Biosynthesis of the phycocyanin ß-subunit in Escherichia coli BL21 and its antioxidant activity and application in the preservation of fresh-cut apples.

In this study, the biosynthesis of phycocyanin ß-subunit (CpcB) in Escherichia coli BL21 was investigated, and its antioxidant activity and application in anti-browning of fresh-cut apples was explored. Four genes (cpcB, cpeS, hox1 and pcyA) involved in the biosynthesis of CpcB were cloned and transformed into E. coli BL21 by constructing recombinant plasmid pETDuet-5. The positive transformant was screened by ampicillin resistance. The analysis of SDS-PAGE and zinc fluorescence spectrum showed that CpcB was successfully expressed in E. coli BL21 with a molecular weight of 21 kDa. The purified CpcB had a maximum absorption peak at 615 nm, and its maximum florescence emission wavelength was 640 nm. It exhibited a stronger ability to scavenge four free radicals than Vc. The color change in fresh-cut apples was obviously delayed by the CpcB treatment. These results suggest that CpcB may be used as a potential anti-browning agent for food preservation.

The role of ultrasonographic findings for PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor-2-negative breast cancer.

To determine whether ultrasound (US) features of breast cancer are associated with Breast Imaging and Reporting Data System molecular subtype, histologic grade, and hormone receptor status as well as to assess the predictive value of these features. Retrospective analysis of the medical records of 220 consecutive patients with invasive breast cancer was reviewed according to the PIK3CA-mutated molecular tumor subtype. US findings of all patients were analyzed. Breast tumors harboring a PIK3CA-mutation were large and exhibited liquefied necrosis and posterior echo attenuation in the nodule. Moreover, such tumors were lobulated and calcified. The aspect ratio of the PIK3CA-mutant was more likely >1. The average nodule elasticity (7.479 ± 0.993 m/s) was measured using US shear wave elastography. Microcalcification was easier to detect inside the nodule using a fluorescence technique. Measurement of the nodule blood flow spectrum showed that the internal blood flow resistance index of nodules was lower than that of other types of breast cancer. The sonographic features of PIK3CA-mutated breast cancers were strongly associated with extensive and liquefied necrosis. The ability to predict molecular subtypes, particularly using US to detect the triple-negative subtype, may play an important role in early management and treatment.

Initial decomposition mechanisms and the inverse effects of temperature and PH2 on the thermodynamic stability of UH3.

The thermodynamic stability of uranium hydrides is of broad interest and fundamental importance for understanding the hydriding corrosion of uranium, and the storage and isotope separation of hydrogen. Based on the first-principles calculations, we reveal the initial decomposition mechanism, interpret the experimental pyrolysis results, and discuss the inverse effects of temperature and hydrogen pressure (PH2) on the thermodynamic stability of ß-UH3. The decomposition mechanism of ß-UH3 is found to be closely related to the changes of U-H bonding properties in UH12 cages. Specifically, at the beginning it is difficult to break the first U-H covalent bond in each UH12 cage, which brings in the existence of a concave region in the experimental PH2-C-T curve; however, it boosts the itinerant character of U-5f electrons. Thereafter, the formation energy of H-vacancies in the degraded UH11 cages is almost changeless when the H/U atom ratio decreases, resulting in the van't Hoff plateau of the PH2-C-T curve. Based on the above mechanisms, we propose a theoretical method to evaluate the thermodynamic stability of ß-UH3. The calculated PH2-C-T curve is consistent with experiment, showing that temperature promotes ß-UH3 decomposition and PH2 plays an opposite role. Moreover, this method is independent of experimental calibration and is applied to discuss the isotope effect of hydrogen in ß-UH3. This work provides new insight and a practical method for the scientific studies of uranium hydride, which is also essential to industrial applications in hydrogen isotope separation.

