Analysis of serum peptidome profiles of non-metastatic and metastatic feline mammary carcinoma using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Feline mammary carcinoma (FMC) is a common aggressive and highly metastatic cancer affecting female cats. Early detection is essential for preventing local and distant metastasis, thereby improving overall survival rates. While acquiring molecular data before surgery offers significant potential benefits, the current protein biomarkers for monitoring disease progression in non-metastatic FMC (NmFMC) and metastatic FMC (mFMC) are limited. The objective of this study was to investigate the serum peptidome profiles of NmFMC and mFMC using liquid chromatography-tandem mass spectrometry. A cross-sectional study was conducted to compare serum peptidome profiles in 13 NmFMC, 23 mFMC and 18 healthy cats. The liquid chromatography-tandem mass spectrometry analysis was performed on non-trypsinized samples. RESULTS: Out of a total of 8284 expressed proteins observed, several proteins were found to be associated with human breast cancer. In NmFMC, distinctive protein expressions encompassed double-stranded RNA-binding protein Staufen homolog 2 (STAU2), associated with cell proliferation, along with bromodomain adjacent to zinc finger domain 2A (BAZ2A) and gamma-aminobutyric acid type A receptor subunit epsilon (GABRE), identified as potential treatment targets. Paradoxically, positive prognostic markers emerged, such as complement C1q like 3 (C1QL3) and erythrocyte membrane protein band 4.1 (EPB41 or 4.1R). Within the mFMC group, overexpressed proteins associated with poor prognosis were exhibited, including B-cell lymphoma 6 transcription repressor (BCL6), thioredoxin reductase 3 (TXNRD3) and ceruloplasmin (CP). Meanwhile, the presence of POU class 5 homeobox (POU5F1 or OCT4) and laminin subunit alpha 1 (LAMA1), reported as metastatic biomarkers, was noted. CONCLUSION: The presence of both pro- and anti-proliferative proteins was observed, potentially indicating a distinctive characteristic of NmFMC. Conversely, proteins associated with poor prognosis and metastasis were noted in the mFMC group.

Detection of 11 carbamate pesticide residues in raw and pasteurized camel milk samples using liquid chromatography tandem mass spectrometry: Method development, method validation, and health risk assessment.

This study aimed to use ultra-high-performance liquid chromatography coupled to a triple-quadrupole mass spectrometer to detect 11 carbamate pesticide residues in raw and pasteurized camel milk samples collected from the United Arab Emirates. A method was developed and validated by evaluating limits of detection, limits of quantitation, linearity, extraction recovery, repeatability, intermediate precision, and matrix effect. Due to the high protein and fat content in camel milk, a sample preparation step was necessary to avoid potential interference during analysis. For this purpose, 5 different liquid-liquid extraction techniques were evaluated to determine their efficiency in extracting carbamate pesticides from camel milk. The established method demonstrated high accuracy and precision. The matrix effect for all carbamate pesticides was observed to fall within the soft range, indicating its negligible effect. Remarkably, detection limits for all carbamates were as low as 0.01 µg/kg. Additionally, the coefficients of determination were >0.998, demonstrating excellent linearity. A total of 17 camel milk samples were analyzed, and only one sample was found to be free from any carbamate residues. The remaining 16 samples contained at least one carbamate residue, yet all detected concentrations were below the recommended maximum residue limits set by Codex Alimentarius and the European Union pesticide databases. Nonetheless, it is worth noting that the detected levels of ethiofencarb in 3 samples were close to the borderline of the maximum residue limit. To assess the health risk for consumers of camel milk, the hazard index values of carbofuran, carbaryl, and propoxur were calculated. The hazard index values for these 3 carbamate pesticides were all below 1, indicating that camel milk consumers are not at risk from these residues.

Evaluation of plasma and urine pharmacokinetics of tranexamic acid for equine medication control.

