Determination of flurbiprofen in rat plasma using ultra-high performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to determine flurbiprofen in rat plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used in negative ion mode. Acetonitrile precipitation was selected to prepare samples. Flurbiprofen and internal standard flurbiprofen-d5 were analyzed on an Acquity UPLC BEH C18 column with the mobile phase consisting of acetonitrile and water, and a gradient procedure was used for separation. The retention time of flurbiprofen was 0.67 min, and the whole running time was only 1.2 min. The detection was performed on a triple quadrupole tandem mass spectrometer using multiple reaction monitoring mode via an ESI source with optimized mass spectrometry parameters. The calibration curve was linear in the range of 25.0-1.00 × 104 ng/mL (r ≥ 0.99). The within-run and between-run relative standard deviations were not more than 13.9%. The within-run and between-run relative errors were from -9.0% to 3.4%. There was no significant matrix effect, and recovery was high. This method was fully validated, including whole blood stability in rat plasma, and successfully applied to the pharmacokinetic study in which 100% incurred sample reanalysis met the criteria.

Regulation of the pigment production by changing Cell morphology and gene expression of Monascus ruber in high-sugar synergistic high-salt stress fermentation.

AIMS: Extreme environment of microbial fermentation is the focus of research, which provides new thinking for the production and application of Monascus pigments (MPs). In this work, the high-sugar synergistic high-salt stress fermentation (HSSF) of MPs was investigated. METHODS AND RESULTS: The Monascus fungus grew well under HSSF conditions with 35 g L-1 NaCl and 150 g L-1 glucose, and the extracellular yellow pigment and intracellular orange pigment yield in HSSF was 98% and 43% higher than that in conventional fermentation, respectively. Moreover, the mycelial morphology was maintained in a better status with more branches and complete surface structure, indicating good biocatalytic activity for pigment synthesis. Four extracellular yellow pigments (Y1, Y2, Y3, and Y4) were transformed into each other, and ratio of the relative content of intracellular orange pigments to yellow pigments (O/Y) significantly (P < 0.05) changed. Moreover, the ratio of unsaturated fatty acids to saturated fatty acids (unsaturated/saturated) was significantly (P < 0.05) increased, indicating that the metabolism and secretion of intracellular and extracellular pigment might be regulated in HSSF. The pigment biosynthesis genes mppB, mppC, mppD, MpPKS5, and MpFasB2 were up-regulated, whereas the genes mppR1, mppR2, and mppE were down-regulated, suggesting that the gene expression to regulate pigment biosynthesis might be a dynamic change process in HSSF. CONCLUSIONS: The HSSF system of MPs is successfully performed to improve the pigment yields. Mycelial morphology is varied to enhanced pigment secretion, and gene expression is dynamically regulated to promote pigment accumulation in HSSF.

Transcription factors: switches for regulating growth and development in macrofungi.

Macrofungi (or mushrooms) act as an extraordinarily important part to human health due to their nutritional and/or medicinal value, but the detailed researches in growth and development mechanisms have yet to be explored further. Transcription factors (TFs) play indispensable roles in signal transduction and affect growth, development, and metabolism of macrofungi. In recent years, increasing research effort has been employed to probe the relationship between the development of macrofungi and TFs. Herein, the present review comprehensively summarized the functional TFs researched in macrofungi, including modulating mycelial growth, fructification, sclerotial formation, sexual reproduction, spore formation, and secondary metabolism. Meanwhile, the possible effect mechanisms of TFs on the growth and development of some macrofungi were also revealed. Specific examples of functional characterizations of TFs in macrofungi (such as Schizophyllum commune and Coprinopsis cinerea) were described to a better comprehension of regulatory effect. Future research prospects in the field of TFs of macrofungi are discussed. We illustrated the functional versatility of the TFs in macrofungi based on specific examples. A systematical realization of the interaction and possible mechanisms between TFs and macrofungi can supply possible solutions to regulate genetic characteristics, which supply novel insights into the regulation of growth, development and metabolism of macrofungi. KEY POINTS: • The functional TFs researched in macrofungi were summarized. • The possible effect mechanisms of TFs in macrofungal were described. • The multiple physiological functions of TFs in macrofungi were discussed.