The Potential Roles of Exosomes Carrying APP and Tau Cleavage Products in Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia throughout the world. It is characterized by major amyloid plaques and neurofibrillary tangles (NFTs), which are composed of amyloid-ß (Aß) peptide and hyperphosphorylated Tau (p-Tau), respectively. Exosomes, which are secreted by cells, are single-membrane lipid bilayer vesicles found in bodily fluids and they have a diameter of 30-150 nm. Recently, they have been considered as critical carriers and biomarkers in AD, as they facilitate communication between cells and tissues by delivering proteins, lipids, and nucleic acids. This review demonstrates that exosomes are natural nanocontainers that carry APP as well as Tau cleavage products secreted by neuronal cells and that their formation is associated with the endosomal-lysosomal pathway. Moreover, these exosomes can transfer AD pathological molecules and participate in the pathophysiological process of AD; therefore, they have potential diagnostic and therapeutic value for AD and might also provide novel insights for screening and prevention of the disease.

Synthesis and Biological Evaluation of Gentiopicroside Derivatives as Novel Cyclooxygenase-2 Inhibitors with Anti-Inflammatory Activity.

Purpose: Nonsteroidal anti-inflammatory drugs cause a series of adverse reactions. Thus, the search for new cyclooxygenase-2 selective inhibitors have become the main direction of research on anti-inflammatory drugs. Gentiopicroside is a novel selective inhibitor of cyclooxygenase-2 from Chinese herbal medicine. However, it is highly hydrophilic owing to the presence of the sugar fragment in its structure that reduces its oral bioavailability and limits efficacy. This study aimed to design and synthesize novel cyclooxygenase-2 inhibitors by modifying gentiopicroside structure and reducing its polarity. Materials and Methods: We introduced hydrophobic acyl chloride into the gentiopicroside structure to reduce its hydrophilicity and obtained some new derivatives. Their in vitro anti-inflammatory activities were evaluated against NO, TNF-α, PGE2, and IL-6 production in the mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide. The in vivo inhibitory activities were further tested against xylene-induced mouse ear swelling. Molecular docking predicted that whether new compounds could effectively bind to target protein cyclooxygenase-2. The inhibitory activity of new compounds to cyclooxygenase-2 enzyme were verified by the in vitro experiment. Results: A total of 21 novel derivatives were synthesized, and exhibit lower polarities than the gentiopicroside. Most compounds have good in vitro anti-inflammatory activity. The in vivo activity results demonstrated that 8 compounds were more active than gentiopicroside. The inhibition rate of some compounds was higher than celecoxib. Molecular docking predicted that 6 compounds could bind to cyclooxygenase-2 and had high docking scores in accordance with their potency of the anti-inflammatory activity. The confirmatory experiment proved that these 6 compounds had significant inhibitory effect against cyclooxygenase-2 enzyme. Structure-activity relationship analysis presumed that the para-substitution with the electron-withdrawing groups may benefit the anti-inflammatory activity. Conclusion: These gentiopicroside derivatives especially PL-2, PL-7 and PL-8 may represent a novel class of cyclooxygenase-2 inhibitors and could thus be developed as new anti-inflammatory agents.

The Mechanism Underlying the Regulation of Long Non-coding RNA MEG3 in Cerebral Ischemic Stroke.

Cerebral ischemic stroke is one of the leading causes of morbidity and mortality worldwide, and rapidly increasing annually with no more effective therapeutic measures. Thus, the novel diagnostic and prognostic biomarkers are urgent to be identified for prevention and therapy of ischemic stroke. Recently, long noncoding RNAs (lncRNAs), a major family of noncoding RNAs with more than 200 nucleotides, have been considered as new targets for modulating pathological process of ischemic stroke. In this review, we summarized that the lncRNA-maternally expressed gene 3 (MEG3) played a critical role in promotion of neuronal cell death and inhibition of angiogenesis in response to hypoxia or ischemia condition, and further described the challenge of overcrossing blood-brain barrier (BBB) and determination of optimal carrier for delivering lncRNA' drugs into the specific brain regions. In brief, MEG3 will be a potential diagnostic biomarker and drug target in treatment and therapy of ischemic stroke in the future.

Quantitative Analysis of the Multicomponent and Spectrum-Effect Correlation of the Antispasmodic Activity of Shaoyao-Gancao Decoction.

