Proteome overview of exosome derived from plasma of cows infected with Mycobacterium bovis.

Bovine tuberculosis (bTB), primarily caused by Mycobacterium bovis (M. bovis), is a globally zoonotic disease with significant economic impacts. Plasma exosomes have been extensively used for investigating disease processes and exploring biomarkers. While mass spectrometry (MS)-based proteomic analysis of plasma exosomes has been employed for human tuberculosis (TB) studies, it has not yet been applied to bTB. Therefore, a comprehensive proteomic overview of plasma exosomes from M. bovis-infected cows is essential. In this study, we presented an extensive proteomic analysis of plasma exosomes from 89 M. bovis-infected cows across three farms, using data dependent acquisition (DDA) mode. Our analysis encompasses 239,894 spectra, 6,011 peptides and 835 proteins. The proteomic overview revealed both consistencies and differences among individual cows, supplements 595 proteins to the bovine exosome library, and enriches tuberculosis and related pathways. Additionally, six pathways were validated as immune response pathways, and three proteins (CATHL1, H1-1, and LCN2) were identified as potential indicators of bTB. This study is the first to investigate the exosome proteome of plasma from cows infected with M. bovis, providing a valuable dataset for exploring candidate bTB markers and understanding the mechanisms of host defense against M. bovis.

Determination of lekethromycin in plasma and tissues of pneumonia-infected rats by ultra-high performance liquid chromatography-tandem mass spectrometry.

Lekethromycin (LKMS), a novel semi-synthetic macrolide lactone, had the characteristics of high plasma protein binding rate, fast absorption, slow elimination, and wide distribution in rat pharmacokinetics studies. A reliable analytical ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based method was established by using tulathromycin and TLM (CP-60, 300) as internal standards for detection of LKMS and LKMS-HA, respectively. Samples preparation and UPLC-MS/MS conditions were optimized for complete and accurate quantification. Tissue samples were extracted with 1% formic acid in acetonitrile and purified by PCX cartridges. According to FDA and EMA guidelines for bioanalytical method, several rat characteristic tissues were selected for method validation, such as muscle, lung, spleen, liver, kidney, and intestines. The transitions m/z 402.900 > 158.300, m/z 577.372 > 158.309, m/z 404.200 > 158.200, and m/z 577.372 > 116.253 were monitored and quantified for LKMS, LKMS-HA, tulathromycin and TLM, respectively. According to the ratio with IS peak aera, the accuracy and precision of LKMS were 84.31%-112.50% with RSD 0.93%-9.79% and LKMS-HA were 84.62%-103.96% with RSD 0.73%-10.69%, and the method had been established and complied with FDA, EU, and Japanese guidelines. Finally, this method was applied to detect LKMS and LKMS-HA in plasma and tissues of pneumonia-infected rats that were intramuscularly administered and treated with LKMS intramuscular injection of 5 mg/kg BW and 10 mg/kg BW, and the characteristics of pharmacokinetics and tissue distribution were compared with normal rats.

Bioequivalence of two tablet formulations of cefpodoxime proxetil in beagle dogs.

The pharmacokinetic profiles and bioequivalence of two cefpodoxime proxetil tablets were investigated in Beagle dogs. A single-dose, four-way complete replication and crossover design was used in the present study. A total of 28 healthy Beagle dogs (half male and female) with an average body weight of 11.1 kg were randomly allocated to this study. A whole reference or test tablet containing the equivalent of 100 mg of cefpodoxime was administered orally to each dog. Serial plasma samples were collected, and cefpodoxime concentrations were determined by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Then a non-compartmental method was used to calculate the pharmacokinetic parameters of both tablet formulations. The average bioequivalence (ABE) or reference-scaled average bioequivalence (RSABE) methods were used to determine the 90% confidence interval (CI) of AUCINF_obs and Cmax. No significant differences were observed for both parameters between both tablets. The test formulation was bioequivalent to the reference one because the 90% CI ranges of Cmax and AUCINF_obs were all between 80 and 125%.

Design and characterization of novel oxyntomodulin derivatives with potent dual GLP-1/glucagon receptor activation and prolonged antidiabetic effects.

AIMS: To investigate the combination of dimerization and PEGylation to enhance the receptor activation and in vivo stability of Oxyntomodulin (OXM). MAIN METHODS: All LDM peptides were produced by using standard method of solid phase synthesis. The in vitro effects of LDM peptides were assessed by glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GcgR) binding test and Proteolytic stability test. Subsequently, saline, Liraglutide and three doses of LDM-3 treated groups were subjected to the evaluation of aute and long-term efficacy. KEY FINDINGS: Five long-acting OXM conjugates, termed LDM-1 to LDM-5, were designed using cysteine (Cys)-specific modification reaction including the activated PEG, bisMal-NH2, and OXM-Cys, and all prepared with high purity. LDM-3 exhibited greater GLP-1R and GcgR activation and ameliorative serum stability. In addition, LDM-3 was identified with enhanced insulinotropic and glycemic abilities in the gene knockout mice. The prolonged glucose-lowering effects of the LDM-3 were proved by hypoglycemic duration test and multiple oral glucose tolerance tests (OGTTs) in the diet-induced obesity (DIO) mice. Furthermore, the pharmacokinetic tests in Sprague Dawley (SD) rat and cynomolgus monkey exhibited the lifespans of LDM-3 at 90 nmol·kg-1 were 101.5 h and 119.4 h, respectively. Nevertheless, consecutive 8-week administration of LDM-3 improved the cumulative body weight gain, food intake, % HbA1c, glucose tolerance and the pancreatic of the obese mice. SIGNIFICANCE: LDM-3, as a dual GLP-1R and GcgR agonist, holds potential to be developed as a promising therapeutic candidate for both diabetes and obesity.