This study aimed to evaluate the pharmacokinetics (PK) of tranexamic acid (TXA) in horses and estimate its irrelevant plasma and urine concentrations using the pharmacokinetic/pharmacodynamic (PK/PD) approach by applying the Pierre-Louis Toutain model. TXA was intravenously administered to eight thoroughbred mares, and plasma and urine TXA concentrations were quantified by liquid chromatography/tandem mass spectrometry. The quantified data were used to calculate the PK parameters of TXA in horses. The plasma elimination curves were best-fitted to a three-compartment model. Using the Toutain model approach, irrelevant plasma and urine TXA concentrations were estimated to be 0.0206 and 0.997 µg/mL, respectively. The typical values of clearance, steady-state volume of distribution, and steady-state urine-to-plasma ratio were 0.080 L/kg/h, 0.86 L/kg, and 49.0, respectively. The obtained irrelevant concentrations will be useful for establishing relevant regulatory screening limits for effective control of TXA use in horse racing and equestrian sports.

MEASUREMENT OF PERSISTENT ORGANIC POLLUTANTS, PERFLUORINATED COMPOUNDS, AND TOXIC METALS IN THE BLOOD OF HUMBOLDT PENGUINS (SPHENISCUS HUMBOLDTI) AT PUNTA SAN JUAN, PERU USING DRIED BLOOD SPOTS.

The Humboldt penguin (Spheniscus humboldti) population at the Punta San Juan Marine Protected Area in Peru is considered critical to the long-term sustainability of this endangered species in Peru. Exposure of the rookery to environmental toxicants is a mounting concern because of regional growth of industries and human populations. Whole blood samples were collected from 30 free-ranging penguins in 2011 as part of a broader population health monitoring program. Dried blood spots (DBS) containing 50 µl of blood were prepared and analyzed to assess exposure to five groups of environmental contaminants. Concentrations of elements arsenic, cadmium, iron, lead, mercury, selenium, and thallium were analyzed using inductively coupled plasma mass spectrometry. Persistent organic pollutant concentrations were measured using gas chromatography-tandem mass spectrometry to analyze organochlorine pesticides (OCP; p,p'-DDT, p,p'-DDE, ß-hexachlorocyclohexane, t-nonachlor, and oxychlordane), polychlorinated biphenyls (congeners 138 and 153), and polybrominated flame retardants (polybrominated biphenyl-153 and polybrominated diphenyl ether congeners 47 and 99). Per- and polyfluoroalkyl substances, including perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid were measured using liquid chromatography-tandem mass spectrometry. Results revealed low levels of exposure to these selected contaminants, at levels not considered to be of concern for wildlife health. DBS methodology was considered effective in a field-based setting for quantification of whole blood concentrations of environmental contaminants in penguins.

Cellular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein.

The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

Effector protein Hcp2a of avian pathogenic Escherichia coli interacts with the endoplasmatic reticulum associated RPL23 protein of chicken DF-1 fibroblasts.

The type VI secretion system (T6SS) is a secretion apparatus widely found in pathogenic Gram-negative bacteria and is important for competition among various bacteria and host cell pathogenesis. Hcp is a core component of functional T6SS and transports toxic effectors into target cells by assembling to form tube-like structures. Studies have shown that Hcp simultaneously acts as an effector to influence cellular physiological activities; however, the mechanism of its activity in host cells remains unclear. To investigate the target of effector protein Hcp2a in a chicken fibroblast cell line, we first detected the subcellular localization of Hcp2a in DF-1 cells by indirect immunofluorescence assay. The results showed that Hcp2a protein was localized in the endoplasmic reticulum of DF-1 cells. We also used a streptavidin-biotin affinity pull-down assay combined with LC-MS/MS to screen DF-1 cell lysates for proteins that interact with Hcp2a and analyze the cellular functional pathways affected by them. The results showed that Hcp2a interacted with 52 DF-1 cellular proteins that are involved in multiple intracellular pathways. To further explore the mechanism of Hcp2a protein targeting the endoplasmic reticulum of DF-1 cells, we screened three endoplasmic reticulum-associated proteins (RSL1D1, RPS3A, and RPL23) from 52 prey proteins of Hcp2a for protein-protein molecular docking analysis. The docking analysis showed that the effector protein Hcp2a and the RPL23 protein had good complementarity. Overall, we propose that Hcp2a has strong binding activity to the RPL23 protein in DF-1 cells and this may help Hcp2a anchor to the endoplasmic reticulum in DF-1 cells.