Biofilm Formation and Control of Foodborne Pathogenic Bacteria.

Biofilms are microbial aggregation membranes that are formed when microorganisms attach to the surfaces of living or nonliving things. Importantly, biofilm properties provide microorganisms with protection against environmental pressures and enhance their resistance to antimicrobial agents, contributing to microbial persistence and toxicity. Thus, bacterial biofilm formation is part of the bacterial survival mechanism. However, if foodborne pathogens form biofilms, the risk of foodborne disease infections can be greatly exacerbated, which can cause major public health risks and lead to adverse economic consequences. Therefore, research on biofilms and their removal strategies are very important in the food industry. Food waste due to spoilage within the food industry remains a global challenge to environmental sustainability and the security of food supplies. This review describes bacterial biofilm formation, elaborates on the problem associated with biofilms in the food industry, enumerates several kinds of common foodborne pathogens in biofilms, summarizes the current strategies used to eliminate or control harmful bacterial biofilm formation, introduces the current and emerging control strategies, and emphasizes future development prospects with respect to bacterial biofilms.

Preliminary Study on Rapid and Simultaneous Detection of Viable Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella by PMA-mPCR in Food.

Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are major foodborne pathogens that are widespread in nature and responsible for several outbreaks of food safety accidents. Thus, a rapid and practical technique (PMA-mPCR) was developed for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella in pure culture and in a food matrix. To eliminate false positive results, propidium monoazide (PMA) was applied to selectively suppress the DNA amplification of dead cells. The results showed the optimum concentration of PMA is 5.0 µg/mL. The detection limit of this assay by mPCR was 103 CFU/mL in the culture broth, and by PMA-mPCR was 104 CFU/mL both in pure culture and a food matrix (milk and ground beef). In addition, the detection of mixed viable and dead cells was also explored in this study. The detection sensitivity ratio of viable and dead counts was less than 1:10. Therefore, the PMA-mPCR assay proposed here might provide an efficient detection tool for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella and also have great potential for the detection and concentration assessment of VBNC cells.

Cold-adaptive mechanism of psychrophilic bacteria in food and its application.

Psychrophilic bacteria are a type of microorganisms that normally grow in low-temperature environments. They are usually found in extremely cold environments. However, as people's demand for low-temperature storage of food becomes higher, psychrophilic bacteria have also begun to appear in cold storage and refrigerators, which has become a food safety hazard. In this paper, the optimal cooling strategies of psychrophilic bacteria are reviewed from the aspects of the cell membrane, psychrophilic enzymes, antifreeze proteins, cold shock proteins, gene regulation, metabolic levels and antifreeze agents, and the principle of psychrophilic mechanism is briefly described. The application of thermophilic bacteria and its products adapted to cold environments in food fields are analyzed. The purpose of this paper is to provide ideas for future research on psychrophilic bacteria based on the mechanism and application of psychrophilic bacteria.

Research on the drug resistance mechanism of foodborne pathogens.

Foodborne diseases caused by foodborne pathogens are one of the main problems threatening human health and safety. The emergence of multi-drug resistant strains due to the abuse of antibiotics has increased the difficulty of clinical treatment. Research on the drug resistance mechanism of foodborne pathogens has become an effective method to solve multi-drug resistant strains. In this paper, the four main drug resistance mechanisms, including reduced cell membrane permeability, efflux pump mechanism, target site mutation mechanism, and enzymatic hydrolysis, were used to systematically analyze the drug resistance of Salmonella, Listeria monocytogenes, and Escherichia coli. And the new methods were discussed that may be used to solve the drug resistance of foodborne pathogens such as CRISPR and bacteriophages. This review provided a certain theoretical basis for the treatment of foodborne diseases.

The application and research progress of bacteriophages in food safety.