Shaoyao-Gancao Decoction (SGD) is a well-known classic traditional Chinese medicine (TCM) with antispasmodic, anti-inflammatory, and analgesic effects. This preparation has been widely used to treat spasticity diseases in the clinic. To date, the material basis of SGD remains unclear, and the spectrum-effect correlation of its antispasmodic activity has not been reported yet. In this study, high-performance liquid chromatography (HPLC) was used to establish the fingerprint and determine the multiple components of SGD. The common peaks of fingerprints were evaluated by the similarity with the chromatographic fingerprints of the TCM. Meanwhile, the multiple components were quantified and analysed using the heatmap and box size analysis. Furthermore, data on the antispasmodic effect were extracted through in vitro smooth muscle contraction assay. Grey relational analysis combined with partial least square regression was used to study the spectrum-effect correlation of SGD. Finally, the potential antispasmolytic components were validated using an isolated tissue experiment. The HPLC fingerprint was established, and 20 common peaks were identified. The similarities of 15 batches of SGD were all above 0.965. The HPLC method for simultaneous determination of the multiple components was accurate and reliable. The contents of albiflorin, paeoniflorin, liquiritin, and glycyrrhizic acid were higher than the other components in SGD. The heatmap and box size also showed that X3 (albiflorin), X4 (paeoniflorin), X5 (liquiritin), X11 (liquirtigenin), and X16 (glycyrrhizic acid) could be used as quality indicators in the further establishment of quality standards. The spectrum-effect correlation results indicated that X4, X11, and X16 were highly correlated with antispasmolytic activity. Verification tests showed that paeoniflorin (11.7-29.25 µg/mL) and liquirtigenin (17.19-28.65 µg/mL) could significantly reduce the maximum contractile (P < 0.01). These compounds exerted concentration-dependent spasmolytic effects with the inhibitory response for acetylcholine (Ach)-evoked contraction. Thus, SGD had a significant antispasmodic effect, which resulted from the synergistic activity of its multiple components. These findings can be used for the pharmacodynamics study of SGD and are of great significance for the determination of quality markers and quality control.

Synthesis, and anti-inflammatory activities of gentiopicroside derivatives.

A series of 26 novel derivatives have been synthesized through structural modification of gentiopicroside, a lead COX-2 inhibitor. And their in vivo and in vitro anti-inflammatory activities have been investigated. The in vitro anti-inflammatory activities were evaluated against NO, PGE2, and IL-6 production in the mouse macrophage cell line RAW264.7 stimulated by LPS. Results showed that most compounds had good inhibitory activity. The in vivo inhibitory activities were further tested against xylene-induced mouse ear swelling. Results demonstrated that several compounds were more active than the parent compound gentiopicroside. The inhibition rate of the most active compound P23 (57.26%) was higher than positive control drug celecoxib (46.05%) at dose 0.28 mmol·kg-1. Molecular docking suggested that these compounds might bind to COX-2 and iNOS. Some of them, e.g P7, P14, P16, P21, P23, and P24, had high docking scores in accordance with their potency of the anti-inflammatory activitiy, that downregulation of the inflammatory factors, NO, PGE2, and IL-6, was possibly associated with the suppression of iNOS and COX-2. Therefore, these gentiopicroside derivatives may represent a novel class of COX-2 and iNOS inhibitors.

Free-Radical-Promoted Remote Unactivated C(sp3)-H Dehydrogenative Coupling Reaction of Free Alcohols with Quinone and Chromone.

An efficient free-radical-promoted unactivated C(sp3)-H dehydrogenative coupling reaction of free alcohols at the δ position with quinone and chromone has been developed. This reaction has a good functional group tolerance and substrate scope; various alcohols reacted with quinones and chromones to give the corresponding C(sp2)-H alkylation products in moderate to good yields. A gram-scale experiment can be successfully operated. This protocol provides a sustainable and practical strategy for the late-stage functionalization of alcohols with quinones and chromones by constructing the challenging δ-selective C(sp3)-C(sp2) bond.

Engineered production of pyridoxal 5'-phosphate in Escherichia coli BL21.

