Objective:To investigate the alteration of right ventricular function after catheter ablation in patients with persistent atrial fibrillation(PAF) and concomitant heart failure with preserved ejection fraction (HFpEF).Methods:The prospective observational study was performed in patients with HFpEF-PAF and undergoing first-time radiofrequency ablation procedures in the First Affiliated Hospital of Nanjing University between May to December 2019. Right ventricular functional parameters were measured before and 5 days, 1, 3, 6 and 12 months after the ablation by transthoracic echocardiography, respectively, including the right ventricular fractional area change (RVFAC), tricuspid annular plane systolic excursion(TAPSE), tricuspid annular diameter (TVAD), tricuspid annular peak systolic speed(TDI-S′) and longitudinal strain of right ventricular free wall (RVFLS). Meanwhile, routine ECG and Holter recordings were performed at each follow-up time point.Results:In this study, atrial fibrillation (AF) recurrence occurred in 4 patients at the 3rd month after ablation, and 7 patients failed to follow up due to the Covid-19. Finally, 19 patients were followed up for the evaluation of cardiac function after catheter ablation. Compared with pre-ablation, right ventricular structural and functional paramters(RVFAC, TAPSE, TVAD, TDI-S′, RVFLS) improved significantly at all stages of follow-up( all P<0.05). Patients with atrial fibrillation recurrence had lower RVFLS and TDI-S′ at the baseline( P=0.039, P=0.019). Conclusions:Right ventricular function could improve in HFpEF-PAF patients who maintain sinus rhythm after radiofrequency ablation.

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.
Asparaginase/genetics* ; Bacillus subtilis/genetics* ; Industrial Microbiology ; Protein Engineering ; Rhizomucor/enzymology* ; Sequence Alignment

2-Hydroxybutyric acid (2-HBA) is an important intermediate for synthesizing biodegradable materials and various medicines. Chemically synthesized racemized 2-HBA requires deracemization to obtain optically pure enantiomers for industrial application. In this study, we designed a cascade biosynthesis system in Escherichia coli BL21 by coexpressing L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) for production of optically pure (S)-2-HBA from bulk chemical L-threonine (L-Thr). To coordinate the production rate and the consumption rate of the intermediate 2-oxobutyric acid in the multi-enzyme cascade catalytic reactions, we explored promoter engineering to regulate the expression levels of TD and FDH, and developed a recombinant strain P21285FDH-T7V7827 with a tunable system to achieve a coordinated multi-enzyme expression. The recombinant strain P21285FDH-T7V7827 was able to efficiently produce (S)-2-HBA with the highest titer of 143 g/L and a molar yield of 97% achieved within 16 hours. This titer was approximately 1.83 times than that of the highest yield reported to date, showing great potential for industrial application. Our results indicated that constructing a multi-enzyme-coordinated expression system in a single cell significantly contributed to the biosynthesis of hydroxyl acids.
Escherichia coli/genetics* ; Formate Dehydrogenases ; Hydroxybutyrates ; Threonine Dehydratase

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.
Aminobutyrates ; Escherichia coli/genetics* ; Leucine Dehydrogenase/genetics* ; Threonine Dehydratase

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.
Aminolevulinic Acid/metabolism* ; Corynebacterium glutamicum/metabolism* ; Fermentation ; Metabolic Engineering ; Rhodobacter capsulatus/enzymology* ; Rhodopseudomonas/enzymology*

Objective:To explore the curative effects and securities of ticagrelor and clopidogrel in the antiplatelet therapy for coronary heart disease in the aged patients, and to provide a more rational strategy for antiplatelet therapy.Methods:Computer search was performed in Cochrane Central Register of Controlled Trial, Ovid-Medline, EMBase, PubMed, CNKI, Wanfang, VIP and CBM, and the randomized controlled trial (RCT) of ticagrelor and clopidogrel in the treatment of coronary heart disease in the old people were collected.The patients in ticagrelor group were given ticagrelor and other drugs, and the patients in clopidogrel group were given clopidogrel and other drugs.RevMan 5.3software was used to perform Meta-analysis, and the adverse events related to the effectiveness such as the major adverse cardiovascular events (MACE) , myocardial infarction (MI) , stent thrombosis, death, stroke and the adverse events related to safety (bleeding event) were observed.Results:According to inclusion and exclusion criteria, a total of 15RCT were included.The results of Meta-analysis showed that the incidences of MACE (RR=0.59, 95％CI:0.46-0.76, Z=4.08, P＜0.01) , MI (RR=0.48, 95％CI:0.28-0.81, Z=2.74, P=0.006) , stent thrombosis (RR=0.16, 95％CI:0.06-0.48, Z=3.30, P=0.0010) , and death (RR=0.52, 95％CI:0.30-0.89, Z=2.41, P=0.02) in ticagrelor group were decreased compared with clopidogrel group;there was no significant difference in stroke rate (RR=0.76, 95％CI:0.39-1.47, Z=0.81, P=0.42) between two groups, and the incidence of bleeding event in ticagrelor group was increased compared with clopidogrel group (RR=1.57, 95％CI:1.20-2.05, Z=3.28, P=0.001) .Conclusion:For the aged patients with coronary heart disease, ticagrelor is superior to clopidogrel;but the incidence of bleeding is increased.

OBJECTIVETo investigate the effect and safety of acupuncture with regulating menstruation to promote pregnancy for diminished ovarian reverse (DOR).