The abuse of antibiotics and the emergence of drug-resistant bacteria aggravate the problem of food safety. Finding safe and efficient antibiotic substitutes is an inevitable demand for ensuring the safety of animal-derived food. Bacteriophages are a kind of virus that can infect bacteria, fungi or actinomycetes. They have advantages of simple structure, strong specificity and nontoxic side effects for the human body. Bacteriophages can not only differentiate live cells from dead ones but also detect bacteria in a viable but nonculturable state. These characteristics make bacteriophages more and more widely used in the food industry. This paper describes the concept and characteristics of bacteriophages, and introduces the application of bacteriophages in preharvest production, food processing, storage and sales. Several methods of using bacteriophages to detect foodborne pathogens are listed. Finally, the advantages and limitations of bacteriophages in the food industry are summarized, and the application prospect of bacteriophages in the food industry is discussed.

The diagnostic tools for viable but nonculturable pathogens in the food industry: Current status and future prospects.

Viable but nonculturable (VBNC) microorganisms have been recognized as pathogenic contaminants in foods and environments. The failure of VBNC cells to form the visible colonies hinders the ability to use conventional media for their detection. Efficient and rapid detection of pathogens in the VBNC state is a prerequisite to ensure the food safety and public health. Despite their nonculturability, VBNC cells have distinct characteristics, such as morphology, metabolism, chemical composition, and gene and protein expression, that have been used as the basis for the development of abundant diagnostic tools. This review covers the current status and advances in various approaches for examining microorganisms in the VBNC state, including but not limited to the methodological aspects, advantages, and drawbacks of each technique. Existing methods, such as direct viable count, SYTO/PI dual staining, and propidium monoazide quantitative polymerase chain reaction (PCR), as well as some techniques with potential to be applied in the future, such as digital PCR, enhanced-surface Raman spectroscopy, and impedance-based techniques, are summarized in depth. Finally, future prospects for the one-step detection of VBNC bacteria are proposed and discussed. We believe that this review can provide more optional methods for researchers and promote the development of rapid, accurate detecting methods, and for inspectors, the diagnostic tools can provide data to undertake risk analysis of VBNC cells.

Investigation of the mycelial morphology of Monascus and the expression of pigment biosynthetic genes in high-salt-stress fermentation.

Extreme environments, for example high-salt-stress condition, that can induce secondary metabolite biosynthesis in fungi are a promising and effective strategy for producing natural Monascus pigments used as food colourants and nutraceutical supplements. In this study, the relationship between the mycelial morphology and expression of pigment biosynthetic genes in high-salt-stress fermentation (HSF) with Monascus ruber CGMCC 10910 was investigated. The Monascus fungus grew well under HSF conditions with 35 g/l NaCl, and the intracellular yellow pigment yield in HSF was 40% higher than that in conventional batch fermentation (CBF). Moreover, the mycelial morphology was maintained in a better state, with a hyphal diameter of 5-6 µm in HSF, indicating good biocatalytic activity for pigment synthesis. The rate of the relative content of intracellular orange pigments to yellow pigments (O/Y) significantly (p < 0.05) changed, and the extracellular yellow pigments were transformed into each other, indicating that the pigment biosynthesis pathway was changed to promote yellow pigment accumulation in HSF. The pigment biosynthesis genes MpPKS5, MpFasB2, mppE, mppD and mppB were significantly (p < 0.05) up-regulated by approximately 58.4-106.1%, whereas the regulatory genes mppR1 and mppR2 were significantly (p < 0.05) down-regulated by approximately 23.2% and 59.0% in HSF. Notably, the mppE gene was highly correlated with (r > 0.95, p < 0.05) hyphal diameter. These findings indicated that the cultivation of the Monascus fungus under high-salt-stress conditions was beneficial for pigment biosynthesis by controlling the mycelial morphology to regulate gene expression. This study first described the relationship between the mycelial morphology and expression of pigment biosynthetic genes in Monascus during fermentation. KEY POINTS: • High-salt-stress fermentation (HSF) was first performed to improve Monascus pigment yield. • Pigment biosynthesis was enhanced by maintaining the mycelial morphology in an improved state in HSF. • Gene expression was up-/downregulated to promote yellow pigment accumulation in HSF. • The mycelial morphology was highly related to the expression of pigment biosynthetic genes in HSF.

