Diagnostic Value of Targeted Next-generation Sequencing in Pulmonary Mycobacterial Infections.

OBJECTIVE: This study aimed to explore the diagnostic value of novel technique-targeted next-generation sequencing (tNGS) of bronchoalveolar lavage fluid (BALF) in pulmonary mycobacterial infections. METHODS: This retrospective study was conducted on patients who underwent bronchoscopy and tNGS, smear microscopy, and mycobacterial culture of BALF. Patients with positive Mycobacterium tuberculosis (MTB) culture or GeneXpert results were classified into the tuberculosis case group. Those diagnosed with nontuberculous mycobacteria (NTM)-pulmonary disease (NTM-PD) composed the case group of NTM-PD patients. The control group comprised patients without tuberculosis or NTM-PD. Sensitivity, specificity, and receiver operating characteristic (ROC) curves were used to evaluate the diagnostic performance. RESULTS: For tuberculosis patients with positive mycobacterial culture results, the areas under the ROC curves (AUCs) for tNGS, GeneXpert, and smear microscopy were 0.975 (95% CI: 0.935, 1.000), 0.925 (95% CI: 0.859, 0.991), and 0.675 (95% CI: 0.563, 0.787), respectively. For tuberculosis patients with positive GeneXpert results, the AUCs of tNGS, culture, and smear microscopy were 0.970 (95% CI: 0.931, 1.000), 0.850 (95% CI: 0.770, 0.930), and 0.680 (95% CI: 0.579, 0.781), respectively. For NTM-PD, the AUCs of tNGS, culture, and smear-positive but GeneXpert-negative results were 0.987 (95% CI: 0.967, 1.000), 0.750 (95% CI: 0.622, 0.878), and 0.615 (95% CI: 0.479, 0.752), respectively. The sensitivity and specificity of tNGS in NTM-PD patients were 100% and 97.5%, respectively. CONCLUSION: tNGS demonstrated superior diagnostic efficacy in mycobacterial infection, indicating its potential for clinical application.

Risk factors for lower extremity deep vein thrombosis in acute stroke patients following endovascular thrombectomy: a retrospective cohort study.

Background: Deep vein thrombosis (DVT) in lower extremities as a common complication of acute ischemic stroke (AIS) has long been studied. However, as the therapeutic options for AIS continue to advance, the pathogenic mechanisms behind DVT may change. Endovascular thrombectomy (EVT) has replaced intravenous thrombolysis and become the preferred treatment for AIS patients with large vessel occlusions. Therefore, it is important to update our understanding of DVT and its management. This study aimed to determine the prevalence and risk factors of DVT in AIS patients following EVT. Methods: In this retrospective study, 245 AIS patients who had received EVT were recruited between January 2020 and December 2021. Within 10 days (median 4 days) of thrombectomy, DVT was diagnosed by ultrasonography. Demographic characteristics, clinical findings, and therapeutic procedures were compared between patients with and without DVT using univariate analysis. Cutoff points were defined for EVT time and plasma D-dimer concentration. Multivariable logistic regression was then used to determine the independent risk factors for DVT and evaluate their predictive power. Results: The prevalence of DVT in AIS patients after EVT was 27.3%. Multifactorial logistic regression analysis showed that age (OR 1.036, 95% CI 1.001-1.073; P = 0.045), female sex (OR 3.015, 95% CI 1.446-6.289; P = 0.003), lower limb muscle strength less than grade three (OR 7.015, 95% CI 1.887-26.080; P = 0.004), longer EVT time (OR 1.012, 95% CI 1.004-1.020; P = 0.003), and higher D-dimer levels (OR 1.350, 95% CI 1.150-1.585; P < 0.001) were independently associated with higher DVT risk in AIS patients following EVT. The cutoff points for operative time of EVT and plasma D-dimer were 65.5 min and 1.62 mg/L, respectively, above which the risk for DVT was dramatically increased with OR > 4 in AIS patients. Conclusion: AIS patients are at increased risk of developing DVT following EVT particularly if they have undergone prolonged thrombectomy procedures and exhibit high plasma levels of D-dimers. However, the results of our study need to be validated by a multicenter prospective study with a larger population of stroke patients.

