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 investigate the protective effect of Toll-like receptor (TLR)2/TLR9 agonists,Pam2 CSK4(Pam)and CpG ODN (CpG)on mice infected with Acinetobacter baumannii (Ab)in the lungs.Methods Female C57 mice (6～8 weeks old)were randomly divided into PBS,Pam,CpG and Pam+CpG groups.In 24 h after intranasal immunization with different doses of the corresponding agonists,the mice were given a lethal dose of Ab infection in the lungs,and the survival rates of the mice were observed.A sublethal dose lung infection model of Ab was then established,and the bacterial colonization in the blood,lungs,liver,kidneys and spleen was measured respectively in the mice after infection.HE staining was used to observe the pathological damages in the lungs and kidneys.The protective effect of the agonists in the immunized mice against Ab was examined at 1,3 and 7 d after immunization to explore the protective time window.Pam+CpG was used to stimulate A549 cells and RAW264.7 cells to investigate the killing or phagocytic effects on Ab.Results Compared to PBS,Pam+CpG treatment significantly improved the survival rate of the mice after a lethal dose of Ab lung infection (P<0.05,P<0.01 ),reduced bacterial colonization in the blood (P<0.01 ),lungs (P<0.01 ),liver (P<0.01 ),kidneys (P<0.01 )and spleen (P<0.01 )in the mice after sublethal challenge,and alleviated pathological damage caused by infection. Immunization at 1 or 3 d before infection significantly improved the survival rate (P<0.05 ),and the protective effect was the best in 3 d after immunization.Furthermore,compared to single PBS,Pam and CpG immunization,Pam+CpG significantly promoted the killing and phagocytic effects of A549 epithelial cells and RAW264.7 cells,respectively,against Ab (P<0.01 ).Conclusion Combined application of TLR2/TLR9 agonists exerts a significant protective effect on both lethal and sublethal infections of Ab,which might be by its promoting the killing or phagocytic effect of lung epithelial cells and macrophages against Ab.

Objective To construct lipid nanoparticles(lipopeptide-lipid nanoparticle,LP-LNP)with novel lipopeptides as adjuvants,and initially explore their synergistic effect on mRNA vaccines.Methods Two novel lipopeptides,SS-10 and SQ18,were designed and synthesized.Microfluidic technology was used to encapsulate lipopeptides in different proportions,as well as mRNAs encoding enhanced green fluorescent protein(eGFP),firefly luciferase(F-luc),and ovalbumin(OVA)into lipid nanoparticles to construct an mRNA delivery system with novel lipopeptides as adjuvants(LP-LNP).The particle size and polydispersity coefficient of LP-LNP were measured using dynamic light scattering.The activation effect on Toll-like receptors 2(TLR2)was detected using HEK-BlueTM mTLR2 reporter cells to screen the optimal lipopeptide ratio.The preferred LP-LNP-eGFP-mRNA was transfected into HEK293T cells,and the expression of eGFP was observed under a fluorescence microscope.In vivo imaging was used to investigate the expression level of LP-LNP-F-luc-mRNA in mice.Flow cytometry was used to evaluate the ability of LP-LNP-OVA-mRNA to induce the maturation of dendritic cells(DCs)in draining lymph nodes and cross-presentation of antigens after immunization.Results Lipopeptides SQ18 and SS-10 were incorporated into LNP at 0.50％and 0.75％molar ratios,respectively,to obtain LP-LNP with uniform particle size,high encapsulation efficiency,and good in vitro safety.The ability of this formulation to activate TLR2 was significantly stronger than the positive control Pam2CSK4(P＜0.01).The preferred LP-LNP obtained effective in vitro transfection,and LP-LNP prepared with SQ18 at 0.50％molar ratio had significantly better in vivo transfection efficiency than traditional LNP(P＜0.01),and significantly promoted the maturation of DCs in draining lymph nodes and cross-presentation of antigens(P＜0.05).Conclusion LP-LNP with novel lipopeptides as adjuvants can enhance the delivery capacity of mRNA and further improve the immune effect of mRNA vaccines.

