RÉSUMÉ
l-threonine as an important precursor substance of l-isoleucine and improving its accumulation in Escherichia coli became an important idea to construct a chassis strain with high l-isoleucine production. Meanwhile, the effect of l-threonine metabolic pathway disruption in E. coli for the improved production of l-isoleucine remains unrevealed. In the present study, a mutant strain of E. coli was engineered by inactivating specific metabolic pathways (e.g., Δtdh, ΔltaE, and ΔyiaY) that were associated with l-threonine metabolism but unrelated to l-isoleucine synthesis. This was done with the aim to reduce the breakdown of l-threonine and, thereby, increase the production of l-isoleucine. The results obtained demonstrated a 72.3% increment in l-isoleucine production from 4.34 to 7.48 g·L-1 in the mutant strain compared with the original strain, with an unexpected 10.3% increment in bacterial growth as measured at OD600. Transcriptome analysis was also conducted on both the mutant strain NXU102 and the original strain NXU101 in the present study to gain a comprehensive understanding of their physiological attributes. The findings revealed a notable disparity in 1294 genes between the two strains, with 658 genes exhibiting up-regulation and 636 genes displaying down-regulation. The activity of tricarboxylic acid (TCA) cycle-related genes was found to decrease, but oxidative phosphorylation-related genes were highly up-regulated, which explained the increased activity of the mutant strain. For instance, l-lysine catabolism-related genes were found to be up-regulated, which reconfigured the carbon flow into the TCA cycle. The augmentation of acetic acid degradation pathway-related genes assisted in the reduction in acetic acid accumulation that could retard cell growth. Notably, substantial up-regulation of the majority of genes within the aspartate pathway could potentially account for the increased production of l-isoleucine in the present study. In this paper, a chassis strain with an l-isoleucine yield of 7.48 g·L-1 was successfully constructed by cutting off the threonine metabolic pathway. Meanwhile, transcriptomic analysis revealed that the cutting off of the threonine metabolic pathway induced perturbation of genes related to the pathways associated with the synthesis of l-isoleucine, such as the tricarboxylic acid cycle, glycolysis, and aspartic acid pathway.
RÉSUMÉ
γ-Aminobutyric acid (GABA) is a kind of non-proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process. The activity of wild-type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strains Escherichia coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B-T62S and Gad B-Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production. Practical Application : Enzymatic properties of the GAD from Escherichia coli and GAD site-specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B-T62S and Gad B-Q309A mutants were improved compared to Gad B. The kinetic parameters Km and Vmax of Gad B, Gad B-T62S, and Gad B-Q309A mutants were 11.3 ± 2.1 mM and 32.1 ± 2.4 U/mg, 7.3 ± 2.5 mM and 76.1 ± 3.1 U/mg, and 7.2 ± 3.8 mM and 87.3 ± 1.1 U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strains E. coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S, and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively.