Exploring a high-urease activity Bacillus cereus for self-healing concrete via induced CaCO3 precipitation.

The structural integrity and esthetic appeal of concrete can be compromised by concrete cracks. Promise has been shown by microbe-induced calcium carbonate precipitation (MICP) as a solution for concrete cracking, with a focus on urease-producing microorganisms in research. Bacillus cereus was isolated from soil and employed for this purpose in this study due to its high urease activity. The strain exhibited strong tolerance for alkaline media and high salt levels, which grew at a pH of 13 and 4% salt concentration. The repair of concrete cracks with this strain was evaluated by assessing the effects of four different thickeners at varying concentrations. The most effective results were achieved with 10 g/L of sodium carboxymethyl cellulose (CMC-Na). The data showed that over 90% repair of cracks was achieved by this system with an initial water penetration time of 30 s. The study also assessed the quantity and sizes of crystals generated during the bacterial mineralization process over time to improve our understanding of the process. KEY POINTS: • MICP using Bacillus cereus shows potential for repairing concrete cracks. • Strain tolerates alkaline media and high salt levels, growing at pH 13 and 4% salt concentration. • Sodium carboxymethyl cellulose (CMC-Na) at 10 g/L achieved over 90% repair of cracks.

Correction: Yang et al. Construction and Evaluation of Chitosan-Based Nanoparticles for Oral Administration of Exenatide in Type 2 Diabetic Rats. Polymers 2022, 14, 2181.

The authors wish to make the following corrections to this paper [...].

IPTG-induced high protein expression for whole-cell biosynthesis of L-phosphinothricin.

BACKGROUND: Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase (E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second E. coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase (E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT. MAIN METHODS AND MAJOR RESULTS: We compared the effects of different concentrations of IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U g-1 and 896.23 U g-1 , respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U g-1 and 109.70 U g-1 , respectively. The 200 mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L-1 ·h-1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L-1 ·h-1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production. CONCLUSIONS AND IMPLICATIONS: We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and IPTG is more environmentally friendly. Our data implicated that IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations.

Characteristics and influencing factors of soil preferential flow in typical stands of Karst area in southwest China.

The study of preferential flow phenomena in Karst areas and the identification of the main factors influencing preferential flow are of great importance for the recovery of local vegetation. The distribution of the dyeing solution in the vertical and horizontal directions was examined by field staining tracer test and image processing technique. We analyzed the total dyeing area ratio, matrix flow depth, preferential flow ratio, and length index as pre-ferential flow characteristic parameters, and 14 factors affecting preferential flow using grey correlation analysis. The results showed that there were two main types of preferential flow, funnel-shaped and dendritic, with lateral water movement occurring in the soil of typical Karst stands. The mean value of the dyeing area ratio of the understory in Karst areas was 19.4%, and that of the matrix flow depth, preferential flow ratio, and length index was 4.96 cm, 62.9%, and 385.5%, respectively. Among the 14 environmental factors influencing preferential flow, the initial soil moisture content had the strongest influence on the dyeing area ratio, the available potassium content had the most significant influence on the matrix flow depth, and available phosphorous content had the most significant influence on both the preferential flow ratio and the length index. The high degree of development and spatial variability of preferential flow under typical forest stands in Karst areas was strongly influenced by physical properties such as initial soil water content, while soil nutrient also exerts important influence on preferential flow.

A robust localization approach for Percutaneous Nephrolithotomy (PCNL): A single-center retrospective study.

