HD and PD groups. The survival rate for more than 1 year in HD patients is higher than that in PD patients. HD is an independent protective factor of survival, and increasing age, comorbidities of chronic obstructive pulmonary disease, and serum albumin <35 g/L are independent risk factors affecting the survival in the oldest old dialysis patients.

Objective:To investigate the effect of pre-dialysis blood pressure (Pre-BP) on all-cause and cardiovascular disease (CVD) mortality in patients on maintenance hemodialysis (MHD).Methods:This single-center, retrospective cohort study enrolled patients undergoing first-time hemodialysis between January 1, 2007, and June 30, 2021, from the dialysis registry of the First Affiliated Hospital, Zhejiang University School of Medicine. General information and laboratory parameters were collected. Pre-dialysis systolic blood pressure (Pre-SBP) and pre-dialysis diastolic blood pressure (Pre-DBP) were calculated and averaged at 4-6 months after dialysis. The mean Pre-SBP and Pre-DBP values were used as continuous variables, and restricted cubic spline (RCS) curves were used to assess the relationship between Pre-BP and mortality risk. Patients were subsequently divided into six groups for Pre-DBP and six groups for Pre-SBP combined with Pre-DBP. Survival analyses were performed using the Kaplan-Meier method. All-cause and CVD mortality were compared between groups using the log-rank test. Multivariate Cox regression models were used to analyze the associations between Pre-BP and all-cause and CVD mortality.Results:A total of 1 213 patients were enrolled. By the end of follow-up, 175 patients (14.4%) had died, of whom 62 (35.4%) died from CVD. Kaplan-Meier survival curves showed that the Pre-DBP<65 mmHg group (1 mmHg=0.133 kPa) had a significantly lower cumulative survival rate ( χ2=90.52, P<0.001) and a significantly higher CVD mortality rate ( χ2=35.54, P<0.001) than the other groups. The combined Pre-SBP and Pre-DBP analysis showed that the Pre-SBP≥150 mmHg and Pre-DBP<80 mmHg groups had a significantly lower cumulative survival rate ( χ2=45.58, P<0.001) and a significantly higher CVD mortality rate ( χ2=30.13, P<0.001) than the other groups. Multivariate Cox regression model analysis showed that compared with other groups, the risk of MHD all-cause mortality was increased in the Pre-DBP<65 mmHg group and the Pre-SBP≥150 mmHg and Pre-DBP<80 mmHg group [ HR (95% CI)=1.927 (1.195-3.109), 3.298 (1.567-6.939), both P<0.05]. Conclusion:In patients undergoing MHD, Pre-DBP<65 mmHg or Pre-SBP≥150 mmHg and Pre-DBP<80 mmHg were independent risk factors for all-cause mortality, with a low cumulative survival rate and a high risk of CVD mortality.

Objective:To compare the clinical effects of plasma exchange（PE）combined with continuous veno-venous hemodiafiltration（CVVHDF）at different time points in children with Kawasaki disease shock syndrome（KDSS）.Methods:Thirty-five children with KDSS admitted to the intensive care unit of Hunan Children's Hospital from January 2018 to December 2022 were enrolled.According to whether PE combined with CVVHDF was performed within 24 hours after reaching the blood purification criteria for KDSS，the patients were divided into the early treatment group(8 cases) and the control group (27 cases).The clinical and laboratory indicators and prognosis were compared between the two groups.Results:There were no statistically significant differences in age，gender，mean arterial pressure，and pediatric critical illness score between the two groups before treatment（ P>0.05）.In the early treatment group,the vasoactive inotropic score (VIS) gradually decreased,and was significantly lower than that in the control group after 24 hours of treatment.The duration of vasopressor use，pediatric logistic organ dysfunction score（PELOD），inflammatory markers，total hospitalization time，and PICU stay were all lower in the early treatment group than in the control group（ P<0.05）.The incidence of coronary artery involvement within 6 months post-discharge was lower in the early treatment group than in the control group（ P<0.05）.Among the 12 children who underwent PE combined with CVVHDF，four cases were in the late treatment group.The duration of CVVHDF，PICU stay，and PELOD scores were lower in the early treatment group than in the late treatment group（ P<0.05）.Additionally，the concentrations of interleukin-6，tumor necrosis factor-α，heparin-binding protein，and N-terminal pro-brain natriuretic peptide before PE were lower in the early treatment group than in the late treatment group（ P<0.05）. Conclusion:Early PE combined with CVVHDF treatment for KDSS patients can reduce inflammatory response，shorten the course of the disease，and reduce the duration of vasopressor use.However，most patients' conditions can be controlled with active conventional treatment.

