FMAlign2: a novel fast multiple nucleotide sequence alignment method for ultralong datasets.

MOTIVATION: In bioinformatics, multiple sequence alignment (MSA) is a crucial task. However, conventional methods often struggle with aligning ultralong sequences. To address this issue, researchers have designed MSA methods rooted in a vertical division strategy, which segments sequence data for parallel alignment. A prime example of this approach is FMAlign, which utilizes the FM-index to extract common seeds and segment the sequences accordingly. RESULTS: FMAlign2 leverages the suffix array to identify maximal exact matches, redefining the approach of FMAlign from searching for global chains to partial chains. By using a vertical division strategy, large-scale problem is deconstructed into manageable tasks, enabling parallel execution of subMSA. Furthermore, sequence-profile alignment and refinement are incorporated to concatenate subsets, yielding the final result seamlessly. Compared to FMAlign, FMAlign2 markedly augments the segmentation of sequences and significantly reduces the time while maintaining accuracy, especially on ultralong datasets. Importantly, FMAlign2 enhances existing MSA methods by conferring the capability to handle sequences reaching billions in length within an acceptable time frame. AVAILABILITY AND IMPLEMENTATION: Source code and datasets are available at https://github.com/malabz/FMAlign2 and https://zenodo.org/records/10435770.

Serological investigation and isolation of Salmonella abortus equi in horses in Xinjiang.

BACKGROUND: Salmonella enterica subspecies enterica serovar abortus equi (S. abortus equi) is one of the main pathogens that causes abortion in pregnant horses and donkeys, which was highly infectious and greatly restricts the healthy development of the horse industry. OBJECTIVES: In order to investigate the prevalence and biological characteristics of S. abortus equi in different regions and breeds of horses in Xinjiang. METHODS: This study conducted ELISA detection of S. abortus equi antibodies on serum samples of 971 horses collected from three large-scale horse farms and five free-range horse farms in Yili Prefecture and Bayingol Mongolian Autonomous Prefecture of Xinjiang from 2020 to 2023. On this basis, bacterial isolation, culture, identification, and drug sensitivity tests were conducted on 42 samples of aborted foal tissues and 23 mare vaginal swabs. RESULTS: The results showed that the positive rate of S. abortus equi antibody was as high as 20.91% in 971 horse serum samples. Among them, the positive rate in the Ili region (29.09%) was significantly higher than that in the Bayingole region (11.24%), and the positive rate in mares (22.45%) was higher than that in stallions (14.05%). In terms of horse breeds, the positive rates of self-propagating thoroughbred horses, half-bred horses, Ili horses and Yanqi horses were 43.22%, 28.81%, 14.72% and 11.24% respectively. In addition, S. abortus equi was more susceptible to juvenile and elderly horses, with positive rates of 70.00%and 41.86%, respectively, both of which were significantly higher than young (10.97%) and adult (19.79%) horses. Further, 9 strains of S. abortus equi were obtained through bacterial isolation, culture and identification, which were resistant to five antibiotics (Clarithromycin, Clindamycin, penicillin, Sulfamethoxazole and Rifampicin), and sensitive to 13 antimicrobial agents (Amoxicillin, Ciprofloxacin and Gentamicin, et al.). CONCLUSION: There was a high infection rate of S. abortus equi in Ili Prefecture and self-propagating thoroughbred horses, and juvenile or old mares were more susceptible, which will provide scientific basis for the prevention of S. abortus equi infection in different regions and breeds of horses in Xinjiang.

Individualized Delivery of Vancomycin by Model-Informed Bayesian Dosing Approach to Maintain an AUC24 Target in Critically Ill Patients.