BACKGROUND: Percutaneous Nephrolithotomy (PCNL) represents the gold standard treatment method for cases with large kidney stones. As a critical step in performing PCNL, the procedure of establishing a safe and accurate nephrostomy tract will dramatically impact the treatment quality of patients with large-sized kidney stones. OBJECTIVE: This work attempts to describe a new and improved process of establishing an accurate nephrostomy tract and clinically evaluate the effectiveness and safeness of this proposed methodology. METHODS: This work represents a retrospective single-center study carried out between August 2013 and November 2019. The collected samples consist of 937 patients who were operated on using PCNL coupled with our proposed procedure. Briefly, a preoperative B-ultrasonography was firstly performed to decide the puncture point in a simulated surgical position where was marked with ureteral catheter segments (2-3 cm). A computed tomography (CT) scan was followed to correct the anchor points in the simulated surgical position. After this, an accurate puncture operation was performed under the real-time guidance of intraoperative B ultrasound. RESULTS: Examining this study, 851 subjects with renal stones and 86 subjects with ureteropelvic junction stones were included for the PCNL operation project. All samples were grouped with Guy's grading system: grade I, II, III, and IV patients there were 0.00%, 42.69%, 51.01%, and 6.30%, respectively. Among these patients, the average age was 48.49 ± 10.80 years old, with a male to female ratio of around 1.73:1. CONCLUSIONS: This study showed that our developed method warrants an accurate and safe PCNL operation that involves the process of establishing the nephrostomy tract. Other advantageous attributes of this new PCNL process include negligible radiation exposure, lesser complications, and low failure rates. More importantly, this new localization approach is particularly attractive for hospitals that are new to the field of adopting PCNL considering its safeness, effectiveness, and learnability.

Increased CAP37 Expression in Stable Chronic Obstructive Pulmonary Disease.

OBJECTIVE: Cationic antimicrobial protein of 37 kDa (CAP37), a neutrophil-derived protein originally identified for its antimicrobial activity, is now known to have many regulatory effects on host cells. However, its role in the pathogenesis of chronic obstructive pulmonary disease (COPD) has not been studied. We therefore investigated the expression of CAP37 in COPD and its effects on airway structural cells, including bronchial epithelial cells, smooth muscle cells, and fibroblasts. METHODS: CAP37 was detected in the lung tissue, sputum, and plasma of COPD patients and the control subjects, as well as in the neutrophils stimulated by cigarette smoke extract (CSE). BEAS-2B cells, human bronchial smooth muscle cells (HBSMCs), and MRC-5 cells were treated with CAP37 or an anti-CAP37 antibody plus CAP37. Interleukin (IL)-6 and IL-8 were detected in the BEAS-2B cells. The cell proliferation was analyzed in the HBSMCs. Collagens were also detected in the MRC-5 cells. RESULTS: The expression of CAP37 was increased in the lung tissue and sputum supernatant of the COPD patients compared with the control subjects. The sputum supernatant CAP37 levels were inversely correlated with the forced expiratory volume in the first second percentage predicted in COPD. CAP37 was induced by CSE stimulation in the peripheral blood neutrophils from healthy non-smokers. CAP37 induced expression of IL-6 and IL-8 in BEAS-2B cells, and collagen expression of lung fibroblasts (MRC-5 cells). However, CAP37 did not significantly alter the proliferation of the HBSMCs. CONCLUSION: Our findings indicated that neutrophil-derived CAP37 may be involved in airway inflammation and fibrosis in COPD via affecting the bronchial epithelial cells, and fibroblasts, thus suggesting a possible role of CAP37 in the development and progression of COPD.

Strain Screening and Particle Formation: a Lysinibacillus boronitolerans for Self-Healing Concrete.

