Flow field-flow fractionation coupled with multidetector: A robust approach for the separation and characterization of resistant starch.

The unique properties of resistant starch (RS) have made it applicable in the formulation of a broad range of functional foods. The physicochemical properties of RS play a crucial role in its applications. Recently, flow field-flow fractionation (FlFFF) has attracted increasing interest in the separation and characterization of different categories of RS. In this review, an overview of the theory behind FlFFF is introduced, and the controllable factors, including FlFFF channel design, sample separation conditions, and the choice of detector, are discussed in detail. Furthermore, the applications of FlFFF for the separation and characterization of RS at both the granule and molecule levels are critically reviewed. The aim of this review is to equip readers with a fundamental understanding of the theoretical principle of FlFFF and to highlight the potential for expanding the application of RS through the valuable insights gained from FlFFF coupled with multidetector analysis.

A myxobacterial GH19 lysozyme with bacteriolytic activity on both Gram-positive and negative phytopathogens.

Myxobacteria, as predatory bacteria, have good application potential in the biocontrol of pathogenic microorganisms. Extracellular enzymes are thought to play an important role in their predation and also provide resources for discovering new antibacterial molecules. We previously isolated a myxobacterium, Corallococcus silvisoli c25j21 GDMCC 1.1387, which is predatory to plant pathogenic bacteria. In this study, we identified an endolysin-like GH19 glycoside hydrolase, C25GH19B, from the genome of c25j21. After its heterologous expression and purification from E. coli, the enzymatic properties of C25GH19B were characterized. C25GH19B showed lysozyme activity with the optimal reaction conditions at 40 °C and pH 4.5-5.0. Moreover, C25GH19B showed bacteriolytic activity against both Gram-positive and Gram-negative plant pathogenic bacteria. Our research provides not only a candidate enzyme for the development of novel biocontrol agents but also an experimental basis for further study on the function and mechanisms of extracellular enzymes in myxobacterial predation.

Study on effects of preparation method on the structure and antioxidant activity of protein-Tremella fuciformis polysaccharide complexes by asymmetrical flow field-flow fractionation.

Oxidation is an essential biological process for human life. In this study, low density lipoprotein-Tremella fuciformis polysaccharide (LDL-TFP) complexes were prepared by electrostatic and covalent methods. The effects of preparation method on the structure and antioxidant activity of LDL-TFP complexes were investigated by asymmetrical flow field-flow fractionation (AF4) coupled with ultraviolet-visible (UV/Vis), multiangle light scattering (MALS), and differential refractive index (dRI) detectors. The results showed that the electrostatic LDL-TFP complexes had a spherical structure, while the covalent LDL-TFP complexes had a rod-like structure as indicated by the ratio of Rg (radius of gyration) to Rh (hydrodynamic radius). Moreover, the results revealed that the antioxidant activity of the LDL-TFP complexes on the HepG2 could be related to the structure of LDL-TFP complexes. The antioxidant activity of LDL-TFP complexes formed by LDL modified with phospholipase A2 was further enhanced. This study would help expand the application of TFP.

Co-Infection with Common Respiratory Pathogens and SARS-CoV-2 in Patients with COVID-19 Pneumonia and Laboratory Biochemistry Findings: A Retrospective Cross-Sectional Study of 78 Patients from a Single Center in China.