INTRODUCTION: Monitoring of AUC24 was updated recommendation in the guideline for the therapeutic drug monitoring (TDM) of vancomycin in Chinese pharmacological society published in 2020. Vancomycin pharmacokinetic profiles are diverse and unique in critically ill patients because of the drastic variability of the patients' physiological parameters, while the study for population pharmacokinetic (PPK) models in Chinese critically ill patients has been rarely reported. The objectives of this study were to construct a PPK model to describe the pharmacokinetic characteristics of vancomycin in critically ill patients and to individualize vancomycin dosing by model-informed Bayesian estimation for maintenance of AUC24 target at 400-650 mg h/L recommended by the 2020 guideline. METHODS: Vancomycin with different dosing was administered intravenously over 1 h for critically ill patients, TDM was started at 48 h or 72 h since initiation of vancomycin therapy for patients. Blood samples were collected from patients for trough concentrations or Cmax. Vancomycin concentrations were determined by high-performance liquid chromatography method with ultraviolet detection. PPK model was performed using the nonlinear mixed-effect model (NONMEM®). Individual PK parameters for critically ill patients treated with vancomycin were estimated using a post hoc empirical Bayesian method based on the final PPK model. AUC24 was calculated as the total daily dose divided by the clearance (L/h). RESULTS: The PPK of vancomycin was determined by a one-compartment model with creatinine clearance as fixed effects. The PK estimates in the final model generally agreed with the median estimates and were contained within the 95% CI generated from the bootstrap results, indicating good precision and stability in the final model. The visual predictive check plots showed the adequate predictive performance of the final PK model and supported a good model fit. The model-informed Bayesian estimation was used to predict the AUC24 of critically ill patient by the acquired TDM results, and the dosing adjustment by maintenance of AUC24 at 400-650 mg h/L had made a great therapeutic effect for the case. CONCLUSION: This study established a PPK model of vancomycin in Chinese critically ill patients, and individualized dosing of vancomycin by model-informed Bayesian estimation to maintain an AUC24 target at 400-650 mg h/L has been successfully applied in clinic. This result supports the continued use of model-informed Bayesian estimation to vancomycin treatment in critically ill patients.

Screening of polysaccharides from the differently processed products of Angelica sinensis with the best liver protection effect on chicken and the intervention mechanism study based on tandem mass tag proteomics and multiple reaction monitoring approach.

The incidence of colibacillosis in poultry is on the rise, significantly affecting the chicken industry. Ceftiofur sodium (CS) is frequently employed to treat this disease, resulting in lipopolysaccharide (LPS) buildup. Processing plays a vital role in traditional Chinese veterinary medicine. The potential intervention in liver injury by polysaccharides from the differently processed products of Angelica sinensis (PDPPAS) induced by combined CS and LPS remains unclear. This study aims to investigate the protective effect of PDPPAS on chicken liver injury caused by CS combined with LPS buildup and further identify the polysaccharides with the highest hepatoprotective activity in chickens. Furthermore, the study elucidates polysaccharides' intervention mechanism using tandem mass tag (TMT) proteomics and multiple reaction monitoring (MRM) methods. A total of 190 1-day-old layer chickens were randomly assigned into 12 groups, of which 14 chickens were in the control group and 16 in other groups, for a 10-day trial. The screening results showed that charred A. sinensis polysaccharide (CASP) had the most effective and the best hepatoprotective effect at 48 h. TMT proteomics and MRM validation results demonstrated that the intervention mechanism of the CASP high-dose (CASPH) intervention group was closely related to the protein expressions of FCER2, TBXAS1, CD34, AGXT, GCAT, COX7A2L, and CYP2AC1. Conclusively, the intervention mechanism of CASPH had multitarget, multicenter regulatory features.

Determination of cyclic adenosine phosphate and protein content in dormant chlamydospore and nondormant chlamydospore of Arthrobotrys flagrans.

Arthrobotrys flagrans, a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and nondormant chlamydospore of A. flagrans to investigate the differences in cyclic adenosine monophosphate (cAMP) and protein content between the two types of spores. cAMP and soluble proteins were extracted from the nondormant chlamydospore and dormant chlamydospore of two isolates of A. flagrans. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in nondormant spores (p < 0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of nondormant spores (p < 0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of nondormant spores, but the difference was not significant (p > 0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus A. flagrans.

Development and efficacy evaluation of a novel water-in-oil-in-water adjuvant for an inactivated foot-and-mouth disease vaccine.