Objective:To summarize the current status of living space for elderly patients with chronic diseases.Methods:Literature on living space for elderly patients with chronic diseases was electronically searched in PubMed, Web of Science, Cochrane Library, Embase, China Biology Medicine disc, China National Knowledge Infrastructure, VIP, and WanFang Data. The search period was from database establishment to April 13, 2024.Results:A total of 33 papers were included. Elderly patients with chronic diseases generally had limited living space, and the assessment tool was mainly the Life Space Assessment. The main influences on living space included demographic, disease-related, psychological, and other factors.Conclusions:Elderly patients with chronic diseases generally have limited living space and have a single assessment tool with complex and diverse influencing factors. Healthcare professionals should develop specific living space assessment tools and individualized interventions based on influencing factors to help patients with chronic diseases improve their living space limitations.

Objective:To compare the clinical effects of plasma exchange（PE）combined with continuous veno-venous hemodiafiltration（CVVHDF）at different time points in children with Kawasaki disease shock syndrome（KDSS）.Methods:Thirty-five children with KDSS admitted to the intensive care unit of Hunan Children's Hospital from January 2018 to December 2022 were enrolled.According to whether PE combined with CVVHDF was performed within 24 hours after reaching the blood purification criteria for KDSS，the patients were divided into the early treatment group(8 cases) and the control group (27 cases).The clinical and laboratory indicators and prognosis were compared between the two groups.Results:There were no statistically significant differences in age，gender，mean arterial pressure，and pediatric critical illness score between the two groups before treatment（ P>0.05）.In the early treatment group,the vasoactive inotropic score (VIS) gradually decreased,and was significantly lower than that in the control group after 24 hours of treatment.The duration of vasopressor use，pediatric logistic organ dysfunction score（PELOD），inflammatory markers，total hospitalization time，and PICU stay were all lower in the early treatment group than in the control group（ P<0.05）.The incidence of coronary artery involvement within 6 months post-discharge was lower in the early treatment group than in the control group（ P<0.05）.Among the 12 children who underwent PE combined with CVVHDF，four cases were in the late treatment group.The duration of CVVHDF，PICU stay，and PELOD scores were lower in the early treatment group than in the late treatment group（ P<0.05）.Additionally，the concentrations of interleukin-6，tumor necrosis factor-α，heparin-binding protein，and N-terminal pro-brain natriuretic peptide before PE were lower in the early treatment group than in the late treatment group（ P<0.05）. Conclusion:Early PE combined with CVVHDF treatment for KDSS patients can reduce inflammatory response，shorten the course of the disease，and reduce the duration of vasopressor use.However，most patients' conditions can be controlled with active conventional treatment.

Objective:To summarize the current status of living space for elderly patients with chronic diseases.Methods:Literature on living space for elderly patients with chronic diseases was electronically searched in PubMed, Web of Science, Cochrane Library, Embase, China Biology Medicine disc, China National Knowledge Infrastructure, VIP, and WanFang Data. The search period was from database establishment to April 13, 2024.Results:A total of 33 papers were included. Elderly patients with chronic diseases generally had limited living space, and the assessment tool was mainly the Life Space Assessment. The main influences on living space included demographic, disease-related, psychological, and other factors.Conclusions:Elderly patients with chronic diseases generally have limited living space and have a single assessment tool with complex and diverse influencing factors. Healthcare professionals should develop specific living space assessment tools and individualized interventions based on influencing factors to help patients with chronic diseases improve their living space limitations.