Objective:To clarify the transitional components in the blood of compound Banlangen Granules; To explore the mechanism of drugs in the treatment of epidemic encephalitis B, hepatitis and parotitis.Methods:The transitional components in blood of compound Banlangen Granules were analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS). The regulatory targets and pathways of compound Banlangen Granules in the treatment of epidemic encephalitis B, hepatitis and parotitis were analyzed based on UPLC-MS/MS and network pharmacology.Results:A total of 9 blood components were identified, of which 8 were prototype components, including sucrose, o-aminobenzoic acid, uridine, adenosine, guanosine, indole-3-acetonitrile-2 murine-S-β-D-glucopyranoside and salicylic acid. Through network pharmacological analysis, it was concluded that compound Banlangen Granules may treat epidemic encephalitis B, hepatitis and parotitis by regulating lipid and atherosclerosis, insulin resistance, IL-17 and other signal pathways.Conclusion:The 9 blood components of compound Banlangen Granules may treat epidemic encephalitis B, hepatitis and parotitis by regulating lipid and atherosclerosis, insulin resistance, IL-17 and other signal pathways.

Objective:To investigate the pregnancy outcome and the maternal and infant follow-up in the puer-peral period(42 days after delivery)of cesarean section with SARS-CoV-2 Omicron variant in different periods of pregnancy.Methods:330 pregnant women who were diagnosed with SARS-CoV-2 infection and delivered by ce-sarean section in Guangdong Women and Children Hospital from November 20,2022 to January 25,2023 were selected as the research subjects.They were divided into the early(80 cases),middle(151 cases)and late(99 cases)pregnancy group according to the gestational age of infection.The general information,pregnancy compli-cations and comorbidities,pregnancy outcomes,and postpartum maternal and infant follow-up of each group of pregnant women were compared.Results:The clinical classification of pregnant women was asymptomatic and mild,with 9 cases of asymptomatic and 321 cases of mild.The incidence of gestational diabetes mellitus(GDM)in the early and late group was higher than that in the middle group(P＜0.05).There was no statistically significant difference in the incidence of placental abruption,fetal growth restriction,fetal distress,amniotic fluid contamina-tion,preterm labor,postpartum hemorrhage,macrosomia,low-birth-weight infants and the maternal intensive care unit(MICU)transfer rate,newborn birth weight,neonatal asphyxia rate and NICU transfer rate among the three groups(P＞0.05).The incidence of preterm rupture of membranes in the middle group was higher than that in the late group(P＜0.05).Puerperal follow-up results showed that there was no statistically significant difference in the incidence of poor maternal uterine incision healing,poor uterine involution,vaginal microecological imbal-ance and neonatal acute upper respiratory tract infection,pneumonia,digestive system disease,cardiovascular system disease among the three groups(P＞0.05).Conclusions:The majority of pregnant women with Omicron infection during pregnancy are mild.Omicron infection in different periods of pregnancy has no significant effect on pregnancy outcome and puerperal follow-up of cesarean section.No evidence was found that GDM and prema-ture rupture of membranes are significantly associated with Omicron infection in different periods of pregnancy.

Objective:To investigate the pregnancy outcome and the maternal and infant follow-up in the puer-peral period(42 days after delivery)of cesarean section with SARS-CoV-2 Omicron variant in different periods of pregnancy.Methods:330 pregnant women who were diagnosed with SARS-CoV-2 infection and delivered by ce-sarean section in Guangdong Women and Children Hospital from November 20,2022 to January 25,2023 were selected as the research subjects.They were divided into the early(80 cases),middle(151 cases)and late(99 cases)pregnancy group according to the gestational age of infection.The general information,pregnancy compli-cations and comorbidities,pregnancy outcomes,and postpartum maternal and infant follow-up of each group of pregnant women were compared.Results:The clinical classification of pregnant women was asymptomatic and mild,with 9 cases of asymptomatic and 321 cases of mild.The incidence of gestational diabetes mellitus(GDM)in the early and late group was higher than that in the middle group(P＜0.05).There was no statistically significant difference in the incidence of placental abruption,fetal growth restriction,fetal distress,amniotic fluid contamina-tion,preterm labor,postpartum hemorrhage,macrosomia,low-birth-weight infants and the maternal intensive care unit(MICU)transfer rate,newborn birth weight,neonatal asphyxia rate and NICU transfer rate among the three groups(P＞0.05).The incidence of preterm rupture of membranes in the middle group was higher than that in the late group(P＜0.05).Puerperal follow-up results showed that there was no statistically significant difference in the incidence of poor maternal uterine incision healing,poor uterine involution,vaginal microecological imbal-ance and neonatal acute upper respiratory tract infection,pneumonia,digestive system disease,cardiovascular system disease among the three groups(P＞0.05).Conclusions:The majority of pregnant women with Omicron infection during pregnancy are mild.Omicron infection in different periods of pregnancy has no significant effect on pregnancy outcome and puerperal follow-up of cesarean section.No evidence was found that GDM and prema-ture rupture of membranes are significantly associated with Omicron infection in different periods of pregnancy.