Pyridoxal 5'-phosphate (PLP) is the coenzyme of more than 140 enzymes and is widely used in various fields. In this study, to enhance the production of PLP in Escherichia coli BL21, the recombinant strain E. coli BL21/pETDuet-1-pdxj-zwf-dxs was constructed. The concentration of PLP in this strain was 82.69 mg/L, which was increased by 1.38-fold as compared to that in E. coli BL21. Glucose, yeast extract, and pH had an obvious impact on the concentration of PLP, and their optimal levels were 34.89 g/L, 31.17 g/L, and 10.07, respectively. The concentration of PLP under the optimal condition reached 2.23 g/L. The time-course analysis showed that the highest concentration of PLP was 2.32 g/L in recombinant strain after the induction for 12 h by 0.1 mM IPTG in a 1 L shake flask, which was increased by 38.76-fold as compared to that in E. coli BL21. This study provides a good basis for the efficient production of PLP in E. coli BL21.

Chaperone-assisted soluble expression and characterization of chitinase chiZJ408 in Escherichia coli BL21 and the chitin degradation by recombinant enzyme.

The chaperone-assisted soluble expression and characterization of high molecular weight chitinase chiZJ408 in Escherichia coli BL21 were investigated. Chitin degradation products by chitinase chiZJ408 were analyzed. The results indicated that the introduction of the chaperone GroELS promoted the correct folding of chitinase chiZJ408 and increased its soluble expression by 14.9% (p < 0.05) in E. coli BL21. The optimal pH and temperature of chitinase chiZJ408 were respectively 6.0 and 50 °C. Chitinase chiZJ408 was stable at pHs of 4.0 ∼ 7.0 and at below 40 °C. Mg2+and Ca2+ had a significant impact on improving the activity of chitinase chiZJ408. Chitinase chiZJ408 was demonstrated to be exochitinase that cleaved the ß-1,4-glycosidic bond of the chitin chain to generate only N,N'-diacetylchitobiose. This study broadens our understanding of chitinase and provides a basis for solving the problem of inclusion body formed by long fragment gene expression in E. coli.

Enhancing the biosynthesis of riboflavin in the recombinant Escherichia coli BL21 strain by metabolic engineering.

In this study, to construct the riboflavin-producing strain R1, five key genes, ribA, ribB, ribC, ribD, and ribE, were cloned and ligated to generate the plasmid pET-AE, which was overexpressed in Escherichia coli BL21. The R1 strain accumulated 182.65 ± 9.04 mg/l riboflavin. Subsequently, the R2 strain was constructed by the overexpression of zwf harboring the constructed plasmid pAC-Z in the R1 strain. Thus, the level of riboflavin in the R2 strain increased to 319.01 ± 20.65 mg/l (74.66% increase). To further enhance ribB transcript levels and riboflavin production, the FMN riboswitch was deleted from E. coli BL21 with CRISPR/Cas9 to generate the R3 strain. The R4 strain was constructed by cotransforming pET-AE and pAC-Z into the R3 strain. Compared to those of E. coli BL21, the ribB transcript levels of R2 and R4 improved 2.78 and 3.05-fold, respectively. The R4 strain accumulated 437.58 ± 14.36 mg/l riboflavin, increasing by 37.17% compared to the R2 strain. These results suggest that the deletion of the FMN riboswitch can improve the transcript level of ribB and facilitate riboflavin production. A riboflavin titer of 611.22 ± 11.25 mg/l was achieved under the optimal fermentation conditions. Ultimately, 1574.60 ± 109.32 mg/l riboflavin was produced through fed-batch fermentation with 40 g/l glucose. This study contributes to the industrial production of riboflavin by the recombinant E. coli BL21.

Enhancing the production of physiologically active vitamin D3 by engineering the hydroxylase CYP105A1 and the electron transport chain.

In this study, the conversion of vitamin D3 (VD3) to its two active forms 25(OH)VD3 and 1α, 25(OH)2VD3 was carried out by engineering the hydroxylase CYP105A1 and its redox partners Fdx and Fdr. CYP105A1 and Fdx-Fdr were respectively expressed in E. coli BL21(DE3) and purified. The electron transport chain Fdx-Fdr had higher selectivity for the coenzyme NADH than NADPH. HPLC analysis showed that CYP105A1 could hydroxylate the C25 and C1α sites of VD3 and convert VD3 to its active forms. Finally, a one-bacterium-multi-enzyme system was constructed and used in whole-cell catalytic experiments. The results indicated that 2.491 mg/L of 25(OH)VD3 and 0.698 mg/L of 1α, 25(OH)2VD3 were successfully produced under the condition of 1.0% co-solvent DMSO, 1 mM coenzyme NADH and 35 g/L biocatalyst loading. This study contributes to a basis for the industrial production of active VD3 in future.

