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.

Group A Streptococcus (GAS) is a very important pathogen, especially for children.On a global scale, GAS is an important cause of morbidity and mortality.But the burden of disease caused by GAS is still unknown in China and also has not obtained enough attention.For this purpose, the expert consensus is comprehensively described in diagnosis, treatment and prevention of GAS diseases in children, covering related aspects of pneumology, infectiology, immunology, microbiology, cardiology, nephrology, critical care medicine and preventive medicine.Accordingly, the consensus document was intended to improve management strategies of GAS disease in Chinese children.

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.
Bacillus subtilis/genetics* ; Bacterial Proteins/genetics* ; Gene Editing ; Metabolic Engineering ; Promoter Regions, Genetic

Objective To study the pharmacokinetics of raltitrexed using different ways of drug delivery, including femoral venous infusion, hepatic artery perfusion, hepatic artery injection of lipiodol suspension, hepatic artery perfusion followed by embolization with Gelfoam. Methods According to the administration way of raltitrexed, a total of 40 New Zealand rabbit models with VX2 liver tumor were randomly divided into group A (femoral venous perfusion), group B (hepatic arterial perfusion), group C (hepatic artery injection of lipiodol suspension), and group D(hepatic artery perfusion followed by embolization with Gelfoam). Drug concentration in plasma were determined by using LC-MS/MS method and the pharmacokinetic parameters were calculated. Results After administration of raltitrexed, the Tmax was 5 minutes in all 4 groups. In group A, B, C and D, the values were (5.88±1.39), (7.31±2.60), (9.86±5.10) and (7.19±2.27) respectively, with group C having the longest t1/2 value, which was significantly different with that of group A (P＜0.05); the (ng·ml-1·h-1) values were (2 056.40± 139.17), (1 389.21±180.28), (911.84±105.62) and (1 133.41±181.42)respectively, with the value of group A being obviously higher than that of group B, C and D (P＜0.05) and the value of group C being the lowest; the AUC0-t(ng· ml-1·h-1) values were (5 482.72±1 007.07), (4 156.99±1 475.77), (2 785.13±1 107.36) and (3 903.64±947.25) respectively, with the value of group A being remarkably higher than that of group B, C and D (P＜0.05) and the value of group C being the lowest. Conclusion Compared with the femoral vein infusion way, the ways of hepatic artery infusion, hepatic artery lipiodol suspension injection and hepatic artery perfusion followed by embolization with Gelfoam may promote more raltitrexed to deposit in the tumor area, thus, the curative effect is enhanced, the drug concentration in plasma is lowered and the side effects are alleviated.

Objective To analyze the epidemiological features of leprosy in China from 2011 to 2015,and to provide scientific evidences for prevention and treatment strategies.Methods An epidemiological analysis and a trend analysis were conducted based on the national leprosy surveillance data from 2011 to 2015.Results The leprosy detection rate in China decreased from 0.085 per 100 000 in 2011 to 0.049 per 100 000 in 2015,with an average annual decline rate being 12.9％.A total of 4 775 leprosy cases were newly detected during 2011-2015,including 106 (2.2％) children,1 499 (31.4％) females,518 (10.8％) floating people,4 041 (84.6％) multibacillary cases and 1 134 (23.7％) cases with grade 2 disabilities.From 2011 to 2015,328 relapsed cases were reported,including 153 (46.6％) cases recurring after combined chemotherapy.The prevalence rate of leprosy in China decreased from 0.407 per 100 000 in 2011 to 0.235 per 100 000 in 2015,with an average annual decline rate being 12.9％.By the end of 2015,there had been 3 230 registered leprosy cases and 124 counties with a prevalence rate above 1 per 100 000.Conclusions The detection rate and prevalence rate of leprosy in China were both decreasing continuously from 2011 to 2015.The high-epidemic provinces were Yunnan,Guizhou,Sichuan and Guangdong.However,leprosy control in middle-and low-epidemic provinces can not be ignored.

Objective To investigate the mechanism and clinical value of sacral plexus perfusion method in the treatment of rachi lumbocrural pain.Methods 80 cases of patients with rachi lumbocrural pain in our hospital from May 2014 to May 2016 were selected, they were randomly divided into sacral plexus perfusion method treatment group ( study group) and infrared short medium long frequency therapeutic instrument combined with acupuncture and massage therapy group (control group) two groups, 40 cases in each group.The main clinical symptoms scores, main clinical signs scores, thoracolumbar spine flexion, VAS scores, clinical efficacy of the two groups were statistically analyzed.Results The low back pain, cold limbs, numbness, leg redicular pain scores of the study group were significantly lower, the difference was statistically significant (P<0.05), the both sides L3 transverse tip tenderness scores, VAS score were significantly lower, the difference was statistically significant (P<0.05), the thoracolumbar flexion was significantly higher, the difference was statistically significant (P<0.05), the total treatment efficiency 92.5%(37/40) was significantly higher than the control group 67.5%(27/40), the difference was statistically significant (P<0.05).Conclusion The clinical value of sacral plexus perfusion method in the treatment of rachi lumbocrural pain is higher than infrared short medium long frequency therapeutic instrument combined with acupuncture and massage therapy, it can more effectively improve the clinical symptoms and signs, relieve the pain, enhance the thoracolumbar flexion and total treatment efficiency of patients.

ObjectiveTo study the epidemiological characteristics of leprosy in China,2001-2010.MethodsData were collected from the database of the national system of leprosy surveillance.A descriptive and comparative analysis was performed.ResultsTotally,15 507 new leprosy cases were detected from 2001 to 2010 with an average case detection rate of 0.118 per 100 000 population.Among these cases,2.7％ were children under 15 years,86.5％ multibacillary,and 22.5％ suffered from grade 2 disability.From 2001 to 2010,a total of 1506 relapse cases were detected,and relapse occurred after multi-drug therapy(MDT) in 464 of these cases.There were significant differences between low and high endemic areas in the proportion of children under 15 years,females,immigrant patients among newly detected patients as well as the proportion of cases of relapse after MDT among all the relapse cases.By the end of 2010,the registered leprosy cases were 6032 with a prevalence rate of 0.450 per 100 000 population,among whom 2886 were under MDT.ConclusionsThe leprosy case detection rate continued to decrease in China from 2001 to 2010 with an unequal distribution.The pocket areas were in Yunnan province,Guizhou province,Sichuan province,Guangdong province,Hunan province and Tibet Autonomous Region.It is warranted to enhance the control of leprosy and reduce the prevalence of disability due to leprosy.