Efficient synthesis of L-methionine by engineering the one carbon module of Escherichia coli / 生物工程学报

L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.

Development and practice of national first-class undergraduate course "bioengineering equipment" / 生物工程学报

As a strategic emerging industry of China, the biotechnology industry develops rapidly in recent years, which significantly increased the demand for creative and capable talents. As a core curriculum of bioengineering specialty, biotechnology equipment plays an important role in fostering such talents. To address the problems in biotechnology equipment course teaching such as limited equipment availability, limited engineering practice, and lack of learning motivations, curriculum reform and optimization were performed based on curriculum resource development, virtual reality-physical combined engineering training, and boosting learning motivations. The optimized teaching contents focus on fostering morality, intelligence, and creative practice abilities by connecting new requirements of social development, introducing new progress in biotechnology research, as well as new practices in research and development (R & D). Measures such as teaching methods innovation, assessment and evaluation methods optimization, cutting-edge R & D progress, diverse resources integration, and online-offline combined teaching, were developed to boost the learning motivation and foster the innovation competence of students. By above exploration and practice, the practice and innovation competence of students were significantly enhanced.

Advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli / 生物工程学报

L-homoserine and its derivatives (O-succinyl-L-homoserine and O-acetyl-L-homoserine) are precursors for the biosynthesis of L-methionine, and various C4 compounds (isobutanol, γ-butyrolactone, 1, 4-butanediol, 2, 4-dihydroxybutyric acid) and L-phosphinothricin. Therefore, the fermentative production of L-homoserine and its derivatives became the research hotspot in recent years. However, the low fermentation yield and conversion rate, and the unclear regulation mechanism for the biosynthesis of L-homoserine and its derivatives, hamper the development of an efficient production process for L-homoserine and its derivatives. This review summarized the advances in the biosynthesis of L-homoserine and its derivatives by metabolic engineering of Escherichia coli from the aspects of substrate uptake, redirection of carbon flow at the key nodes, recycle of NADPH and export of target products. This review may facilitate subsequent metabolic engineering and biotechnological production of L-homoserine and its derivatives.

Treating nonspecific low back pain by SET sling exercise therapy combined with tropical herb-Rattan Moxibustion liquid external rubbing therapy

Objective: To explore the clinical effects of tropical Eucommia ulmoides compound-Rattan Moxibustion liquid external rubbing therapy which is from medicine of Li Nationality in Hainan combined with SET sling exercise therapy technique and lumbar-abdominal muscle training program on treating nonspecific low back pain (NLBP) as well as its influence on serum 5-hydroxytryptamine and beta-endorphin levels. Methods: From June 2016 to June 2018, 42 NLBP patients were admitted to the outpatient and ward of Rehabilitation Department of the First Affiliated Hospital of Hainan Medical College, who were randomly divided into control group (n=21) and experimental group (n=21). On the basis of traditional massage manipulation, the control group adopts lumbar and abdominal muscles training, while Rattan Moxibustion liquid external rubbing therapy combined with SET sling exercise therapy was used in the experimental group. The treatment was given once every other day, three times a week and 20 min each time for 8 weeks. Then ODI index and VAS scale of low back pain, as well as serum 5-HT and beta-EP levels were observed and detected. Results: The improvement in low back pain index ODI index of the experimental group were better than that of the control group (control group from 21.23±1.57 to 4.78±0.86 while experiment group from 21.27±1.36 to 2.75±0.64 after 8 weeks' treatment)(P<0.05). VAS scale decreased significantly in the experiment group compared with control group (control group from 6.62±1.51 to 2.71±0.92 while experiment group from 6.71±1.55 to 1.83±0.79 after 8 weeks' treatment)(P<0.05). Serum 5-HT in the experimental group were significantly improved compared with those in the control group [control group from (70.15±20.64) ng/mL to (127.34±40.59) ng/mL while experiment group from (68.72±19.18) ng/mL to (142.65±38.72) ng/mL after treatment](P<0.05) and beta-EP level ameliorates compared with control group [control group from (180.63±51.87) pg/mL to (225.76±57.63) pg/mL while experiment group from (172.39±50.72) pg/mL to (251.26±62.89) pg/mL after treatment](P<0.05). Conclusions: Tropical herb-Rattan Moxibustion liquid external rubbing therapy combined with SET sling exercise therapy technology shows significant effects on NLBP. The mechanism may be related to the improved serum 5-HT and beta-EP levels.