BACKGROUND This retrospective study aimed to investigate co-infections with common respiratory pathogens and SARS-CoV-2 and laboratory biochemistry findings in patients with COVID-19 in the Zhuzhou area of China, in order to provide a reference for the disease assessment and clinical treatment of COVID-19. MATERIAL AND METHODS The clinical data of COVID-19 patients admitted to the hospital of Zhuzhou City from January 28 to March 15, 2020, as well as laboratory test results for respiratory pathogens and biochemical indicators, were collected to conduct correlation analyses. All patients were diagnosed based on fluorescence-based PCR assay for SARS-CoV-2. RESULTS Eleven of the 78 patients (14.1%) were co-infected with other respiratory pathogens, among which Mycoplasma pneumoniae (n=5, 45.5%) and respiratory syncytial virus (n=4, 36.4%) were the most frequent. There were 8 patients co-infected with 1 other pathogen and 3 patients co-infected with 2 other pathogens. Compared with mono-infected COVID-19 patients, patients with co-infections had significantly higher levels of procalcitonin (P=0.002). CONCLUSIONS The findings showed that Mycoplasma pneumonia and respiratory syncytial virus were the most common co-infections in patients with COVID-19 pneumonia. Increased levels of PCT in patients with COVID-19 pneumonia were associated with co-infection.

Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design.

Lytic polysaccharide monooxygenases (LPMOs) oxidatively break down the glycosidic bonds of crystalline polysaccharides, significantly improving the saccharification efficiency of recalcitrant biomass, and have broad application prospects in industry. To meet the needs of industrial applications, enzyme engineering is needed to improve the catalytic performance of LPMOs such as enzyme activity and stability. In this study, we engineered the chitin-active CjLPMO10A from Cellvibrio japonicus through a rational disulfide bonds design. Compared with the wild-type, the variant M1 (N78C/H116C) exhibited a 3-fold increase in half-life at 60°C, a 3.5°C higher T 50 15 , and a 7°C rise in the apparent Tm. Furthermore, the resistance of M1 to chemical denaturation was significantly improved. Most importantly, the introduction of the disulfide bond improved the thermal and chemical stability of the enzyme without causing damage to catalytic activity, and M1 showed 1.5 times the specific activity of the wild-type. Our study shows that the stability and activity of LPMOs could be improved simultaneously by selecting suitable engineering sites reasonably, thereby improving the industrial adaptability of the enzymes, which is of great significance for applications.

Myxococcus xanthus R31 Suppresses Tomato Bacterial Wilt by Inhibiting the Pathogen Ralstonia solanacearum With Secreted Proteins.

The pathogenic bacterium Ralstonia solanacearum caused tomato bacterial wilt (TBW), a destructive soil-borne disease worldwide. There is an urgent need to develop effective control methods. Myxobacteria are microbial predators and are widely distributed in the soil. Compared with other biocontrol bacteria that produce antibacterial substances, the myxobacteria have great potential for biocontrol. This study reports a strain of Myxococcus xanthus R31 that exhibits high antagonistic activity to R. solanacearum. Plate test indicated that the strain R31 efficiently predated R. solanacearum. Pot experiments showed that the biocontrol efficacy of strain R31 against TBW was 81.9%. Further study found that the secreted protein precipitated by ammonium sulfate had significant lytic activity against R. solanacearum cells, whereas the ethyl acetate extract of strain R31 had no inhibitory activity against R. solanacearum. Substrate spectroscopy assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of secreted proteins showed that some peptidases, lipases, and glycoside hydrolases might play important roles and could be potential biocontrol factors involved in predation. The present study reveals for the first time that the use of strain M. xanthus R31 as a potential biocontrol agent could efficiently control TBW by predation and secreting extracellular lyase proteins.

A myxobacterial LPMO10 has oxidizing cellulose activity for promoting biomass enzymatic saccharification of agricultural crop straws.

Myxobacteria are soil microorganisms with the ability to break down biological macromolecules due to the secretion of a large number of extracellular enzymes, but there has been no research report on myxobacterial lytic polysaccharide monooxygenases (LPMOs). In this study, two LPMO10s, ViLPMO10A and ViLPMO10B, from myxobacterium Vitiosangium sp. GDMCC 1.1324 were characterized. Of which, ViLPMO10B is a C1-oxidizing cellulose-active LPMO. Moreover, ViLPMO10B could decrease the degree of polymerization of crop straws cellulose and synergize with commercial cellulase to promote the saccharification. When the weight ratio of commercial cellulase to ViLPMO10B was 9:1, the conversion efficiency of corn stalk, sugarcane bagasse, and rice straw into reducing sugar was improved by 17%, 16%, and 22%, respectively, compared with commercial cellulase without ViLPMO10B. These results indicate that ViLPMO10B has the potential to be a component of a high-efficient cellulase cocktail and has application value in the saccharification of agricultural residual biomasses.