To develop a novel water-in-oil-in-water (W/O/W) adjuvant and evaluate the effect on foot-and-mouth disease (FMD) inactivated vaccine, in this study, we prepared the novel nano-emulsion adjuvant based on QS-21 (BEA) which is composed of the mixture of mineral oil Marcol52, surfactant Tween80, oleate polyoxyethylene ether ester, polyoxyethylene palmitic acid ester and span80, cosurfactant polyethylene glycol and QS-21. The two-step emulsification method formed the W/O/W nano-emulsion with two films and three-phase structures. The effective particle diameter of the BEA was about 184 nm, and it has good thermal stability. Then, BEA was emulsified as an adjuvant to prepare for the inactivated FMDV vaccine, and BALB/c mice and pigs were immunized to evaluate its safety and immunization effect. The results showed that the inactivated BEA-FMDV vaccine significantly increased BALB/c mice and pigs' antibodies and cytokine IFN-γ in serum. Meanwhile, the pig-neutralizing antibodies were higher than control group. Safety tests found no symptoms of FMD or significant toxic reactions. After 28 days of immunization, the protection rate can reach 93.3%. The BEA vaccine had good stability at 4 °C, no stratification after 180 days, and the content of 146S in the vaccine did not decrease. In conclusion, the BEA prepared in this study is suitable for FMDV inactivated vaccine and is an effective adjuvant.

HAlign 3: Fast Multiple Alignment of Ultra-Large Numbers of Similar DNA/RNA Sequences.

HAlign is a cross-platform program that performs multiple sequence alignments based on the center star strategy. Here we present two major updates of HAlign 3, which helped improve the time efficiency and the alignment quality, and made HAlign 3 a specialized program to process ultra-large numbers of similar DNA/RNA sequences, such as closely related viral or prokaryotic genomes. HAlign 3 can be easily installed via the Anaconda and Java release package on macOS, Linux, Windows subsystem for Linux, and Windows systems, and the source code is available on GitHub (https://github.com/malabz/HAlign-3).

WMSA: a novel method for multiple sequence alignment of DNA sequences.

MOTIVATION: Multiple sequence alignment (MSA) is a fundamental problem in bioinformatics. The quality of alignment will affect downstream analysis. MAFFT has adopted the Fast Fourier Transform method for searching the homologous segments and using them as anchors to divide the sequences, then making alignment only on segments, which can save time and memory without overly reducing the sequence alignment quality. MAFFT becomes slow when the dataset is large. RESULTS: We made a software, WMSA, which uses the divide-and-conquer method to split the sequences into clusters, aligns those clusters into profiles with the center star strategy and then makes a progressive profile-profile alignment. The alignment is conducted by the compiled algorithms of MAFFT, K-Band with multithread parallelism. Our method can balance time, space and quality and performs better than MAFFT in test experiments on highly conserved datasets. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at https://github.com/malabz/WMSA/, which is implemented in C/C++ and supported on Linux, and datasets are available at https://github.com/malabz/WMSA-dataset. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Yujin powder improves large intestine dampness-heat syndrome by regulating gut microbiota and serum metabolism.

Large intestine dampness-heat syndrome (LIDHS) is a common syndrome type in animal diarrheal diseases. Yujin powder (YJP) is one of the classic prescriptions for treating damp-heat diarrhea. The aim of this study was to investigate the regulatory effects of YJP on gut microbiota and serum metabolism in LIDHS rats using 16S rRNA sequencing and nontargeted metabolomics. The LIDHS rat model was induced through a high-sugar and high-fat diet, exposure to a high-temperature and high-humidity environment, and infection with Escherichia coli. The results demonstrated that the administration of YJP resulted in a decrease in the abundance of Desulfovibrio, Parabacteroides, Bacteroides, Allobaculum, Escherichia, Butyricimonas, Parasutterella, and Blautia and an increase in Ruminococcus, Akkermansia, Roseburia, and Lachnoclostridium. A total of 25 potential biomarkers were identified in three groups of rats. These metabolites were primarily involved in glycerophospholipid metabolism, taurine and hypotaurine metabolism, glycerol ester metabolism, arachidonic acid metabolism, primary bile acid synthesis, and tryptophan metabolism. Our study demonstrated that YJP has the potential to alleviate LIDHS by modulating gut microbial and serum metabolic homeostasis. These results establish a foundation and offer valuable guidance for the utilization of YJP in the treatment of LIDHS.