Quantitative evaluation and significance of ultrasound in bronchoalveolar lavage for lung consolidation in children with severe mycoplasma pneumonia.

BACKGROUND: The aim of this study was to evaluate the effect of lung ultrasound on bronchoalveolar lavage in the treatment of mycoplasma pneumonia in children. METHODS: Thirty children with mycoplasma pneumonia who were treated with medication and bronchial lavage were selected. We collected the results of laboratory and imaging examinations after admission, and the changes in lung consolidation area were examined by ultrasound before, immediately upon completion, and at 1, 3, and 7 days after bronchoalveolar lavage. The effective factors affecting bronchial lavage were analyzed through logistic regression. RESULTS: Univariate regression analysis showed significant differences in the lung consolidation area at admission, before, immediately upon completion, and at 1 and 7 days after lavage, as well as in the disease course and atelectasis (or not). Multivariate analysis showed that there were statistical differences in the lung consolidation area at admission, atelectasis (or not), and disease course: lung consolidation area at admission [odds ratio (OR): 2.31512, 95% confidence interval (CI): (0.00182, 0.031775), P=0.029474], atelectasis [OR: 2.695742, 95% CI: (0.079281, 0.597218), P=0.012629], and disease course [OR: -2.43347, 95% CI: (-0.02568, -0.00211), P=0.022773]. CONCLUSIONS: Lung ultrasound can evaluate the effect of bronchial lavage through lung consolidation and atelectasis, which can provide a reference for clinical treatment.

Recent advances in research on vine tea, a potential and functional herbal tea with dihydromyricetin and myricetin as major bioactive compounds.

Vine tea has been used as an herbal tea by several ethnic minorities for hundreds of years in China. Flavonoids, a kind of indispensable component in a variety of nutraceutical, pharmaceutical and cosmetic applications, are identified to be the major metabolites and bioactive ingredients in vine tea. Interestingly, vine tea exhibits a wide range of significant bioactivities including anti-oxidant, anti-inflammatory, anti-tumor, antidiabetic, neuroprotective and other activities, but no toxicity. These bioactivities, to some extent, enrich the understanding about the role of vine tea in disease prevention and therapy. The health benefits of vine tea, particularly dihydromyricetin and myricetin, are widely investigated. However, there is currently no comprehensive review available on vine tea. Therefore, this report summarizes the most recent studies investigating bioactive constituents, pharmacological effects and possible mechanisms of vine tea, which will provide a better understanding about the health benefits and preclinical assessment of novel application of vine tea.

Enhancing the production of γ-aminobutyric acid in Escherichia coli BL21 by engineering the enzymes of the regeneration pathway of the coenzyme factor pyridoxal 5'-phosphate.

The compound γ-aminobutyric acid (GABA) was widely used in various fields. To enhance the production of GABA in Escherichia coli BL21(DE3), the enzymes of the regeneration pathway of the coenzyme factor pyridoxal 5'-phosphate (PLP) were engineered. The recombinant E. coli strain was screened and identified. The initial concentrations of L-monosodium glutamate (L-MSG) had an obvious influence on the production of GABA. The highest concentration of GABA in recombinant E. coli BL21/pET28a-gadA was 5.54 g/L when the initial L-MSG concentration was 10 g/L, whereas it was 8.45 g/L in recombinant E. coli BL21/pET28a-gadA-SNO1-SNZ1 at an initial L-MSG concentration of 15 g/L. The corresponding conversion yields of GABA in these two strains were 91.0% and 92.7%, respectively. When the initial concentrations of L-MSG were more than 15 g/L, the concentrations of GABA in E. coli BL21/pET28a-gadA-SNO1-SNZ1 were significantly higher as compared to those in recombinant E. coli BL21/pET28a-gadA, and it reached a maximum of 13.20 g/L at an initial L-MSG concentration of 25 g/L, demonstrating that the introduction of the enzymes of the regeneration pathway of PLP favored to enhance the production of GABA. This study provides new insight into producing GABA effectively in E. coli BL21(DE3).