Microbial-induced calcite precipitation is a promising technology to solve the problem of cracks in soil concrete. The most intensively investigated microorganisms are urease-producing bacteria. Lysinibacillus that is used as urease-producing bacteria in concrete repair has rarely been reported. In this study, Lysinibacillus boronitolerans with a high urease activity was isolated from soil samples. This strain is salt- and alkali-tolerance, and at pH 13, can grow to ~OD600 2.0 after 24 h. At a salt concentration of 6%, the strain can still grow to ~OD600 1.0 after 24 h. The feasibility of using this strain in self-healing concrete was explored. The data showed that cracks within ~0.6 mm could be repaired naturally with hydration when spores and substrates were added to the concrete in an appropriate proportion. Moreover, the number and morphology of CaCO3 crystals that were produced by bacteria can be influenced by the concrete environment. An efficiency method to elucidate the process of microbial-induced calcium carbonate crystal formation was established with Particle Track G400. This study provides a template for future studies on the theory of mineralization based on microorganisms. IMPORTANCE The formation of calcium carbonate crystals in concrete by urease-producing bacteria is not understood fully. In this study, a Lysinibacillus boronitolerans strain with a high urease activity was isolated and used to analyze the counts and sizes of the crystals and the relationship with time. The data showed that the number of crystal particles increases exponentially in a short period with sufficient substrate, after which the crystals grow, precipitate or break. In concrete, the rate-limiting steps of calcium carbonate crystal accumulation are spore germination and urease production. These results provided data support for the rational design of urease-producing bacteria in concrete repair.

Construction and Evaluation of Chitosan-Based Nanoparticles for Oral Administration of Exenatide in Type 2 Diabetic Rats.

Oral delivery of therapeutic peptides has been a daunting challenge due to poor transport across the tight junctions and susceptibility to enzymatic degradation in the gastrointestinal tract. Numerous advancement in nanomedicine has been made for the effective delivery of protein and peptide. Owing to the superior performance of chitosan in opening intercellular tight junctions of epithelium and excellent mucoadhesive properties, chitosan-based nanocarriers have recently garnered considerable attention, which was formulated in this paper to orally deliver the GLP-1 drug (Exenatide). Against this backdrop, we used chitosan (CS) polymers to encapsulate the exenatide, sodium tripolyphosphate (TPP) as the cross-linking agent and coated the exterior with sodium alginate (ALG) to impart the stability in an acidic environment. The chitosan/alginate nanoparticles (CS-TPP-ALG) functioned as a protective exenatide carrier, realized efficient cellular uptake and controlled release, leading to a steady hypoglycemic effect and a good oral bioavailability in vivo. Trimethyl chitosan (TMC), a chitosan derivative with stronger positive electrical properties was additionally selected as a substitute for chitosan to construct the TMC-TPP-ALG nanoparticle, and its oral peptide delivery capacity was explored in terms of both characterization and pharmacodynamics studies. Overall, our study demonstrated that functional chitosan/alginate nanoparticles can protect proteins from enzymatic degradation and enhance oral absorption, which presents important research value and application prospects.

Comparison of Application of MD-VF-Auto SEM Method and Plankton Gene Multiplex PCR System in the Diagnosis of Drowning.

OBJECTIVES: To compare the application effect of microwave digestion - vacuum filtration - automated scanning electron microscopy (MD-VF-Auto SEM) method and plankton gene multiplex PCR system in the diagnosis of drowning. METHODS: Lung, liver and kidney tissue of 10 non-drowning cases and 50 drowning cases were prepared for further MD-VF-Auto SEM method analysis and plankton gene multiplex PCR system analysis. The positive detection rate of the two methods in each tissue was calculated. RESULTS: The positive rate of the MD-VF-Auto SEM method detecting diatoms in drowning cases was 100%, and few diatoms were detected in the liver and kidney tissues of 6 non-drowning cases. By using the plankton gene multiplex PCR system, the diatom positive rate of drowning cases was 84%, and all the non-drowning cases were negative. There were significant differences in the positive rate of the liver, kidney tissues between MD-VF-Auto SEM method and plankton gene multiplex PCR system (P<0.05), as well as the total positive rate of cases. However, no significant differences were found in the positive rates of lung tissues (P>0.05). CONCLUSIONS: MD-VF-Auto SEM method is more sensitive than plankton gene multiplex PCR system in diatom test. But the plankton gene multiplex PCR system can also detect plankton other than diatoms. Combination of the two methods can provide a more reliable basis for the diagnosis of drowning.

Maternal Ureaplasma exposure during pregnancy and the risk of preterm birth and BPD: a meta-analysis.