Efficacy and Mechanism of Danlu Tongdu Tablets on Lumbar Disc Herniation with Kidney Deficiency and Stasis Syndrome / 中国实验方剂学杂志

Objective: To observe short-term and long-term efficacies of Danlu Tongdu tablets on lumbar disc herniation (LDH) with kidney deficiency and stasis syndrome, and its effect on nucleus pulposus reabsorption and immunoinflammatory factors. Method: One hundred and sixty patients were randomly divided into control group (80 cases) and observation group by random number table. Patients in control group (80 cases) got acupoint massage and acupuncture, 1 time/day. In addition to the therapy of control group, patients in observation group were also given Danlu Tongdu tablets, 4 tablets/time, 3 times/days. A course of treatment was 12 weeks, and a 9-month follow-up was recorded. Lumbago and leg pain were recorded by visual simulation (VAS) before treatment and at the first, second, third month after treatment. Before and after treatment, Japanese orthopaedic association (JOA), symptoms and signs were scored. During the 9-month follow-up, relapse rate and relapse time were recorded, and VAS and JOA were scored. Absorption of nucleus pulposus was assessed, and levels of interleukin-1 (IL-1), IL-17, tumor necrosis factor-α (TNF-α), matrix metalloproteinases-3 (MMP-3) were detected. Result: By rank sum test, the clinical efficacy in observation group was better than that in control group (Z=2.125, PZ=1.924, PPPPPχ2=5.138, Pα and MMP-3 were lower than that in control group (Pχ2=4.668, PPPPConclusion: In addition to acupuncture and massage therapy, Danlu Tongdu tablets can also be used to relieve pain and ameliorate function, improve clinical efficacy, reduce relapse rate, promote nucleus pulposus reabsorption, and relieve inflammation of nerve root.

Development and application of in-situ microscopy in on-line monitoring of cell biomass / 生物工程学报

With the rapid development of modern biotechnology, fermentation process is increasingly important in industrial production. To guarantee the stability of products, fermentation process should be elaborately monitored and controlled. Biomass is an important parameter for on-line monitoring in bioprocesses because biomass can reflect cell growth in a bioreactor directly. In-situ microscope, a non-invasive and image-analysis based technology, can real-time monitor cells in biological process. This review summarizes the development and application of in-situ microscopy in biomass monitoring.

Effect of microparticles on echinocandin B production by Aspergillus nidulans / 生物工程学报

Anidulafungin is an effective antifungal medicine, which can inhibit activities of candida in vitro and in vivo. Echinocandin B (ECB) is the key precursor of Anidulafungin, thus the price and market prospect of Anidulafungin is directly due to the fermentation titer of ECB. In this study, Aspergillus nidulans was used for ECB fermentation, and the influence of adding microparticles on ECB fermentation was studied, such as talcum powder, Al2O3, and glass beads. The particle size and concentration were the key factors for mycelium morphology and ECB production, and ECB production could reach 1 262.9 mg/L and 1 344.1 mg/L by adding talcum powder of 20 g/L (d50 = 14.2 μm) and 7 glass beads (6 mm), an increase by 33.2% and 41.7%, respectively. The results indicated that the mycelium morphology of filamentous microorganisms and the product yield of fermentation could be improved by adding microparticles remarkably, and it provide an important method for the fermentative optimization of filamentous microorganisms.

Establishment of multiple regression model for virulence factors of Saccharomyces albicans by random amplified polymorphic DNA bands / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To research the relationship between the virulence factors of Saccharomyces albicans (S. albicans) and the random amplified polymorphic DNA (RAPD) bands of them, and establish the regression model by multiple regression analysis.</p><p><b>METHODS</b>Extracellular phospholipase, secreted proteinase, ability to generate germ tubes and adhere to oral mucosal cells of 92 strains of S. albicans were measured in vitro; RAPD-polymerase chain reaction (RAPD-PCR) was used to get their bands. Multiple regression for virulence factors of S. albicans and RAPD-PCR bands was established.</p><p><b>RESULTS</b>The extracellular phospholipase activity was associated with 4 RAPD bands: 350, 450, 650 and 1 300 bp (P < 0.05); secreted proteinase activity of S. albicans was associated with 2 bands: 350 and 1 200 bp (P < 0.05); the ability of germ tube produce was associated with 2 bands: 400 and 550 bp (P < 0.05).</p><p><b>CONCLUSION</b>Some RAPD bands will reflect the virulence factors of S. albicans indirectly. These bands would contain some important messages for regulation of S. albicans virulence factors.</p>

Effects of sarcoplasmic reticulum Ca(2+)-ATPase gene transfer in a minipig model of chronic ischemic heart failure / 中华心血管病杂志