Optimization of Ethanol Extraction Technology for Yujin Powder Using Response Surface Methodology with a Box-Behnken Design Based on Analytic Hierarchy Process-Criteria Importance through Intercriteria Correlation Weight Analysis and Its Safety Evaluation.

Here, we aimed to optimize the ethanol extraction technology for Yujin powder (YJP) and evaluate its safety. The ultrasonic-assisted ethanol reflux extraction method refluxing was used to extract YJP. The parameters were optimized through a combination of single-factor and response surface methodology (RSM). The comprehensive Y value score calculated using the content of 13 active ingredients in YJP ethanolic extracts (YEEs) and the yield of the dry extract were used as measuring criteria. RSM with a Box-Behnken design using three factors and three levels was adopted to optimize the ethanol extraction technology for YJP. Finally, acute and subchronic toxicity tests were performed to evaluate its safety. The results revealed the best technological parameters: a liquid-material ratio of 24:1, an ethanol concentration of 69%, assistance of ultrasound (40 °C, 50 kHZ, 30 min), reflux time of 53 min, and reflux temperature of 50 °C. In acute toxicity tests, the maximum administration dosage in mice was 28.21 g/kg, which is higher than 10 times the clinical dosage. Adverse effects in the acute and subchronic toxicity tests were not observed. All clinical indexes were normal. In conclusion, the RSM based on AHP-CRITIC weight analysis could be used to optimize the ethanol extraction technology for YJP and YEEs prepared under the above conditions and ensure high safety.

Formulation enhanced the stability of Foot-and-mouth virus and prolonged vaccine storage.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that affects cloven-hoofed animals. Vaccination is the most effective measure to control FMD. However, FMDV particles are prone to dissociation, leading to insufficient potency of vaccine. Based on this characteristic, a combination of twenty percentage trehalose, 500 mM NaCl and 3 mM CuSO4·5H2O was developed to increase viral stability. Heating-resistance test showed that FMDV infectivity was maintained when formulated with formulation. Additionally, the half-life of FMDV inactivation was prolonged remarkably. Sequencing analysis demonstrated that viral genome could not be altered in serial passages. Vaccine stability was monitored for up to 1 year at 4 °C, with a higher level of 146S content remained. This study suggested that the formulation could protect FMDV against massive structural breakdown and extend the shelf life of vaccine. Our findings could provide strategy to develop more solutions for the stabilization of viral vaccine.

A new method providing complementary explanation of the blood-enriching function and mechanism of unprocessed Angelica sinensis and its four kinds of processed products based on tissue-integrated metabolomics and confirmatory analysis.

Angelica sinensis (AS) is a common Traditional Chinese Medicine used for tonifying blood in China. Unprocessed AS and its four kinds of processed products (ASs) are used to treat blood deficiency syndrome in the country. The different blood-tonifying mechanisms of ASs remain unclear. In this work, a novel method integrating metabolomics and hematological and biochemical parameters was established to provide a complementary explanation of blood supplementation mechanism of ASs. Our results revealed that different ASs exhibited various blood supplementation effect, and that AS parched with alcohol demonstrated the best blood supplementation effect. Eight metabolites from liver tissue and 12 metabolites from spleen tissue were considered to be potential biomarkers. These biomarkers were involved in four metabolic pathways. Correlation analysis results showed that l-aspartic acid and l-alanine (spleen tissue), linoleic acid, and l-cystathionine (liver tissue) exhibited a high positive or negative correlation with the aforesaid biochemical indicators. The blood-supplementation effect mechanism of ASs were related to four metabolic pathways. l-Aspartic acid and l-alanine (spleen tissue), linoleic acid, and l-cystathionine (liver tissue) were the four key metabolites associated with the blood supplementation effect of ASs. This study gives a complementary explanation of the blood supplementation effect and mechanism of action of ASs.

Enhanced stability of foot-and-mouth disease vaccine antigens with a novel formulation.