Production of diacylglycerols through glycerolysis with SBA-15 supported Thermomyces lanuginosus lipase as catalyst.

BACKGROUND: In this study, SBA-15 was functionalized by silane coupling reagents, then lipase from Thermomyces lanuginosus (TLL) was immobilized onto the parent and the organically modified SBA-15 for diacylglycerol (DAG) production through glycerolysis. RESULTS: Diacylglycerol content of 54.77 ± 0.63%, and triacylglycerol (TAG) conversion of 77.75 ± 1.24%, were obtained from the parent SBA-15 supported TLL-mediated glycerolysis reaction in a solvent-free system. However, poor performance was unexpectedly observed when co-solvents were introduced into the reaction system. After organic modification, the functionalized SBA-15 supported TLL samples all exhibited reasonable performance, producing DAG content over 40 wt% and TAG conversion over 70 wt%. Higher DAG content, up to 59.19 ± 1.10%, was observed from the phenyl group-modified SBA-15 supported TLL. The operational stability of the immobilized TLL samples in glycerolysis was also improved after organic functionalization. The phenyl group-modified SBA-15 supported TLL showed good reusability in the present glycerolysis reaction, and 95.21 ± 4.87% of the initial glycerolysis activity remained after five cycles of reuse. CONCLUSION: The organic modification of SBA-15 improved the catalytic performance of its supported TLL in glycerolysis, in terms of TAG conversion, DAG content, and reusability. © 2019 Society of Chemical Industry.

Label-free colorimetric aptasensor for IgE using DNA pseudoknot probe.

The development of simple and low-cost approaches to the detection of immunoglobulin E (IgE) would provide a method for the early diagnosis and prevention of atopic diseases. The current methods of detection are generally tedious, multi-step processes and are limited by the high cost of the labeled proteins. We describe here a label-free structure-switching colorimetric method for the simple measurement of IgE using DNA pseudoknot probes and gold nanoparticles. In the absence of a target the IgE aptamer probe adopts a pseudoknot conformation that dissociates a capture probe from the unmodified gold nanoparticles. However, when IgE binds to the aptamer probe, the pseudoknot is resolved, leading to a favorable hybridization between the 3' terminal loop of the aptamer probe and the capture probe; this induces the aggregation of the gold nanoparticles. As a result, the colorimetric IgE sensor using this structure-switching mechanism is sensitive, specific and convenient, and the assay works even when challenged with complicated biological matrixes such as vaginal fluids. The proposed method is expected to be of great clinical value for IgE detection and could be used, after appropriate design, for sensing applications of other structured aptamers.

Intraspecific protoplast fusion of Brettanomyces anomalus for improved production of an extracellular ß-glucosidase.

Improvement of production of an extracellular ß-glucosidase with high activity by Brettanomyces anomalus PSY-001 was performed by using recursive protoplast fusion in a genome-shuffling format. The initial population was generated by ultraviolet irradiation, ultrasonic mutagenesis and, then, subjected to recursive protoplast fusion. Mutant strains exhibiting significantly higher ß-glucosidase activities in liquid media were isolated. The best mutant strain showed increased cell growth in a flask culture, as well as increased ß-glucosidase production. A recombinant strain, F3-25, was obtained after three rounds of genome shuffling and its production of ß-glucosidase activity reached 4790 U L-1, which was a nearly eightfold increase compared to the original strain B. anomalus PSY-001. The subculture experiments indicated that F3-25 was genetically stable.

Bactericidal efficacy of plasma-activated water against Vibrio parahaemolyticus on Litopenaeus vannamei.