Oroxylin A Inhibits Allergic Airway Inflammation in Ovalbumin (OVA)-Induced Asthma Murine Model.

Oroxylin A, a natural flavonoid isolated from the medicinal herb Scutellaria baicalensis Georgi, has been reported to have anti-inflammatory property. In this study, we aimed to investigate the protective effects and mechanism of oroxylin A on allergic inflammation in OVA-induced asthma murine model. BABL/c mice were sensitized and airway-challenged with OVA to induce asthma. Oroxylin A (15, 30, and 60 mg/kg) was administered by oral gavage 1 h before the OVA treatment on day 21 to 23. The results showed that oroxylin A attenuated OVA-induced lung histopathologic changes, airway hyperresponsiveness, and the number of inflammatory cells. Oroxylin A also inhibited the levels of IL-4, IL-5, IL-13, and OVA-specific IgE in BALF. Furthermore, oroxylin A significantly inhibited OVA-induced NF-κB activation. In conclusion, these results suggested that oroxylin A inhibited airway inflammation in OVA-induced asthma murine model by inhibiting NF-κB activation. These results suggested that oroxylin A was a potential therapeutic drug for treating allergic asthma.

Simultaneous determination of cyromazine and dicyclanil in animal edible tissues using UPLC-MS/MS.

Cyromazine and dicyclanil are both used as insect growth regulators. This paper describes an easy and innovative simultaneous extraction method for residues of cyromazine and dicyclanil in food of animal origin, and a confirmation procedure using UHPLC-MS/MS. The sample was extracted, deproteinised by 1% trichloracetic acid in water-acetonitrile, followed by selective defatting using hexane based on the degree of matrix complexity; cleaned-up on an mixed-mode cation exchange (MCX) cartridge; and quantified by using matrix-matched calibration. The mean recoveries were all between 62.0% and 99.2%, and the RSDs were all below 9.94%. The present method was rapid, sensitive and reliable, which was applied to the quantitative analysis of these residues in animal tissues.

Detection of the staphylococcal multiresistance gene cfr in Escherichia coli of domestic-animal origin.

OBJECTIVES: To investigate the presence and the genetic environment of the multiresistance gene cfr in Escherichia coli found in domestic animals. METHODS: A total of 1230 E. coli isolates, collected from pigs, chickens and ducks, were screened by PCR for the cfr gene. The location of the cfr gene was determined by Southern blotting, the transferability of cfr gene was tested by conjugation and transformation, and the regions flanking the cfr gene were sequenced by a modified random primer walking strategy. The location of the cfr promoter sequence was analysed by mapping the cfr transcription start site using rapid amplification of 5' cDNA ends (5' RACE). RESULTS: Only a single strain from the nasal swab of a pig harboured the cfr gene. Southern blotting indicated that the cfr gene was located on a ~110 kb plasmid, designated pEC-01. A cfr-carrying segment of 1545 bp with a sequence identical to that of the cfr-harbouring plasmid pSCFS1 was flanked by two IS26 elements in the same orientation. The IS26 transposition created a new hybrid promoter in which the -35 region was part of the left inverted repeat of IS26 while the -10-like sequence was part of the original cfr upstream region. CONCLUSIONS: To the best of our knowledge, this is the first report of the cfr gene in a naturally occurring E. coli strain. Continued surveillance of the presence of the cfr gene in Gram-negative bacteria of domestic-animal origin is warranted.

Simultaneous determination of 13 quinolones in eggs using column high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry and depletion of pefloxacin methanesulfonate in eggs.

An efficient method was developed for simultaneous determination of 13 quinolones--namely, enoaxacin (ENO), marbofloxacin (MAR), ciprofloxacin (CIP), norfloxacin (NOR), ofloxacin (OFL), pefloxacin methanesulfonate (PEF), danofloxacin (DAN), enrofloxacin (ENR), lomefloxacin (LOM), difloxacin (DIF), sarafloxacin (SAR), oxolinic acid (OXO), and flumequine (FLU)--in eggs by column liquid chromatography/electrospray ionization-tandem mass spectrometry. Samples were extracted with a phosphoric acid-phosphate buffer followed by purification with a solid-phase extraction cartridge. Recoveries for the 13 quinolones were 67-93% with intraday and interday coefficients of variation ranging from 4 to 9% and 2 to 18%, respectively. The limit of determination was 0.05 microg/kg for OXO and FLU; 0.1 microg/kg for MAR, OFL, CIP, LOM, DAN, SAR, DIF, NOR, and ENR; and 0.2 microg/kg for ENO and PEF. The method was also applied to study the depletion of PEF in eggs. The concentration of PEF increased and reached a maximum value on the third day, and then decreased rapidly until it could not be detected on day 32; its metabolite NOR was detectable on the second day, and then reached a maximum on the sixth day, after which it could not be detected until day 15.