Hygrophorus subsection Hygrophorus (Hygrophoraceae, Agaricales) in China.

Hygrophorus subsect. Hygrophorus has been relatively well-studied in Europe and North America, but studies on the taxa in Asia, particularly in China, are still limited. In this study, phylogenetic overviews of genus Hygrophorus, based on the nuclear large subunit (LSU) ribosomal RNA gene and of subsect. Hygrophorus, based on the nuclear ribosomal internal transcribed spacer (ITS) regions were generated. Four new species, i.e. H. brunneodiscus, H. fuscopapillatus, H. glutiniceps and H. griseodiscus are described from southern China; and a rarely reported edible species H. hedrychii is described in detail, based upon the materials from north-eastern China. The main characteristics of the species under subsect. Hygrophorus worldwide are summarised in a table.

Study on structure-function of starch by asymmetrical flow field-flow fractionation coupled with multiple detectors: A review.

Starch is one of the most abundant, renewable, non-toxic, and biodegradable polysaccharides, mainly comprising amylose and amylopectin. The physicochemical properties and structure of starch affect the quality of starch-based products. Therefore, the accurate characterization of the physicochemical properties and structure of starch is important to obtain products that meet specific functions. Recently, asymmetrical flow field-flow fractionation coupled with multi-angle light scattering (MALS) and differential refraction detectors (dRI) (AF4-MALS-dRI) has attracted great attention in the separation and characterization of starch. In this review, the structure and functions of starch characterized by AF4-MALS-dRI are summarized. The principle and important characterization parameters of AF4 are introduced. The starch structure-function relationship is deeply investigated owing to utilization of AF4-MALS-dRI. Besides separation of starch, AF4-MALS-dRI can provide more information about starch sample, such as radius of gyration (Rg), hydrodynamic radius (Rh), molar mass (Mw), apparent density (ρ), and conformation (Rg/Rh) of starch molecules. These parameters can be further used to investigate the structure-function relationship of starch.

Reflection and transmission of thermo-elastic waves without energy dissipation at the interface of two dipolar gradient elastic solids.

The reflection and transmission of thermal elastic waves at the interface between two different dipolar gradient elastic solids are studied based on the generalized thermo-elastic theory of Green and Naghdi [(1993). J. Elasticity 31, 189-208] (type II of no energy dissipation). First, some thermodynamic formulas are generalized to a dipolar gradient elastic solid and the function of free energy density is postulated. Second, equations of thermal motion and constitutive relations in a dipolar gradient elasticity are derived. Then the nontraditional interfacial conditions are used to determine the amplitude ratio of the reflection and transmission waves with respect to the incident wave. Some numerical results of the reflection and transmission coefficients in the form of an energy flux ratio are given for different microstructure parameters while thermal parameters are fixed. The numerical results are validated by the consideration of energy conservation. It is found that there are a total of five modes of dispersive waves, namely, coupled MT1 wave, coupled MT2 wave, coupled MT3 wave, SV wave, and one evanescent wave which reduces to the surface waves at an interface, namely, SS wave. The thermal parameters mainly affect the coupled MT2 wave while the microstructure parameters affect not only the coupled waves but also the SS surface waves.

Wave propagation through a flexoelectric piezoelectric slab sandwiched by two piezoelectric half-spaces.