In this study, the antimicrobial mechanism of plasma-activated water (PAW) against Vibrio parahaemolyticus and the effectiveness of PAW in artificially contaminated Litopenaeus vannamei were investigated. The results demonstrated a significant reduction (p < 0.05) in viable counts of V. parahaemolyticus with increasing plasma discharge time (5, 10, 20, and 30 min) and PAW immersion time (3, 5, 10, 20, and 30 s). Specifically, the count of V. parahaemolyticus decreased by 2.1, 2.7, 3.3, and 4.4 log CFU/mL after exposed to PAW 5, PAW 10, PAW 20, and PAW 30 for 30 s, respectively. Significant cell surface wrinkling, accompanied by notable nucleic acid and protein leakage were observed after treatment with PAW. The permeability of the inner and outer cell membranes was significantly increased (p < 0.05), along with an increase in electrical conductivity (p < 0.05). The reactive oxygen species (ROS) within V. parahaemolyticus cells were significantly increased (p < 0.05), while superoxide dismutase (SOD) activity, and the relative expression of the ompW, emrD, and luxS genes were significantly decreased (p < 0.05). A reduction number of 1.3, 1.8, 2.1, and 2.2 log CFU/g of V. parahaemolyticus in artificially contaminated L. vannamei was obtained with PAW for 5 min. The study elucidated that PAW could destroy cell membranes, leading to cell death. The findings would strengthen strategies for V. parahaemolyticus control and provide a potential application of PAW for preserving aquatic products.

[Management of immunocompromised renal transplant patients infected with coronavirus disease 2019].

OBJECTIVE: To analyze the treatment process of a renal transplant patient infected with coronavirus disease 2019 (COVID-19), and discuss the management strategy for the immunocompromised hosts. METHODS: The diagnosis and treatment of a case of transplant patients with COVID-19 admitted to Horgos designated hospital of Xinjiang Uygur Autonomous Region in October 2021 were reviewed. The medical history and laboratory and imaging examination treatment and outcome of this case were analyzed. RESULTS: The recipient was a middle-aged male with a time from renal transplantation of 3 years. The onset was moderate to low fever, accompanied by cough and fatigue. Chest CT showed multiple ground glass shadows under the pleura of both lungs, mainly in both lower lungs, gradually worsening until "white lung" appeared, with early renal and cardiac insufficiency. In the course of treatment, immunosuppressants were reduced and the dosage of glucocorticoid was increased. In the early stage, due to renal insufficiency and hyperkalemia, dialysis was conducted for 3 times. Oral abidol and Lianhua Qingwen capsule were given as antiviral and anti-infection treatment. Special immunoglobulin and convalescent plasma of COVID-19 were used to boost the immunity of patients. The patient was eventually clinically cured. CONCLUSIONS: The clinical manifestations and diagnosis of COVID-19 for the kidney transplantation recipient are not significantly different from other populations, but immunocompromised hosts are more likely to suffer from organ dysfunction. The adjustment of immunosuppressants and glucocorticoids, respiratory support, selection of antibiotics, organ protection, nutritional support and traditional Chinese medicine intervention in the treatment of renal transplant recipients with severe COVID-19 need further discussion.

Comparison of dietary control and atorvastatin on high fat diet induced hepatic steatosis and hyperlipidemia in rats.

BACKGROUND: Treatment with atorvastatin (ATO) or dietary control has been demonstrated to benefit patients with non-alcoholic fatty liver disease (NAFLD) and hyperlipidemia. However, little is known on whether combination of dietary control and ATO treatment could enhance the therapeutic effect. METHODS: We employed a rat model of NAFLD to examine the therapeutic efficacy of dietary control and/or ATO treatment. Sprague-Dawley rats were fed with normal chow diet as normal controls or with high fat diet (HFD) for 12 weeks to establish NAFLD. The NAFLD rats were randomized and continually fed with HFD, with normal chow diet, with HFD and treated with 30 mg/kg of ATO or with normal chow diet and treated with the same dose of ATO for 8 weeks. Subsequently, the rats were sacrificed and the serum lipids, aminotransferase, hepatic lipids, and liver pathology were characterized. The relative levels of fatty acid synthesis and ß-oxidation gene expression in hepatic tissues were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Hepatic expression of hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined by Western blot assay. RESULTS: While continual feeding with HFD deteriorated NAFLD and hyperlipidemia, treatment with dietary control, ATO or ATO with dietary control effectively improved serum and liver lipid metabolism and liver function. In comparison with ATO treatment, dietary control or combined with ATO treatment significantly reduced the liver weight and attenuated the HFD-induced hyperlipidemia and liver steatosis in rats. Compared to ATO treatment or dietary control, combination of ATO and dietary control significantly reduced the levels of serum total cholesterol and low density lipoprotein cholesterol (LDL-C). However, the combination therapy did not significantly improve triglyceride and free fatty acid metabolism, hepatic steatosis, and liver function, as compared with dietary control alone. CONCLUSIONS: ATO treatment effectively improved NAFLD-related hyperlipidemia and inhibited liver steatosis, accompanied by modulating the expression of genes for regulating lipid metabolism. ATO enhanced the effect of dietary control on reducing the levels of serum total cholesterol and LDL-C, but not triglyceride, free fatty acid and hepatic steatosis in HFD-induced fatty liver and hyperlipidemia in rats.