Objective:To compare the differences of long-term survival rates between hemodialysis (HD) and peritoneal dialysis (PD) in the oldest old end-stage renal disease (ESRD) patients, and analyze the influencing factors of mortality.Methods:It was a retrospective cohort study. The clinical data from the oldest old patients (≥80 years old) who underwent HD or PD for the first time and maintained dialysis treatment for ≥3 months in the Zhejiang Dialysis Registration System from January 1, 2008 to December 31, 2021 were collected. The follow-up endpoint was until the patients' death or December 31, 2022. The propensity score matching method was used to match groups. Kaplan-Meier method and log-rank test were used to compare the differences of long-term survival rates between the two groups. Cox regression analysis was used to analyze the risk factors of mortality.Results:A total of 5 880 the oldest old dialysis patients were included in this study, with 5 363 patients in the initial HD group and 517 patients in the initial PD group. After matching, there were 517 patients in the HD group and 517 patients in the PD group. The median survival time of HD group before matching was 39.9 months, with 1-year, 3-year, and 5-year survival rates of 85.4%, 54.9%, and 30.0%, respectively. The median survival time of PD group was 32.9 months, with 1-year, 3-year, and 5-year survival rates of 82.5%, 47.1%, and 22.3%, respectively. After matching, the median survival time of HD group was 40.3 months, and the 1-year, 3-year, and 5-year survival rates were 86.1%, 57.8%, and 29.1%, respectively. The survival rate of PD group remained unchanged. The difference of 1-year survival rate between the two groups was not statistically significant, but the 2-year, 3-year, 4-year, 5-year, and overall survival rates in HD group were higher than those in PD group (Log‐rank test, χ2=4.897, P=0.027; χ2=9.693, P=0.002; χ2=10.194, P=0.001; χ2=7.868, P=0.005; χ2=12.510, P<0.001). Multivariate Cox regression analysis showed that HD (HD/PD, HR=0.794, 95% CI 0.669-0.943, P=0.009), increasing age ( HR=1.069, 95% CI 1.038-1.110, P<0.001), comorbidity with chronic obstructive pulmonary disease ( HR=1.510, 95% CI 1.065-2.139, P=0.021) and serum albumin <35 g/L ( HR=1.393, 95% CI 1.165-1.665, P<0.001) were independent correlated factors of mortality. Conclusions:There is no significant difference in the 1-year survival rate between HD and PD groups. The survival rate for more than 1 year in HD patients is higher than that in PD patients. HD is an independent protective factor of survival, and increasing age, comorbidities of chronic obstructive pulmonary disease, and serum albumin <35 g/L are independent risk factors affecting the survival in the oldest old dialysis patients.

Objective:To investigate the effect of pre-dialysis blood pressure (Pre-BP) on all-cause and cardiovascular disease (CVD) mortality in patients on maintenance hemodialysis (MHD).Methods:This single-center, retrospective cohort study enrolled patients undergoing first-time hemodialysis between January 1, 2007, and June 30, 2021, from the dialysis registry of the First Affiliated Hospital, Zhejiang University School of Medicine. General information and laboratory parameters were collected. Pre-dialysis systolic blood pressure (Pre-SBP) and pre-dialysis diastolic blood pressure (Pre-DBP) were calculated and averaged at 4-6 months after dialysis. The mean Pre-SBP and Pre-DBP values were used as continuous variables, and restricted cubic spline (RCS) curves were used to assess the relationship between Pre-BP and mortality risk. Patients were subsequently divided into six groups for Pre-DBP and six groups for Pre-SBP combined with Pre-DBP. Survival analyses were performed using the Kaplan-Meier method. All-cause and CVD mortality were compared between groups using the log-rank test. Multivariate Cox regression models were used to analyze the associations between Pre-BP and all-cause and CVD mortality.Results:A total of 1 213 patients were enrolled. By the end of follow-up, 175 patients (14.4%) had died, of whom 62 (35.4%) died from CVD. Kaplan-Meier survival curves showed that the Pre-DBP<65 mmHg group (1 mmHg=0.133 kPa) had a significantly lower cumulative survival rate ( χ2=90.52, P<0.001) and a significantly higher CVD mortality rate ( χ2=35.54, P<0.001) than the other groups. The combined Pre-SBP and Pre-DBP analysis showed that the Pre-SBP≥150 mmHg and Pre-DBP<80 mmHg groups had a significantly lower cumulative survival rate ( χ2=45.58, P<0.001) and a significantly higher CVD mortality rate ( χ2=30.13, P<0.001) than the other groups. Multivariate Cox regression model analysis showed that compared with other groups, the risk of MHD all-cause mortality was increased in the Pre-DBP<65 mmHg group and the Pre-SBP≥150 mmHg and Pre-DBP<80 mmHg group [ HR (95% CI)=1.927 (1.195-3.109), 3.298 (1.567-6.939), both P<0.05]. Conclusion:In patients undergoing MHD, Pre-DBP<65 mmHg or Pre-SBP≥150 mmHg and Pre-DBP<80 mmHg were independent risk factors for all-cause mortality, with a low cumulative survival rate and a high risk of CVD mortality.