Measurement of the sound velocity of shock compressed water.

The sound velocities of water in the Hugoniot states are investigated by laser shock compression of precompressed water in a diamond anvil cell. The obtained sound velocities in the off-Hugoniot region of liquid water at precompressed conditions are used to test the predictions of quantum molecular dynamics (QMD) simulations and the SESAME equation-of-state (EOS) library. It is found that the prediction of QMD simulations agrees with the experimental data while the prediction of SESAME EOS library underestimates the sound velocities probably due to its improper accounting for the ionization processes.

Recent advances in research on vine tea,a potential and functional herbal tea with dihydromyricetin and myricetin as major bioactive compounds / 药物分析学报

Vine tea has been used as an herbal tea by several ethnic minorities for hundreds of years in China.Flavonoids,a kind of indispensable component in a variety of nutraceutical,pharmaceutical and cosmetic applications,are identified to be the major metabolites and bioactive ingredients in vine tea.Interest-ingly,vine tea exhibits a wide range of significant bioactivities including anti-oxidant,anti-inflammatory,anti-tumor,antidiabetic,neuroprotective and other activities,but no toxicity.These bioactivities,to some extent,enrich the understanding about the role of vine tea in disease prevention and therapy.The health benefits of vine tea,particularly dihydromyricetin and myricetin,are widely investigated.However,there is currently no comprehensive review available on vine tea.Therefore,this report summarizes the most recent studies investigating bioactive constituents,pharmacological effects and possible mechanisms of vine tea,which will provide a better understanding about the health benefits and preclinical assessment of novel application of vine tea.

Electrochemical Determination of Nicotine in Tobacco Products Based on Biosynthesized Gold Nanoparticles.

In this work, gold nanoparticles were biosynthesized via Plectranthus amboinicus leaf extract as the reducing agent. A series of techniques were used for sample analysis. The biosynthesized gold nanoparticles (bAuNPs) are a uniform size with a spherical shape. The FTIR analysis reveals the presence of many oxygen-containing functional groups on the bAuNP surface. The cyclic voltammetry and electrochemical impedance spectroscopic characterizations reveal that while the bAuNPs have a slightly lower conductivity than chemically synthesized AuNPs (cAuNPs). However, the bAuNPs have a superior electrocatalytic performance toward nicotine reduction. After optimization, the bAuNP-modified SPE could detect nicotine linearly from 10 to 2,000 µM with a low detection limit of 2.33 µM. In addition, the bAuNPs/SPE have been successfully used for nicotine-containing-product analysis.

Application of lung ultrasonography in critically ill patients with COVID-19.

PURPOSE: Lung ultrasonography (LU) is useful to assess lung lesions and variations at bedside. To investigate the results of LU in severe and critical patients with coronavirus disease 2019 (COVID-19), we performed a single-institution study to evaluate the related lung lesions and variations, and prophylactic strategies, in a large referral and treatment center. METHODS: We included 91 adult patients with severe and critical COVID-19, namely 62 males and 29 females, with an average age of 59 ± 11 years, who underwent LU. We collected the following patient information: sex, age, days in hospital, and days in ICU. In the ultrasound examinations, we recorded the presence of discrete B lines, confluent B lines, consolidation, pleural thickening, pleural effusion, and pneumothorax (PTX). RESULTS: Among the 91 severe and critical patients, 59 cases had scattered B lines, 56 cases had confluent B lines, 58 cases had alveolar-interstitial syndrome (AIS), 48 cases had lung consolidation, six cases had pleural thickening, 39 cases had pleural effusion (average depth of the pleural effusion: 1.0 ± 1.5 cm), and 20 patients developed PTX. In the Cox multivariate analysis, there were significant differences in age, hospitalization days, ICU days, and lung consolidation. CONCLUSION: Lung ultrasonography performed at the bedside can detect lung diseases, such as B lines, PTX, pulmonary edema, lung consolidation, pleural effusion, and variations of these findings. Our findings support the use of LU and measurements for estimating factors, and monitoring response to therapy in severe and critical COVID-19 patients.