Study of the factors affecting the extraction of soybean protein by reverse micelles.

In this work, the forward and back extraction of soybean protein by reverse micelles was studied. The reverse micellar systems were formed by anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT), isooctane and KCl solution. The effects of AOT concentration, aqueous pH, KCl concentration and phase volume ratio on the extraction efficiency of soybean protein were tested. Suitability of reverse micelles of AOT and Triton-X-100/AOT mixture in organic solvent toluene for soybean protein extraction was also investigated. The experimental results lead to complete forward extraction at the AOT concentration 120 mmol l(-1), aqueous pH 5.5 and KCl concentration 0.8 mol l(-1). The backward extraction with aqueous phase (pH 5.5) resulted in 100% extraction of soybean protein from the organic phase.

Development and application of a loop-mediated isothermal amplification method on rapid detection Escherichia coli O157 strains from food samples.

We developed and evaluated the specificity and sensitivity of a loop-mediated isothermal amplification (LAMP) method for rapid detection of the food-borne Escherichia coli O157 strains. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on three targets, which were rfbE, stx1 and stx2. The detection limits were found to be 100, 100 and 10 fg DNA/tube for rfbE, stx1 and stx2, respectively. Application of LAMP assays were performed on 417 food-borne E. coli strains, the sensitivity of LAMP assays for the rfbE, stx1 and stx2 was 100, 95.3 and 96.3%, and the negative predictive value was 100, 96.7 and 97.1%, respectively; with a 100% specificity and positive predictive value for all three targets.

Effects of quorum sensing on the biofilm formation and viable but non-culturable state.

Quorum sensing exists widely in all kinds of microorganisms and is a communication channel for microorganisms. Many bacterial processes, including virulence factor expression, biofilm formation, and viable but non-culturable (VBNC) cell resuscitation, are mediated by quorum sensing, and biofilm formation complicates the treatment of various infections. Foodborne pathogens can enter VBNC state in extreme environments, and pathogens in VBNC state can evade traditional detection and resuscitate under appropriate conditions, causing potential harm to human health. The disruption of quorum sensing may decisively help control biofilm formation and VBNC cell resuscitation. This review describes the quorum sensing systems of various bacteria and major fungi, and summarizes the role of bacterial quorum sensing system in biofilm formation and VBNC resuscitation. In addition, the relationship between quorum sensing inhibitors (QSI) with biofilms and VBNC is also discussed.

Organic Modifications of SBA-15 Improves the Enzymatic Properties of its Supported TLL.

In this study, lipase from Thermomyces lanuginosus (TLL) was immobilized onto the parent and organic groups modified SBA-15, and the enzymatic properties of the obtained immobilized TLL samples were investigated. 1) Activity of SBA-15-TLL at 2862.78 ± 293.24 U/g was obtained. 2) Most of the organic groups modification favored a great improvement in activity, and higher activity over 12000 U/g was observed for N-phenylaminomethyl and phenyl group modification. 3) Most of the supported TLL showed better thermostability in air while poor in phosphate buffer, with over 80% vers less than 20% of their initial activity retained after 4 h incubation at 70℃. 4) The n-dodecyl, phenyl and N-phenylaminomethyl group functionalization decreased the sensitivity of immobilized TLL in extreme pH values. 5) The n-octyl and 2-(propoxymethyl)oxirane group modification confered the supported TLL good reusability, and over 60% of their initial activity was retained after five successive cycles of reuse.