RÉSUMÉ
Objective:To investigate the value of N-terminal pro-brain natriuretic peptide (NT-proBNP) combined with bedside echocardiography in diagnosis and treatment of neonatal sepsis with cardiac dysfunction.Methods:A total of 56 children diagnosed with neonatal sepsis in the Neonatal Intensive Care Unit, Liaocheng People′s Hospital from July 2016 to July 2017 were enrolled and divided into 2 groups, namely, the cardiac dysfunction group (26 cases) and the non-cardiac dysfunction group (30 cases). Children with general infection (45 cases) hospita-lized at the same period were taken as the control group.The clinical characteristics, related laboratory indexes and prognosis were compared among 3 groups.The related factors of neonatal sepsis with cardiac dysfunction were analyzed by the multivariate Logistic regression approach, and the value of related indexes in the early prediction neonatal sepsis with cardiac dysfunction was analyzed by using the receiver operating characteristic curve (ROC). Results:The onset age of sepsis patients with cardiac dysfunction [63.0 h (30.5 h, 185.6 h)] was significantly earlier than that of the patients without cardiac dysfunction [65.0 h (34.5 h, 170.6 h)] and the control group [80.0 h (45.5 h, 202.3 h)] ( P<0.05). The main primary site of the disease was the lung, which was not statistically significant among the 3 groups ( P>0.05). The NT-proBNP level and the high sensitivity-C-reactive protein (hs-CRP)/albumin (ALB) ratio in the cardiac dysfunction group [20 230.6 ng/L (15 890.0 ng/L, 35 000.0 ng/L); 0.33(0.29, 0.81)] were significantly higher than those in the control group [7 324.5 ng/L (2 426.5ng/L, 13 890.0 ng/L); 0.06(0, 0.21)] (all P<0.05). The right ventricular diameter and the Tei index of the cardiac dysfunction group [(8.74±2.42) mm; 0.52±0.03] were significantly higher than those in the control group [(8.55±1.41)mm; 0.30±0.04], while the EF of the cardiac dysfunction group [(62.61±2.56)%] was significantly lower than that in the control group [(70.03±0.35)%] (all P<0.05). The ROC curve analysis showed that NT-proBNP and the Tei index could effectively predict sepsis with cardiac dysfunction.Specifically, NT-proBNP had a cutoff value of 12 291.5 ng/L, with sensitivity of 80%, specificity of 79%, and the area under ROC curve (AUC) of 0.81.The Tei index had a cutoff value of 0.45, with sensitivity of 74%, specificity of 77%, and the AUC of 0.78. Conclusions:NT-proBNP can be used as a marker of early cardiac dysfunction.Its combination with the Tei index of bedside echocardiography can quickly diagnose cardiac dysfunction of children with sepsis, better guide clinicians in drug use, improve cardiac function of patients and enhance the treatment effect.
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Objective:To investigate the significance of N-terminal pro-brain natriuretic peptide (NT-proBNP) in the early assessment of neonatal cardiac dysfunction in sepsis.Methods:The children diagnosed with neonatal sepsis and common infection neonates admitted to the department of pediatric neonatal intensive care unit (NICU) of Liaocheng People's Hospital from January 2016 to January 2019 were enrolled. Data of clinical sign, laboratory results, bedside echocardiography and survival data were collected, and the differences of clinical indexes were compared among sepsis patients with and without cardiac dysfunction and common infection. The risk factors of sepsis with cardiac dysfunction were analyzed by multivariate Logistic regression, and the early prediction value of NT-proBNP for neonatal septic cardiac dysfunction was evaluated by the receiver operating characteristic (ROC) curve.Results:There were 112 neonates with sepsis (49 with cardiac dysfunction and 63 without cardiac dysfunction) and 67 children with common infection included