The structural instability of inactivated Foot-and-mouth disease virus antigen hinders the development of vaccine industry. The use of an inexpensive, biocompatible formulation to slow down the degradation of antigen would address the problem. Here, phosphate-buffered saline (PBS) was showed to be effective in stabilizing 146S and hence determined as basic solution buffer. Excipients such as trehalose, sucrose, arginine, cysteine, calcium chloride, BSA and ascorbic acid were found to protect 146S from massive structural breakdown. Using orthogonal test, we confirmed the novel formulation as a combination of 5% (w/v) trehalose, 5% (w/v) sucrose, 0.05 M arginine, 0.01 M cysteine, 0.01 M calcium chloride, 1% (W/V) BSA and 0.001 M ascorbic acid in PBS. The formulation increased vaccine stabilization, with retention rate of 14% after storage at 4 °C for 14 months. Particle size for vaccine was at approximately 220 nm and physicochemical detecting findings were rarely abnormal in morphology and emulsion type. In summary, these results revealed that the novel formulation is beneficial to make the FMD vaccine more stable and effective, reducing the dependence on cold storage and delivery.

[Integrated strategy for mechanism of Baitouweng Decoction in treating dampness-heat diarrhea based on urine metabolomics coupled with network pharmacology].

Baitouweng Decoction is a famous Chinese medicinal decoction that has been used to treat diarrhea over thousands of years. In this study, we investigated the effect and mechanism of Baitouweng Decoction in the treatment of diarrhea. Wistar rats were randomly assigned into 4 groups: control group, dampness-heat diarrhea model group(modeling by complex factors including high-sugar and high-fat diet, improper diet, hot and humid environment, drinking and intraperitoneal injection of Escherichia coli), Baitouweng Decoction(3.6 g·kg~(-1)) group, and self-healing group. A urine metabolomics approach was developed with ultra liquid chromatography-quadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS) for metabolic profiling. The differential metabolites were screened out by the multivariate comparison between groups. Diarrhea-related protein targets and the active compounds of Baitouweng Decoction were used to predict the protein targets of Baitouweng Decoction. Cytoscape 3.2.1 was employed to establish a active component-target protein interaction network. Three protein-protein interaction(PPI) networks of component target proteins, diarrhea-related proteins, and differential metabolite-related proteins were established and then merged by BisoGenet. ClueGO was used to perform the gene enrichment based on the genetic similarity. The results showed that Baitouweng Decoction effectively treated dampness-heat diarrhea in vivo. N-acetylserotonin, L-gamma-glutamylcysteine, glutathione, retinoate, melatonin, indole-3-acetaldehyde, L-cystine, biotin, and L-tryptophan were screened as differential metabolites in dampness-heat diarrhea model group. Tryptophan metabolism, glutathione metabolism, biotin metabolism, retinol metabolism, and cysteine and methionine metabolism were involved in the therapeutic effect of Baitouweng Decoction in vivo. A total of 167 targets were identified as major candidates for diarrhea progression. The gene-set enrichment revealed that the targets were involved in reactive oxygen species production, inflammation, and apoptosis. Baitouweng Decoction can restrain inflammation, production of reactive oxygen, and block apoptosis, thereby contributing to the treatment of dampness-heat diarrhea.

Rapid and visual detection of porcine deltacoronavirus by recombinase polymerase amplification combined with a lateral flow dipstick.

BACKGROUND: Porcine Deltacoronavirus (PDCoV) is a newly emerging Coronavirus that was first identified in 2012 in Hong Kong, China. Since then, PDCoV has subsequently been reported worldwide, causing a high number of neonatal piglet deaths and significant economic losses to the swine industry. Therefore, it is necessary to establish a highly sensitive and specific method for the rapid diagnosis of PDCoV. RESULTS: In the present study, a highly sensitive and specific diagnostic method using recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) was developed for rapid and visual detection of PDCoV. The system can be performed under a broad range of temperature conditions from 10 to 37 °C, and the detection of PDCoV can be completed in 10 min at 37 °C. The sensitivity of this assay was 10 times higher than that of conventional PCR with a lower detection limit of 1 × 102 copies/µl of PDCoV. Meanwhile, the LFD-RPA assay specifically amplified PDCoV, while there was no cross-amplification with other swine-associated viruses, including Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine kobuvirus (PKoV), Foot and mouth disease virus (FMDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV2), Classical swine fever virus (CSFV) and Seneca valley virus (SVV). The repeatability of the test results indicated that this assay had good repeatability. In addition, 68 clinical samples (48 fecal swab specimens and 20 intestinal specimens) were further tested by LFD-RPA and RT-PCR assay. The positive rate of LFD-RPA clinical samples was 26.47% higher than that of conventional PCR (23.53%). CONCLUSIONS: The LFD-RPA assay successfully detected PDCoV in less than 20 min in this study, providing a potentially valuable tool to improve molecular detection for PDCoV and to monitor the outbreak of PDCoV, especially in low-resource areas and laboratories.