Reflection and transmission of plane waves through a flexoelectric piezoelectric slab sandwiched by two piezoelectric half-spaces are studied in this paper. The secular equations in the flexoelectric piezoelectric material are first derived from the general governing equation. Different from the classical piezoelectric medium, there are five kinds of coupled elastic waves in the piezoelectric material with the microstructure effects taken into consideration. The state vectors are obtained by the summation of contributions from all possible partial waves. The state transfer equation of flexoelectric piezoelectric slab is derived from the motion equation by the reduction of order, and the transfer matrix of flexoelectric piezoelectric slab is obtained by solving the state transfer equation. By using the continuous conditions at the interface and the approach of partition matrix, we get the resultant algebraic equations in term of the transfer matrix from which the reflection and transmission coefficients can be calculated. The amplitude ratios and further the energy flux ratios of various waves are evaluated numerically. The numerical results are shown graphically and are validated by the energy conservation law. Based on these numerical results, the influences of two characteristic lengths of microstructure and the flexoelectric coefficients on the wave propagation are discussed.

[Application of asymmetrical flow field-flow fractionation for size characterization of low density lipoprotein in egg yolk plasma].

Home-made asymmetrical flow field-flow fractionation (AF4) system, online coupled with ultraviolet/visible (UV/Vis) detector was employed for the separation and size characterization of low density lipoprotein (LDL) in egg yolk plasma. At close to natural condition of egg yolk, the effects of cross flow rate, sample loading, and type of membrane on the size distribution of LDL were investigated. Under the optimal operation conditions, AF4-UV/Vis provides the size distribution of LDL. Moreover, the precision of AF4-UV/Vis method proposed in this work for the analysis of LDL in egg yolk plasma was evaluated. The intra-day precisions were 1.3% and 1.9% (n=7) and the inter-day precisions were 2.4% and 2.3% (n=7) for the elution peak height and elution peak area of LDL, respectively. Results reveal that AF4-UV/Vis is a useful tool for the separation and size characterization of LDL in egg yolk plasma.

Study on external factors affecting egg yolk plasma by asymmetrical flow field-flow fractionation.

In this work, the ability of asymmetrical flow field-flow fractionation (AF4) coupled with UV-vis, multiangle light scattering (MALS), and quasi-elastic light scattering (QELS) (AF4-UV-MALS-QELS) for monitoring aggregation of components of egg yolk plasma was evaluated. The effect of external factors (i.e., pH, storage conditions, freezing and heat treatments) on the egg yolk plasma was studied. The results reveal that the aggregation mechanism of components of egg yolk plasma during heat and freezing treatment is different. The results suggest that the low density lipoproteins (LDLs) in egg yolk plasma undergo a 'clusters-fusion-gel' process under heat treatment at pH10. The alkaline conditions promote the formation of LDL aggregates. Also, storage conditions play a role in the formation of LDL aggregates. It was found that the hen eggs stored for 7days at room temperature contain less aggregate than those stored at 4°C for the same period. The combination of AF4 with online MALS-QELS provided conformational information in terms of the shape and size distribution of LDL aggregates. AF4-UV-MALS-QELS was proved to be a rapid and gentle method for the separation and characterization of egg yolk plasma aggregates.

Asymmetrical flow field-flow fractionation coupled with multiple detections: A complementary approach in the characterization of egg yolk plasma.

The capability of asymmetrical flow field-flow fractionation (AF4) coupled with UV/VIS, multiangle light scattering (MALS) and quasi-elastic light scattering (QELS) (AF4-UV-MALS-QELS) for separation and characterization of egg yolk plasma was evaluated. The accuracy of hydrodynamic radius (Rh) obtained from QELS and AF4 theory (using both simplified and full expression of AF4 retention equations) was discussed. The conformation of low density lipoprotein (LDL) and its aggregates in egg yolk plasma was discussed based on the ratio of radius of gyration (Rg) to Rh together with the results from bio-transmission electron microscopy (Bio-TEM). The results indicate that the full retention equation is more relevant than simplified version for the Rh determination at high cross flow rate. The Rh from online QELS is reliable only at a specific range of sample concentration. The effect of programmed cross flow rate (linear and exponential decay) on the analysis of egg yolk plasma was also investigated. It was found that the use of an exponentially decaying cross flow rate not only reduces the AF4 analysis time of the egg yolk plasma, but also provides better resolution than the use of either a constant or linearly decaying cross flow rate. A combination of an exponentially decaying cross flow AF4-UV-MALS-QELS and the utilization of full retention equation was proved to be a useful method for the separation and characterization of egg yolk plasma.