in the analysis. The onset time of neonates in septic cardiac dysfunction group was significantly earlier than that of septic non-cardiac dysfunction group and common infection group [hours: 52.9 (0, 180.3) vs. 53.9 (0, 183.6), 81.0 (45.6, 202.4), both P < 0.05]. Compared with the general infection group, albumin (ALB), white blood cell count (WBC), left ventricular ejection fraction (LVEF) in septic cardiac dysfunction group significantly decreased, NT-proBNP, hypersensitive C-reactive protein (hs-CRP)/ALB, pulmonary artery systolic pressure (PASP) significantly increased, while right ventricular (RV) and Tei index significantly increased [ALB (g/L): 24.1±3.8 vs. 27.8±3.6, WBC (×10 9/L): 12.7 (3.7, 18.9) vs. 15.4 (9.9, 23.2), LVEF: 0.626±0.123 vs. 0.700±0.021, NT-proBNP (ng/L): 20 230.6 (15 890.0, 35 000.0) vs. 7 324.5 (2 426.5, 13 890.0), hs-CRP/ALB: 0.33 (0.29, 0.81) vs. 0.06 (0.00, 0.21), PASP (mmHg, 1 mmHg = 0.133 kPa): 52.25±14.12 vs. 41.07±27.73, RV (mm): 10.74±2.42 vs. 8.55±1.41, Tei index: 0.52±0.03 vs. 0.30±0.04, all P < 0.05]. NT-proBNP and Tei index in septic cardiac dysfunction group were significantly higher than those in septic non-cardiac dysfunction group [NT-proBNP (ng/L): 20 230.6 (15 890.0, 35 000.0) vs. 13 057.6 (8 946.0, 35 000.0), Tei index: 0.52±0.03 vs. 0.39±0.02, both P < 0.05], and LVEF was significantly lower than that in septic non-cardiac dysfunction group (0.626±0.123 vs. 0.671±0.086, P < 0.05). Multivariate Logistic regression analysis showed that NT-proBNP, Tei index and hs-CRP/ALB were independent risk factors for cardiac dysfunction in sepsis neonates [odds ratio ( OR) and 95% confidence interval (95% CI) were 8.73 (1.54-5.67), 1.97 (1.26-2.87), 1.87 (1.03-3.40) respectively, all P < 0.05]. ROC curve analysis showed that NT-proBNP, Tei index and hs-CRP/ALB had good predictive value for the occurrence of cardiac dysfunction in septic neonates, the area under ROC curve (AUC) was 0.81 (95% CI was 0.84-0.91), 0.78 (95% CI was 0.65-0.79) and 0.77 (95% CI was 0.61-0.77), respectively. The sensitivity and specificity of NT-proBNP were 80.0% and 79.0% respectively with 12 291.5 ng/L as the cut-off value, the sensitivity and specificity of Tei index were 74.0% and 77.0% respectively with 0.45 as the cut-off value, and the sensitivity and specificity of hs-CRP/ALB were 76.0% and 76.3% respectively with 0.10 as the cut-off value. Conclusions:NT-proBNP can be used as a diagnostic marker of early cardiac dysfunction, and for rapid diagnosis of neonatal cardiac dysfunction in sepsis. The application may guide clinicians to use drugs better to improve cardiac function and treatment effect.
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Objective? To? explore? the? clinical? significance? of? early? oral? intervention? measures? in? the?prognosis?of?premature?infants.? Methods? 151?preterm?infants?admitted?to?neonatal?intensive?care?unit?(NICU)?of?Liaocheng?People's?Hospital?from?January?2015?to?January?2017?were?enrolled.?Premature?infants?were?divided?into?intervention?group?and?control?group?according?to?random?number?table?method?and?with?the?consent?of?legal?guardian.?Both?groups?received?routine?treatment?of?preterm?infants?after?stable?vital?signs.?The?intervention?group?received?the?oral?massage?method?adopted?by?none-nutritive?sucking,?stimulating?swallowing?function?and?SandraFucile?on?the?basis?of?routine?treatment,?once?a?day?for?14?consecutive?days.?Both?groups?were?followed?up?for?6?months.?The?oral?feeding?ability?of?premature?infants?was?evaluated?by?the?proficiency?(PRO),?rate?of?transfer?(RT),?feeding?process?and??non-nutritive?suction?(NNS).?At?40?weeks?of?postmenstrual?age?(PMA),?neonatal?behavioral?neurological?