Screening study of blood-supplementing active components in water decoction of Angelica sinensis processed with yellow rice wine based on response surface methodology.

CONTEXT: Angelica sinensis (Oliv.) Diels (Apiaceae) (syn. Angelica polymorpha Maxim var. sinensis Oliver) processed with yellow rice wine (WAS) has a blood-supplementing effect. OBJECTIVE: To establish an optimal technology for preparing water decoction of WAS (WASD), and screen blood-supplementing fractions. MATERIALS AND METHODS: Ferulic acid and crude polysaccharide were used in optimizing the preparation technology for WASD through response surface methodology. The independent variables were liquid-solid ratio, soaking time, and extraction time. Eighty Kunming mice were randomly divided into normal control, model, and six intervention groups (n = 10). The intervention groups were given different WASD fractions by gavage (5 or 10 g/kg). The model intervention groups received acetylphenyl hydrazine (subcutaneous injection) and cyclophosphamide (intraperitoneal injection). Duration of study, 9 days. The components of blood-supplementing fractions were analyzed. RESULTS: The optimum extraction parameters were liquid-solid ratio, 7.69:1 mL/g; soaking time, 119.78 min; and extraction time, 143.35 min. The optimal OD value was 0.8437. RBC, WBC, and Hb in the water fraction (5, 10 g/kg) and n-butanol fraction (10 g/kg) intervention groups increased significantly compared with the model group (p < 0.05). Polysaccharide and caffeic acid contents of water fraction were 252.565 and 0.346 µg/mg, respectively; ferulic acid was not detected. Caffeic acid and ferulic acid contents of n-butanol fraction were 1.187 and 0.806 µg/mg, respectively, polysaccharide was not detected. CONCLUSIONS: The optimum preparation technology of WASD was obtained, and the water, n-butanol fractions were blood-supplementing fractions. This study provides a theoretical foundation for further application of WAS in the pharmaceutical industry.

Urinary metabolomics analysis reveals the effect of volatile oil from Angelica sinensis on LPS-induced inflammation rats.

Lipopolysaccharide (LPS)-induced inflammation occurs commonly and volatile oil from Angelica sinensis (VOAS) can be used as an anti-inflammatory agent. The molecular mechanisms that allow the anti-inflammatory factors to be expressed are still unknown. In this paper, we applied gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-time-of-flight mass spectrometry (LC-Q/TOF-MS) based on a metabolomics platform coupled with a network approach to analyze urine samples in three groups of rats: one with LPS-induced inflammation (MI); one with intervention with VOAS; and normal controls (NC). Our study found definite metabolic footprints of inflammation and showed that all three groups of rats, MI, intervention with VOAS and NC have distinct metabolic profiles in urine. The concentrations of 48 metabolites differed significantly among the three groups. The metabolites in urine were screened by the GC-MS and LC-Q/TOF-MS methods. The significantly changed metabolites (p < 0.05, variable importance in projection > 1.5) between MI, NC and VOAS were included in the metabolic networks. Finally, hub metabolites were screened, including glycine, arachidonic acid, l-glutamate, pyruvate and succinate, which have high values of degree (k). the Results suggest that disorders of glycine, arachidonic acid, l-glutamate, pyruvate and succinate metabolism might play an important part in the predisposition and development of LPS-induced inflammation. By applying metabolomics with network methods, the mechanisms of diseases are clearly elucidated.

Metabolomics study on promoting blood circulation and ameliorating blood stasis: Investigating the mechanism of Angelica sinensis and its processed products.