Sedimentation field-flow fractionation for characterization of citric acid-modified Hß zeolite particles: Effect of particle dispersion and carrier composition.

In this study, sedimentation field-flow fractionation (SdFFF) was, for the first time, applied for determination of size distribution of Hß zeolite particles modified by citric acid (CA-Hß). Effects of the particle dispersion and the carrier liquid composition (type of dispersing reagent (surfactant) and salt added in the carrier liquid, ionic strength, and pH) on SdFFF elution behavior of CA-Hß zeolite particles were systematically investigated. Also the SdFFF separation efficiency of the particles was discussed in terms of the forces such as van der Waals, hydrophobic, and induced-dipole interactions. Results reveal that the type of salt and pH of the carrier liquid significantly affect the SdFFF separation efficiency of the zeolite particles. It was found that addition of a salt (NaN3) into the carrier liquid affects the characteristic of the SdFFF channel surface. It was found that the use of an acidic medium (pH 3.2) leads to a particle-channel interaction, while the use of a basic medium (pH 10.6) promotes an inter-particle hydrophobic interaction. Result from SdFFF was compared with those from scanning electron microscopy (SEM) and dynamic light scattering (DLS). It seems that, once the experimental conditions are optimized, SdFFF becomes a valuable tool for size characterization of the zeolite particles.

[Simultaneous separation and detection of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate by RP-HPLC and structure confirmation].

A simple, fast and sensitive analytical method for the simultaneous separation and detection of 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B by RP-HPLC and drug quality standard was established. The structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate have been confirmed. Reference European Pharmacopoeia EP7.0 version, British Pharmacopoeia 2012 version, National Drug Standards of China (WS 1-XG-2002), domestic and international interrelated literature were referred to select the composition of mobile phase. The experimental parameters including salt concentration, pH, addition quantities of organic solvent, column temperature and flow rate were optimized. Finally, the assay was conducted on a Durashell-C18 column (250 mm x 4.6 mm, 5 microm) with 0.01 mol x mL(-1) ammonium perchlorate (add ammonia to adjust the pH value to 8.2) -methanol (48 : 52) as mobile phase at the flow rate of 0.8 mL x min(-1), and the detection wavelength was set at 254 nm. The column temperature was 50 degrees C and the injection volume was 10 microL. The MS, NMR, UV and RP-HPLC were used to confirm the structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate. Under the optimized separation conditions, the calibration curves of 18 alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B showed good linearity within the concentration of 0.50-100 microg x mL(-1) (r = 0.999 9). The detection limits for 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B were 0.15, 0.10, 0.10, 0.15 microg x mL(-1) respectively. The method is sensitive, reproducible and the results are accurate and reliable. It can be used for chiral resolution of 18alpha-glycyrrhizinic acid, 18Pbeta-glycyrrhizinic acid, and detection content of principal component and related substances of raw material drug of ammonium glycyrrhizinate. It is concluded that the separation of principal component isomer of raw material drug of ammonium glycyrrhizinate and the validity of the substance's structure assignments of retention time being 1.2 in the European pharmacopoeia EP7.0 version, British pharmacopoeia 2012 version remains open to question. It may be of practical value for the quality control of raw material drug, preparation, and Chinese herbal medicine of ammonium glycyrrhizinate.

[Simultaneous determination of principal components and related substances of raw material drug of ammonium glycyrrhizinate by reversed-phase high performance liquid chromatography].