<p><b>OBJECTIVE</b>Chronic myocardial ischemia (CMI) has become the most important cause of heart failure (HF) all over the world. The aim of the current study was to investigate the effects of Sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) gene transfer on cardiac function and endoplasmic reticulum stress (ERS) associated myocardial apoptosis in a minipig HF animal model induced by CMI.</p><p><b>METHODS</b>HF was induced in minipigs by implantation of ameroid constrictor in the initial segment of left anterior descending (LAD) branch of coronary artery. After confirmation of myocardial perfusion defects and cardiac function impairment by myocardial perfusion imaging and echocardiography, animals were divided into 4 groups (n = 4 each): HF group, HF + enhanced green fluorescent protein (EGFP) group, HF + SERCA2a group, and shamed animals as control group. A total amount of 1 × 10(12) v.g. of rAAV1-EGFP or rAAV1-SERCA2a were injected intramyocardially to each animal of HF + EGFP and HF + SERCA2a groups. Sixty days after gene transfer, protein level and activity of SERCA2a were examined, cardiac functions and changes of serum inflammatory and neuro-hormonal factors were determined. Apoptotic index of the ischemic myocardium, protein levels of ER stress marker glucose regulated protein 78 (GRP 78) and ER stress specific apoptotic marker caspase-12 were also assayed.</p><p><b>RESULTS</b>At the study end, echocardiographic and hemo dynamic measurements indicated a significant improvement of both cardiac systolic and diastolic function in HF + SERCA2a group compared with HF/HF + EGFP groups [LVEF (60.2 ± 8.6)% vs (44.2 ± 7.1)% and (46.8 ± 6.7)%, Ev/Av 1.28 ± 0.24 vs 0.77 ± 0.17 and 0.80 ± 0.21, +dp/dt(max) (2713.9 ± 434.0) mm Hg/s (1 mm Hg = 0.133 kPa) vs (1892.3 ± 434.2) mm Hg/s and (1931.2 ± 397.4) mm Hg/s, -dp/dt(max) (1422.1 ± 334.4) mm Hg/s vs (848.3 ± 308.3) mm Hg/s and (849.5 ± 278.3) mm Hg/s, P < 0.05], along with increase in both SERCA2a protein level (1.13 ± 0.26 vs 0.73 ± 0.17 and 0.64 ± 0.18, P < 0.05) and activity [(16.2 ± 5.5) IU/ml vs (7.9 ± 3.1) IU/ml and (7.5 ± 2.8) IU/ml, P < 0.05] compared with HF/HF + EGFP groups. Serum concentrations of inflammatory factor tumor necrotic factor α [(382.3 ± 114.4) ng/L vs (732.3 ± 201.4) ng/L and (689.8 ± 192.5) ng/L, P < 0.05], neural-hormonal factors brain natriuretic peptide [(142.6 ± 45.3) ng/L vs (422.3 ± 113.6) ng/L and (393.7 ± 103.3) ng/L, P < 0.01], endothelin-1 [(111.4 ± 37.5) ng/L vs (193.5 ± 54.3) ng/L and (201.0 ± 72.1) ng/L, P < 0.05] and angiotensin II [(189.7 ± 65.2) µg/L vs (538.3 ± 135.2) µg/L and (525.5 ± 144.1) µg/L, P < 0.01] were also significantly decreased in HF + SERCA2a group compared with HF/HF + EGFP groups. The apoptotic index [(12.71 ± 4.11)% vs (23.22 ± 7.23)% and (24.31 ± 6.38)%, P < 0.05], protein levels of GRP 78 (1.27 ± 0.33 vs 3.23 ± 1.14 and 4.18 ± 1.13, P < 0.05) and protein level ratios of cleaved caspase-12 to total caspase-12 [(4.62 ± 1.93)% vs (9.71 ± 2.70)% and (10.14 ± 2.81)%, P < 0.05] were also significantly reduced in the ischemic myocardium of HF + SERCA2a group compared with the HF/HF + EGFP groups.</p><p><b>CONCLUSION</b>Overexpression of SERCA2a significantly improved cardiac systolic and diastolic function in this HF model partly through attenuation of ER stress related myocardial apoptosis, suggesting its therapeutic potential for CMI related heart failure.</p>

Studies on chemical constituents of Helwingia chinensis / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the chemical constituents of Helwingia Chinensis.</p><p><b>METHOD</b>Compounds were isolated with silica gel, Sephadex LH - 20 and polyamide chromatography, and their structures were elucidated by means of spectral analysis.</p><p><b>RESULT</b>Six compounds were isolated and identified as cinnamic acid (1), gult-5-en-3beta-ol (2), friedelin (3), alpha-amyrin (4), luteolin-7-O-beta-D-glucoside (5) and 4, 5-dimethoxy-1, 2-benzoquinone (6).</p><p><b>CONCLUSION</b>All these Compounds were obtained from H. chinensis for the first time.</p>

A case report of huge fungal granuloma in deep maxillofacial region and review of the literature / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To report one case of huge fungal granuloma in the deep maxillofacial region with its clinical findings,diagnosis and treatment process to improve our understanding of fungal granuloma and lessen misdiagnosis.METHODS: The literature was reviewed with combination of case report.RESULTS: The clinical and imaging findings of fungal granuloma had no specificity and were very subject to misdiagnosis. Confirmed diagnosis needed histopathological examination and bacterial culture.CONCLUSION: Deepened understanding of this disease can decrease misdiagnosis and benefit prompt and proper treatment.