METHODSAccording to prospective case series, 46 patients were observed and finally 40 cases were included. The acupoints were ① Baihui (GV 20), Shenting (GV 24), Guanyuan (CV 4) and bilateral Benshen (GB 13), Huangshu (KI 16), Dahe (KI 12), Luanchao (Extra), Zusanli (ST 36), Sanyinjiao (SP 6), Taixi (KI 3), Taichong (LR 3) and ② bilateral Shenshu (BL 23) and Ciliao (BL 32). The points in the two groups were used alternately. Acupuncture was given for 3 courses, 12 times as a course and 3 times a week. Before and after treatment, and 3 months after treatment, follicle-stimulating hormone (FSH), follicle-stimulating hormone/luteinizing hormone (FSH/LH), estradiol (E), antral follicle count (AFC) and TCM symptom score were observed. The safety was evaluated.

RESULTSCompared with before treatment, the levels of FSH, FSH/LH decreased, and the levels of Eand AFC increased after treatment and at follow-up (all<0.05). And the TCM symptom scores were significantly lower than those before treatment (both<0.05). The rate of pregnancy after treatment was 15% (6/40). There was no infection and organ injury.

CONCLUSIONAcupuncture with regulating menstruation to promote pregnancy can safely improve the ovarian reserve of patients with DOR.

We constructed plasmid pMTac to overexpress 3-ketosteroid-Δ1-dehydrogenase (KSDD) in Mycobacterium neoaurum JC-12 for improving androst-1,4-diene-3,17-dione (ADD) production. To construct pMTac, pACE promoter on pMF41 was replaced by tac promoter, and then four recombinants were constructed, which were M. neoaurum JC-12/pMF41-gfp, M. neoaurum JC-12/pMTac-gfp, M. neoaurum JC-12/pMF41-ksdd and M. neoaurum JC-12/pMTac-ksdd. Fluorescence detection results show that much more green fluorescent protein (GFP) was expressed in M. neoaurum JC-12/pMTac-ksdd than M. neoaurum JC-12/pMF41-ksdd. The activity of KSDD was 2.41 U/mg in M. neoaurum JC-12/pMTac-ksdd, 6.53-fold as that of M. neoaurum JC-12 and 4.36-fold as that of M. neoaurum JC-12/pMF41-ksdd. In shake flask fermentation, ADD production of M. neoaurum JC-12/pMTac-ksdd was 5.94 g/L, increased about 22.2% compared to the original strain M. neoaurum JC-12 and 12.7% to M. neoaurum JC-12/pMF41-ksdd. AD (4-androstene-3,17-dione) production of JC-12/pMTac-ksdd was 0.17 g/L, decreased 81.5% compared to M. neoaurum JC-12 and 71.2% to M neoaurum JC-12/pMF41-ksdd. In the 5 L fermenter, 20 g/L phytosterols was used as substrate, ADD production of M. neoaurum JC-12/pMTac-ksdd was improved to 10.28 g/L. pMTac is favorable for expressing KSDD in M. neoaurum JC-12, and overexpression of KSDD has beneficial effect on ADD producing, and it is the highest level ever reported using fermentation method in M. neoaurum.
Androstadienes ; metabolism ; Fermentation ; Industrial Microbiology ; Mycobacterium ; Oxidoreductases ; genetics ; metabolism ; Phytosterols ; metabolism ; Plasmids

Objective:To screen the binding peptide against adenovirus type 7(Ad7) and evaluate the relevance with the ade-novirus receptor .Methods:Binding peptide against Ad 7 was screened by panning the phage display 12 peptides library .The antibody against the selected peptide was prepared and was used to study the binding to the membrane by immunofluorescence technique .Re-sults:Using Ad7 as the target protein , GTS09 peptide was selected from the phage display 12 peptides library by biopanning .GTS09-phage complex could significantly bind Ad 7, with the affinity constant up to 1.93 ×1010 L/mol;at the same time, immunofluorescence showed that antibody of GTS09 could specifically bind to membrane of 293 cell.Conclusion: Antibody against GTS09 peptide could specifically bind to membrane of 293 cell,which shows that the peptide may presumably have homology with the cell receptors of Ad 7.

Objective To evaluate left ventricular(LV)myocardial contraction patterns and function when pacing in different right ventricular(RV)sites and discuss echocardiogarphic method to evaluate physiologcal pacing mode.Methods This study included 26 patients with paroxysmal supraventricular tachycardia without organic heart disease.Four pacing modes including right atrium pacing(AAI),RV apex pacing(VVI-RVA),RV septal pacing(VVI-IVS)and RV outflow tract pacing(VVI-RVOT)were performed on the patients in a random order after succussful radiofrequency ablation.The parameters measured in each pacing mode included(1)LV systolic function parameters:LV twist angle(Twist),aortic systolic velocity-time integral(VTIAo)and LV global strain(Gε)；(2)LV contracting pattern:segmental peak systolic strain(Sε),the time to peak value(TPε),and the distribution of segmental Sε,TPε in each layer or wall.The relationship between Sε,TPε of each wall was analyzed.[Results]Pacing from RV sites showed lower Twist,VTIAO and Gε than AAI mode.Gε demonstrated significant difference in three RV sites pacing mode(VVI-RVOT＞VVI-IVS＞VVI-RVA,P＜0.05).Compared with the AAI mode,the distribution of segmental Sε,TPε in the each layer or wall alerted significantly in three RV sites pacing mode,especially in VV1-RVA.The distribution pattern was similar in VVI-RVOT and VVI-IVS.Furthermore,the wall Sε collated negtively with wall TPε(r =-0.51,P＜0.001).[Conclusions]Compared with AAI mode,RV pacing,especially the VVI-RVA induced the alternation of LV contraction patterns and reduction of systolic function.Longitudinal strain parameters can be used to assess the myocardial contraction patterns and function in different pacing mode.