(NBNA)?was?used?to?assess?neonatal?brain?development;?Infanib?was?used?for?early?motor?development?evaluation?at?3?months?and??6?months?after?birth.? Results? Finally,?151?premature?infants?were?enrolled,?including?78?in?the?intervention?group?and?73?in?the?control?group.?The?time?to?complete?oral?feeding?of?the?intervention?group?was?significantly?shorter?than?that?of?the?control?group?(days:?18.1±3.7?vs.?23.4±5.8,?P?<?0.05).?Compared?with?the?control?group,?at?the?time?of?complete?oral?feeding,?the?PMA?of?the?intervention?group?was?significantly?decreased?(weeks:?33.4±0.9?vs.?35.9±1.9,?P <?0.05),?the?feeding?efficiency?was?significantly?increased?(mL/min:?10.6±5.1?vs.?8.1±4.7,?P?<?0.05),?and?PRO?was?significantly?increased?[(95±8)%?vs.?(72±28)%,?P <?0.05],?and?the?body?weight?was?significantly?decreased?(g:?1?836.0±193.0?vs.?2?000.8±204.5,?P?<?0.05).?The?NNS?scores?of?the?intervention?group?and?the?control?group?were?increased?gradually?with?time?(F?values?were?86.21?and?75.23,?respectively,?both?P?<?0.01),?and?the?NNS?scores?of?the?intervention?group?at??10?days?and?14?days?were?significantly?higher?than?those?of?the?control?group?(52.89±6.26?vs.?46.74±6.24,?73.90±7.01? vs.?63.53±6.80,?both?P?<?0.01).?The?NBNA?scores?of?the?two?groups?were?lower,?but?there?was?no?significant?difference?between?the?intervention?group?and?the?control?group?(32.7±3.6?vs.?32.0±4.1,?P?>?0.05).?Infanib?evaluation?at?3?months?of?age?showed?that?the?proportion?of?normal?children?in?the?intervention?group?was?significantly?higher?than?that?in?the?control?group?[67.95%?(53/78)?vs.?49.31%?(36/73),?P?<?0.05],?and?at?6?months?of?age,?the?proportion?of?normal?children?in?the?intervention?group?was?significantly?higher?than?that?in?the?control?group?[84.62%?(66/78)?vs.?58.90%?(43/73),??P <?0.01].? Conclusion? Early?oral?exercise?intervention?can?shorten?the?transition?time?from?tube?feeding?to?full?oral?feeding?in?NICU?premature?infants?and?improve?the?performance?of?infants?during?feeding.
RÉSUMÉ
Objective To study the clinical features,diagnosis,treatment and prognosis of neonatal fulminant myocarditis.Method From January 2016 to August 2016,clinical data of neonates with fulminant myocarditis admitted to the neonatal intense care unit (NICU) were retrospectively collected and analyzed.Result A total of 11 neonates were enrolled,including 6 males and 5 females,and 5 preterms and 6 full term infants.The average gestation age was (37.7 ± 1.6) weeks and their weight on hospital admission was (3 382 ± 675) g.Among the infants,9 got ill in summer and 2 in spring and winter.The onset of illness was within 3 ~ 5 d after birth in 8 cases and 2 ~ 3 weeks in the other 3 cases.The main clinical presentations included fever,anorexia,shortness of breath and lethargy.Various degrees of cardiac dysfunction appeared in all 11 cases,including cardiogenic shock in 10 cases,severe arrhythmias with multiple organ dysfunction in 7 cases,and viral meningitis in 7 cases.10 infants had significantly elevated brain natriuretic peptide (BNP) and troponin Ⅰ,and those with troponin Ⅰ above 20 μg/L had poor prognosis.A comprehensive treatment of limiting liquid volume,high-dose adrenocortical steroids,and IVIG were carried out.Meanwhile,therapy to prevent shock,improve cardiac function,reverse arrhythmia,and mechanical ventilation were used in children with dyspnea.7 cases were cured and 6 patients were followedup for 6 to 12 months.Among the 6 followed-up patients,within 1 ~3 months after discharge,4 cases had normal echocardiogram,and persistently abnormal echocardiogram were found in the other 2 cases and eventually confirmed as dilated cardiomyopathy.4 patients were dead.Conclusion The clinical manifestations of neonatal fulminant myocarditis are unspecific.It's difficult to recognize the early symptoms,missed and delayed diagnosis are common,resulting in high mortality rate.Timely diagnosis and effective treatment can improve the survival rate.