Objective:To investigate the pregnancy outcome and the maternal and infant follow-up in the puer-peral period(42 days after delivery)of cesarean section with SARS-CoV-2 Omicron variant in different periods of pregnancy.Methods:330 pregnant women who were diagnosed with SARS-CoV-2 infection and delivered by ce-sarean section in Guangdong Women and Children Hospital from November 20,2022 to January 25,2023 were selected as the research subjects.They were divided into the early(80 cases),middle(151 cases)and late(99 cases)pregnancy group according to the gestational age of infection.The general information,pregnancy compli-cations and comorbidities,pregnancy outcomes,and postpartum maternal and infant follow-up of each group of pregnant women were compared.Results:The clinical classification of pregnant women was asymptomatic and mild,with 9 cases of asymptomatic and 321 cases of mild.The incidence of gestational diabetes mellitus(GDM)in the early and late group was higher than that in the middle group(P＜0.05).There was no statistically significant difference in the incidence of placental abruption,fetal growth restriction,fetal distress,amniotic fluid contamina-tion,preterm labor,postpartum hemorrhage,macrosomia,low-birth-weight infants and the maternal intensive care unit(MICU)transfer rate,newborn birth weight,neonatal asphyxia rate and NICU transfer rate among the three groups(P＞0.05).The incidence of preterm rupture of membranes in the middle group was higher than that in the late group(P＜0.05).Puerperal follow-up results showed that there was no statistically significant difference in the incidence of poor maternal uterine incision healing,poor uterine involution,vaginal microecological imbal-ance and neonatal acute upper respiratory tract infection,pneumonia,digestive system disease,cardiovascular system disease among the three groups(P＞0.05).Conclusions:The majority of pregnant women with Omicron infection during pregnancy are mild.Omicron infection in different periods of pregnancy has no significant effect on pregnancy outcome and puerperal follow-up of cesarean section.No evidence was found that GDM and prema-ture rupture of membranes are significantly associated with Omicron infection in different periods of pregnancy.

Objective:To investigate the pregnancy outcome and the maternal and infant follow-up in the puer-peral period(42 days after delivery)of cesarean section with SARS-CoV-2 Omicron variant in different periods of pregnancy.Methods:330 pregnant women who were diagnosed with SARS-CoV-2 infection and delivered by ce-sarean section in Guangdong Women and Children Hospital from November 20,2022 to January 25,2023 were selected as the research subjects.They were divided into the early(80 cases),middle(151 cases)and late(99 cases)pregnancy group according to the gestational age of infection.The general information,pregnancy compli-cations and comorbidities,pregnancy outcomes,and postpartum maternal and infant follow-up of each group of pregnant women were compared.Results:The clinical classification of pregnant women was asymptomatic and mild,with 9 cases of asymptomatic and 321 cases of mild.The incidence of gestational diabetes mellitus(GDM)in the early and late group was higher than that in the middle group(P＜0.05).There was no statistically significant difference in the incidence of placental abruption,fetal growth restriction,fetal distress,amniotic fluid contamina-tion,preterm labor,postpartum hemorrhage,macrosomia,low-birth-weight infants and the maternal intensive care unit(MICU)transfer rate,newborn birth weight,neonatal asphyxia rate and NICU transfer rate among the three groups(P＞0.05).The incidence of preterm rupture of membranes in the middle group was higher than that in the late group(P＜0.05).Puerperal follow-up results showed that there was no statistically significant difference in the incidence of poor maternal uterine incision healing,poor uterine involution,vaginal microecological imbal-ance and neonatal acute upper respiratory tract infection,pneumonia,digestive system disease,cardiovascular system disease among the three groups(P＞0.05).Conclusions:The majority of pregnant women with Omicron infection during pregnancy are mild.Omicron infection in different periods of pregnancy has no significant effect on pregnancy outcome and puerperal follow-up of cesarean section.No evidence was found that GDM and prema-ture rupture of membranes are significantly associated with Omicron infection in different periods of pregnancy.