PURPOSE: Perinatal Ureaplasma infection is associated with a variety of adverse outcomes and neonatal diseases. This meta-analysis is to evaluate current evidence evaluating the association between Ureaplasma and adverse pregnancy outcomes and bronchopulmonary dysplasia (BPD) in preterm infants. METHODS: We searched for published articles on Ureaplasma, preterm and BPD in PubMed, the Cochrane Library, and Embase databases posted before August 28, 2021. In addition, the references of these articles were screened. A random/fixed-effect model was used to synthesize predefined outcomes. RESULTS: A total of 19 cohort studies involving 11,990 pregnancy women met our inclusion criteria. Pregnancy Ureaplasma positive increased the risk of preterm birth [odds ratios (OR) 2.76, 95% confidence intervals (CI) 1.63-4.68], BPD (OR 2.39 95% CI 1.73-3.30), chorioamnionitis (OR 2.71, 95% CI 2.02-3.64) and premature rupture of membranes (PROM, OR 2.19, 95% CI 1.34-3.58). CONCLUSIONS: Pregnancy Ureaplasma positive may increase the risk of developing preterm birth, chorioamnionitis, PROM and BPD in the preterm infant. The evidence base is, however, of low quality and well-designed studies are needed.

Comparison of Application of MD-VF-Auto SEM Method and Plankton Gene Multiplex PCR System in the Diagnosis of Drowning / 法医学杂志

OBJECTIVES@#To compare the application effect of microwave digestion - vacuum filtration - automated scanning electron microscopy (MD-VF-Auto SEM) method and plankton gene multiplex PCR system in the diagnosis of drowning.@*METHODS@#Lung, liver and kidney tissue of 10 non-drowning cases and 50 drowning cases were prepared for further MD-VF-Auto SEM method analysis and plankton gene multiplex PCR system analysis. The positive detection rate of the two methods in each tissue was calculated.@*RESULTS@#The positive rate of the MD-VF-Auto SEM method detecting diatoms in drowning cases was 100%, and few diatoms were detected in the liver and kidney tissues of 6 non-drowning cases. By using the plankton gene multiplex PCR system, the diatom positive rate of drowning cases was 84%, and all the non-drowning cases were negative. There were significant differences in the positive rate of the liver, kidney tissues between MD-VF-Auto SEM method and plankton gene multiplex PCR system (P<0.05), as well as the total positive rate of cases. However, no significant differences were found in the positive rates of lung tissues (P>0.05).@*CONCLUSIONS@#MD-VF-Auto SEM method is more sensitive than plankton gene multiplex PCR system in diatom test. But the plankton gene multiplex PCR system can also detect plankton other than diatoms. Combination of the two methods can provide a more reliable basis for the diagnosis of drowning.

Comparison of LAMP, GeneXpert, Mycobacterial Culture, Smear Microscopy, TSPOT.TB, TBAg/PHA Ratio for Diagnosis of Pulmonary Tuberculosis.

OBJECTIVE: To investigate the application value of loop-mediated isothermal amplification (LAMP), GeneXpert, mycobacterial culture, smear microscopy, TSPOT.TB (TSPOT), ratio of TB-specific antigen to phytohemagglutinin (TBAg/PHA ratio) in the detection of mycobacterium tuberculosis in the bronchoalveolar lavage fluid. METHODS: A retrospective analysis was performed on the patients who underwent bronchoscopy from December 2018 to November 2019 in Tongji Hospital. The patients with positive tuberculosis culture or positive GeneXpert in bronchoalveolar lavage fluid were selected as the case group, and those without tuberculosis served as the control group. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of LAMP, GeneXpert, culture, smear microscopy, TSPOT, and TBAg/PHA ratio. RESULTS: For the patients with positive cultures as case, the sensitivity of LAMP, GeneXpert, smear microscopy, TSPOT and TBAg/PHA ratio was 73.49%, 89.16%, 25.30%, 80.00%, 33.85%, respectively, the specificity was 99.00%, 100.00%, 99.00%, 86.00%, 100.00%, respectively, the area under the ROC curve (AUC) was 0.849, 0.938, 0.633, 0.830, 0.669, respectively. For the patients with positive GeneXpert as case, the sensitivity of LAMP, mycobacterial culture, smear microscopy, TSPOT and TBAg/PHA ratio was 73.20%, 74.23%, 22.68%, 68.92%, 29.73%, respectively, the specificity was 99.00%, 100.00%, 99.00%, 86.00%, 100.00%, respectively, the AUC was 0.853, 0.878, 0.623, 0.775, 0.649, respectively. CONCLUSION: The sensitivity of GeneXpert was best. The sensitivity and diagnostic value of LAMP were slightly lower than those of GeneXpert, and were similar to tuberculosis culture. The sensitivity of smear microscopy was low. The specificity of TSPOT was low. When TBAg/PHA ratio >0.2 was used as a diagnostic index, the specificity was improved, but the sensitivity was low.