An analytical method for the simultaneous determination of 18alpha-glycyrrhizic acid, 18beta-glycyrrhizinic acid, related substances A and B and drug quality standard by reversed-phase high performance liquid chromatography (RP-HPLC) was established. The assay was carried out on a Durashell-C18 column (250 mm x 4.6 mm, 5 microm) with 10 mmol/L ammonium perchlorate (the pH value was adjusted to 8.20 with ammonia)-methanol (48:52, v/v) as mobile phase at a flow rate of 0.80 mL/min, and the detection wavelength was set at 254 nm. The column temperature was 50 degrees C and the injection volume was 10 microL. Under the separation conditions, the calibration curves of the analytes showed good linearities within the mass concentrations of 0.50 -100 mg/L (r > 0.999 9). The detection limits for 18alpha-glycyrrhizic acid, 18beta-glycyrrhizinic acid, related substances A and B were 0.15, 0.10, 0.10, 0.15 mg/L, respectively. The average recoveries were between 97.32% and 99.33% (n = 3) with the relative standard deviations (RSDs) between 0.05% and 1.06%. The method is sensitive, reproducible, and the results are accurate and reliable. The method can be used for the determination of principal components and related substances of ammonium glycyrrhizinate for the quality control of raw material drug of ammonium glycyrrhizinate.

[Expression and significance of immunoglobulin free light chains in serum and lung tissues from patients with chronic obstructive pulmonary disease].

OBJECTIVE: To study the association of free immunoglobulin light chain (FLC) with clinical manifestations and lung inflammation in smokers with normal lung function and chronic obstructive pulmonary disease (COPD) patients. METHODS: Thirty-two patients with peripheral lung cancer undergoing surgical resection were enrolled from the Department of Thoracic Surgery,Affiliated Hospital of Xuzhou Medical College. They were divided into non-smoking with normal lung function group (non-smoking group, 10 cases), smoking with normal lung function group (smoking group, 12 cases) and smoking with stable COPD group (COPD group, 10 cases). Their preoperative fasting serum and lung tissues away from cancer were used in the study.Enzyme-linked immunesorbent assays (ELISA) were used to detect the levels of FLC-λ and FLC-κ in serum and lung tissue homogenates. The expression of FLC-λ and FLC-κ in the airway epithelium, alveolar wall and blood vessel wall was detected by immunohistochemistry. The correlation between FLC levels and pulmonary functions were analyzed. RESULTS: The serum levels of FLC-λ and FLC-κ in COPD group and smoking group were (35 ± 11),(38 ± 12) and (26 ± 9),(26 ± 8) mg/L, respectively. They were all significantly increased compared with the non-smoking group [(16 ± 7),(16 ± 5) mg/L]. The differences were all statistically significant (q = 3.590-7.482, P < 0.01), and those of the COPD group were significantly higher than those of the smoking group (q = 3.209-4.198, P < 0.05 and P < 0.01). The concentrations of FLC-λ and FLC-κ in lung tissue homogenates of the COPD group and the smoking group were (1.29 ± 0.31),(1.32 ± 0.30) and (0.86 ± 0.42),(0.85 ± 0.37) µg/mg, respectively. They were all significantly increased compared with those of the non-smoking group [(0.45 ± 0.18),(0.42 ± 0.13) µg/mg],(q = 4.178- 9.795, P < 0.05 and P < 0.01). The levels of FLC-λ and FLC-κ in the lung tissue homogenates from the COPD group were significantly higher than those from the smoking group (q = 4.269-4.349, all P < 0.05). The expression of FLC-λ and FLC-κ was detected in airway epithelium, alveolar wall and blood vessel wall. The levels of FLC-λ and FLC-κ in serum and lung tissue homogenates showed a negative correlation with FEV1 percentage of predicted value (r = -0.476 to -0.591, all P < 0.01). CONCLUSIONS: Expressions of FLC were increased in the serum and the lung tissues of COPD patients and smokers with normal lung function, and closely correlated with airflow limitation. The results suggest that FLC plays a proinflammatory role in the pathogenesis of COPD.