Angelica sinensis (Danggui, DG) parched with alcohol (Jiu Danggui, JDG) and charred DG are the main processed products of DG, which are used to treat blood stasis syndrome (BSS). However, their therapeutic effect and mechanisms are still unclear. Based on an acute rat BSS model, the intervention effects of DG and its processed products (DGPPs) were evaluated by the hemorheology and coagulation function parameters. Meanwhile, plasma and urine metabolites were detected and analyzed by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and multivariate statistical analysis method. The results of hemorheology, coagulation function parameters and metabolomics all showed that the BSS model was successfully established, DGPPs intervention could significantly relieve rats BSS and the therapeutic effect of JDG was best. Moreover, 23 differential metabolites (14 in plasma and nine in urine) were identified that were closely related to the BSS, involving seven potential target metabolic pathways. DGPP intervention showed different degrees of reverse effect on these metabolites. JDG was the most effective owing to extensive regulation effect on differential metabolites. This study provides a reference for understanding the pathological mechanism of BSS and the mechanism of DGPPs, which lays a theoretical foundation for the rational use of DGPPs in clinical practice.

Metabolomics analysis of Pulsatilla decoction on treatment of wetness-heat-induced diarrhea in rats based on UPLC-Q/TOF-MS/MS.

Pulsatilla decoction (PD) is a classical prescription in traditional Chinese medicine that has therapeutic effects on wetness-heat-induced diarrhea (WHD). To investigate the therapeutic effects of PD in the treatment of WHD and elucidate the potential mechanism, we used a metabolomics strategy on the base of ultraperformance liquid chromatography coupled with quadrupole time-of-flight/mass spectrometry (UPLC-Q/TOF-MS/MS) and analyzed the serum samples of 32 rats to identify differential metabolites and pathways associated with the PD treatment of WHD. With variable importance for projection >1.0 in the Orthogonal partial least-squares discriminant analysis (OPLS-DA ) models and FC ≥1.2 or ≤0.8, 67 differential metabolites in the model and control groups and 33 differential metabolites in the model and PD groups were screened. A total of 23 differential metabolites were selected based on Venny analysis. Functional analysis showed that the differential metabolites identified were primarily involved in pentose and glucuronate interconversions, glycerophospholipid metabolism, tryptophan metabolism, starch and sucrose metabolism, and glycerolipid metabolism. This study suggested that PD exerts inhibitory effects on WHD. In particular, the significant roles of PD for treating WHD lie in regulating perturbed energy metabolism, glycerophospholipid metabolism and glycerolipid metabolism, and promoting lysoPC production restoring the function of intestinal tract.

Fructose-1,6-bisphosphate aldolase of Mycoplasma bovis is a plasminogen-binding adhesin.

Mycoplasma bovis is an extremely small cell wall-deficient pathogenic bacterium in the genus Mycoplasma that causes serious economic losses to the cattle industry worldwide. Fructose-1,6-bisphosphate aldolase (FBA), a key enzyme in the glycolytic pathway, is a multifunctional protein in several pathogenic bacterial species, but its role in M. bovis remains unknown. Herein, the FBA gene of the M. bovis was amplified by PCR, and subcloned into the prokaryotic expression vector pET28a (+) to generate the pET28a-FBA plasmid for recombinant expression in Escherichia coli Transetta. Expression of the 34 kDa recombinant rMbFBA protein was confirmed by electrophoresis, and enzymatic activity assays based on conversion of NADH to NAD+ revealed Km and Vmax values of 48 µM and 43.8 µmoL/L/min, respectively. Rabbit anti-rMbFBA and anti-M. bovis serum were generated by inoculation with rMbFBA and M. bovis, and antigenicity and immunofluorescence assay demonstrated that FBA is an immunogenic protein expressed on the cell membrane in M. bovis cells. Enzyme-linked immunosorbent assays revealed equal distribution of FBA in the cell membrane and cytoplasm. Complement-dependent mycoplasmacidal assays showed that rabbit anti-rMbFBA serum killed 44.1% of M. bovis cells in the presence of complement. Binding and ELISA assays demonstrated that rMbFBA binds native bovine plasminogen and in a dose-dependent manner. Fluorescent microscopy revealed that pre-treatment with antibodies against rMbFBA decreased the adhesion of M. bovis to embryonic bovine lung (EBL) cells. Furthermore, adherence inhibition assays revealed 34.4% inhibition of M. bovis infection of EBL cells following treatment with rabbit anti-rMbFBA serum, suggesting rMbFBA participates in bacterial adhesion to EBL cells.