Development of a fermentation strategy to enhance the catalytic efficiency of recombinant Escherichia coli for l-2-aminobutyric acid production.

Microbial fermentation for enzyme production and then whole-cell catalysis for l-2-aminobutyric acid (l-ABA) production have huge potential for industrial application, but the catalytic capacities of cells are directly related to the fermentation process. Using a 50 L fermenter, the effects of initial glycerol concentration in the medium and rotating speed on cell catalytic capacity were investigated. Fermentation cells showed the best catalytic activity when the initial glycerol concentration was 12 g/L and the rotating speed was 250 rpm. Furthermore, we studied the difference between glycerol and glycerol mixtures as fed-batch media in pH-stat fed-batch fermentation. Results showed that glycerol had better catalytic activity than the glycerol mixture, and the effect of fed-batch fermentation was better than batch fermentation. Meanwhile, the enzyme activities of leucine dehydrogenase and formate dehydrogenase reached 129.87 U/g DCW and 437.02 U/g DCW, respectively, and the intracellular NAD(H) concentration reached 14.94 µmol/g DCW. Using the optimized fermentation parameters, amplified fermentation was then carried out in a 5000 L fermenter to demonstrate the industrial production of l-ABA by Escherichia coli BL21.

Development of a Combination Fermentation Strategy to Simultaneously Increase Biomass and Enzyme Activity of D-amino Acid Oxidase Expressed in Escherichia coli.

D-amino acid oxidase (DAAO) is widely used in the industrial preparation of L-amino acids, and cultivating Escherichia coli (E. coli) expressing DAAO for the biosynthesis of L-phosphinothricin (L-PPT) is very attractive. At present, the biomass production of DAAO by fermentation is still limited in large-scale industrial applications because the expression of DAAO during the fermentation process inhibits the growth of host cells, which limits higher cell density. In this study, the factors that inhibit the growth of bacterial cells during a 5-L fed-batch fermentation process were explored, and the fermentation process was optimized by co-expressing catalase (CAT), by balancing the biomass and the enzyme activity, and by adding exogenous D-alanine (D-Ala) to relieve the limitation of DAAO on the cells and optimize fermentation. Under optimal conditions, the DO-STAT feeding mode with DO controlled at 30% ± 5% and the addition of 27.5 g/L lactose mixed with 2 g/L D-Ala during induction at 28 °C resulted in the production of 26.03 g dry cell weight (DCW)/L biomass and 390.0 U/g DCW specific activity of DAAO; an increase of 78% and 84%, respectively, compared with the initial fermentation conditions. The fermentation strategy was successfully scale-up to a 5000-L fermenter.

Efficient racemization of N-phenylacetyl-D-glufosinate for L-glufosinate production.

Most amino acids contain chiral centres and exist as both D-enantiomer and L-enantiomer. The optically pure enantiomer is often more valuable than the racemate. Enzymatic resolution provides an effective strategy to obtain optically pure amino acids but often results in large amounts of unwanted isomer. In this study, optically pure L-glufosinate (L-PPT) was obtained by coupling amidase-mediated hydrolysis of N-phenylacetyl-D,L-glufosinate with racemization of N-phenylacetyl-D-glufosinate (NPDG), which exclusively exhibits effective herbicidal properties compared with its D-enantiomer. To improve the yield of L-PPT, the racemization reaction conditions were optimized, and through single-factor experiments, the optimal reaction temperature, reaction time, and mole ratio of phenylacetic acid to NPDG were determined to be 150°C, 30 minutes, and 1.5, respectively. The response surface methodology was applied to further optimize the racemization conditions, and the final yield of L-PPT reached 96.13% with optimum reaction temperature of 154°C, reaction time of 23 minutes, and phenylacetic acid/NPDG mole ratio of 1.7, respectively. Moreover, adding a small amount of acetic anhydride further raised the yield of L-PPT to 97.02%.