Objective:To investigate the pregnancy outcome and the maternal and infant follow-up in the puer-peral period(42 days after delivery)of cesarean section with SARS-CoV-2 Omicron variant in different periods of pregnancy.Methods:330 pregnant women who were diagnosed with SARS-CoV-2 infection and delivered by ce-sarean section in Guangdong Women and Children Hospital from November 20,2022 to January 25,2023 were selected as the research subjects.They were divided into the early(80 cases),middle(151 cases)and late(99 cases)pregnancy group according to the gestational age of infection.The general information,pregnancy compli-cations and comorbidities,pregnancy outcomes,and postpartum maternal and infant follow-up of each group of pregnant women were compared.Results:The clinical classification of pregnant women was asymptomatic and mild,with 9 cases of asymptomatic and 321 cases of mild.The incidence of gestational diabetes mellitus(GDM)in the early and late group was higher than that in the middle group(P＜0.05).There was no statistically significant difference in the incidence of placental abruption,fetal growth restriction,fetal distress,amniotic fluid contamina-tion,preterm labor,postpartum hemorrhage,macrosomia,low-birth-weight infants and the maternal intensive care unit(MICU)transfer rate,newborn birth weight,neonatal asphyxia rate and NICU transfer rate among the three groups(P＞0.05).The incidence of preterm rupture of membranes in the middle group was higher than that in the late group(P＜0.05).Puerperal follow-up results showed that there was no statistically significant difference in the incidence of poor maternal uterine incision healing,poor uterine involution,vaginal microecological imbal-ance and neonatal acute upper respiratory tract infection,pneumonia,digestive system disease,cardiovascular system disease among the three groups(P＞0.05).Conclusions:The majority of pregnant women with Omicron infection during pregnancy are mild.Omicron infection in different periods of pregnancy has no significant effect on pregnancy outcome and puerperal follow-up of cesarean section.No evidence was found that GDM and prema-ture rupture of membranes are significantly associated with Omicron infection in different periods of pregnancy.

Objective:To investigate the pregnancy outcome and the maternal and infant follow-up in the puer-peral period(42 days after delivery)of cesarean section with SARS-CoV-2 Omicron variant in different periods of pregnancy.Methods:330 pregnant women who were diagnosed with SARS-CoV-2 infection and delivered by ce-sarean section in Guangdong Women and Children Hospital from November 20,2022 to January 25,2023 were selected as the research subjects.They were divided into the early(80 cases),middle(151 cases)and late(99 cases)pregnancy group according to the gestational age of infection.The general information,pregnancy compli-cations and comorbidities,pregnancy outcomes,and postpartum maternal and infant follow-up of each group of pregnant women were compared.Results:The clinical classification of pregnant women was asymptomatic and mild,with 9 cases of asymptomatic and 321 cases of mild.The incidence of gestational diabetes mellitus(GDM)in the early and late group was higher than that in the middle group(P＜0.05).There was no statistically significant difference in the incidence of placental abruption,fetal growth restriction,fetal distress,amniotic fluid contamina-tion,preterm labor,postpartum hemorrhage,macrosomia,low-birth-weight infants and the maternal intensive care unit(MICU)transfer rate,newborn birth weight,neonatal asphyxia rate and NICU transfer rate among the three groups(P＞0.05).The incidence of preterm rupture of membranes in the middle group was higher than that in the late group(P＜0.05).Puerperal follow-up results showed that there was no statistically significant difference in the incidence of poor maternal uterine incision healing,poor uterine involution,vaginal microecological imbal-ance and neonatal acute upper respiratory tract infection,pneumonia,digestive system disease,cardiovascular system disease among the three groups(P＞0.05).Conclusions:The majority of pregnant women with Omicron infection during pregnancy are mild.Omicron infection in different periods of pregnancy has no significant effect on pregnancy outcome and puerperal follow-up of cesarean section.No evidence was found that GDM and prema-ture rupture of membranes are significantly associated with Omicron infection in different periods of pregnancy.