Fermentative production of the unnatural amino acid L-2-aminobutyric acid based on metabolic engineering.

BACKGROUND: L-2-aminobutyric acid (L-ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important pharmaceuticals. To make the biosynthesis of L-ABA environmental friendly and more suitable for the industrial-scale production. We expand the nature metabolic network of Escherichia coli using metabolic engineering approach for the production of L-ABA. RESULTS: In this study, Escherichia coli THR strain with a modified pathway for threonine-hyperproduction was engineered via deletion of the rhtA gene from the chromosome. To redirect carbon flux from 2-ketobutyrate (2-KB) to L-ABA, the ilvIH gene was deleted to block the L-isoleucine pathway. Furthermore, the ilvA gene from Escherichia coli W3110 and the leuDH gene from Thermoactinomyces intermedius were amplified and co-overexpressed. The promoter was altered to regulate the expression strength of ilvA* and leuDH. The final engineered strain E. coli THR ΔrhtAΔilvIH/Gap-ilvA*-Pbs-leuDH was able to produce 9.33 g/L of L-ABA with a yield of 0.19 g/L/h by fed-batch fermentation in a 5 L bioreactor. CONCLUSIONS: This novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of L-ABA.

Multiple-line Chemotherapy and Tyrosine Kinase Inhibitor Treatment in Patients with Advanced Lung Cancer.

AIM: To analyse the clinical outcomes of patients with lung cancer treated with first and multiple-line chemotherapy and tyrosine kinase inhibitor (TKI). PATIENTS & METHODS: The present study included a total of 89 patients of whom lung cancer was histologically confirmed between July 2016 and September 2017. Patients' demographics, chemotherapy/TKI treatment details and clinical outcomes were retrieved. The progression-free survivals (PFS) after first-line and multiple-line treatments were evaluated using Kaplan-Meier analysis with log-rank test. Risk factors for progressive disease (PD) were identified using Cox multivariate regression model. RESULTS: A total of 50 males and 39 females were enrolled. About 90% of the tumors were histologically classified as adenocarcinoma, and 77/89 cases (86.5%) were at TNM stage IV. The median PFS for the first-line treatment was 6.17 months. After first-line treatment, more favourable PFS was observed in patients who had prior surgery of lung cancer (P = 0.002). Multivariate analysis showed that patients who had received no prior surgical treatment for lung cancer were at higher risk of PD (OR, 4.311; 95% CI, 1.836 to 10.120; P = 0.0008). Besides, in patients with driver mutations, those who received no TKI treatment were under higher risk of PD compared to those who had been treated with TKI (OR, 4.876; 95% CI, 1.877 to 12.666; P = 0.0011). The median PFS for the multiple-line treatment was 24.67 months. After multiple-line treatments, favourable PFS was associated with tumor histological types of adenocarcinoma (P = 0.041), genetic lesions at exon 19 of EGFR (P = 0.001) and fourth-line treatment (P = 0.001). Risk factors for PD after multiple-line treatments were no prior surgery for lung cancer (OR, 3.335; 95% CI, 1.158 to 9.605; P = 0.0256), no TKI use in multiple-line treatment (OR, 10.095; 95% CI, 2.405 to 42.378; P = 0.0016), and being treated by first-line treatment alone (OR, 30.421; 95% CI, 4.813 to 192.269; P = 0.0003). CONCLUSION: The present study demonstrated the benefits of TKI in patients with advanced lung cancer, providing insights into the refinement of the management strategy.