Multi-class/residue method for determination of veterinary drug residues, mycotoxins and pesticide in urine using LC-MS/MS technique.

BACKGROUND: Veterinary drugs are widely used in animals to prevent diseases and are a complex set of drugs with very different chemical properties. Multiclass and multi-residue methods for simultaneous detection of residues from veterinary drugs and contaminants in urine are very rare or non-existent. Therefore, the aim of this study was to develop and validate a sensitive and reliable quantitative LC-MS/MS method for simultaneous determination of a wide range of veterinary drug and pesticide residues and mycotoxins in bovine urine. This involved 42 veterinary drug residues (4 thyreostats, 6 anabolic hormones, 2 lactones, 10 beta agonists, 15 antibiotics, 5 sulphonamides), 28 pesticides and 2 mycotoxins. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. Analysis was performed in both positive and negative ionization modes with multiple reaction monitoring transitions over a period of 12 min. RESULTS: The parameters validated included linearity, limit of detection (LOD), limit of quantification (LOQ), detection capability (CCß), decision limit (CCα), stability, accuracy and precision. The process followed guidelines of the regulation 2021/808/EC. The calibration curves were linear with coefficient of correlation (R2) from 0.991 to 0.999. The LODs were from 0.01 to 2.71 µg/L, while the LOQs were from 0.05 to 7.52 µg/L. The CCα and CCß were in range 0.05-12.11 µg/L and 0.08-15.16 µg/L. In addition, the average recoveries of the spiked urine samples were from 71.0 to 117.0% and coefficient of variation (CV) < 21.38% (intraday and interday). CONCLUSION: A new isotopic LC-MS/MS method has been developed, validated and applied for identification and quantification of 72 residues of veterinary drugs and pesticides and other contaminants such as mycotoxins in bovine urine. The most appropriated sample preparation procedures involved sodium acetate buffer, enzymatic hydrolysis using ß-glucuronidase and cleanup solid phase extraction with OASIS SPE cartridges. The parameters were satisfactorily validated fulfilling requirements under Regulation 2021/808/EC. Consequently, the method could be used in routine analysis of bovine urine samples for simultaneous detection of veterinary drug and pesticide residues as well as contaminants such as mycotoxins.

Untargeted metabolomics reveals alternations in metabolism of bovine mammary epithelial cells upon IFN-γ treatment.

BACKGROUND: IFN-γ is a pleiotropic cytokine that has been shown to affect multiple cellular functions of bovine mammary epithelial cells (BMECs) including impaired milk fat synthesis and induction of malignant transformation via depletion of arginine, one of host conditionally essential amino acids. But the molecular mechanisms of these IFN-γ induced phenotypes are still unknown. METHODS: BMECs were treated with IFN-γ for 6 h, 12 h, and 24 h. The metabolomic profiling in BMECs upon IFN-γ induction were assessed using untargeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) metabolomic analysis. Key differentially expressed metabolites (DEMs) were quantified by targeted metabolomics. RESULTS: IFN-γ induction resulted in significant differences in the contents of metabolites. Untargeted analysis identified 221 significantly DEMs, most of which are lipids and lipid-like molecules, organic acids and derivatives, organ heterocyclic compounds and benzenoids. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEMs were enriched in fructose and mannose metabolism, phosphotransferase system (PTS), ß-alanine metabolism, arginine and proline metabolism, methane metabolism, phenylalanine metabolism, and glycolysis/gluconeogenesis. Quantification of selected key DEMs by targeted metabolomics showed significantly decreased levels of D-(-)-mannitol, argininosuccinate, and phenylacetylglycine (PAG), while increased levels in S-hydroxymethylglutathione (S-HMG) and 2,3-bisphospho-D-glyceric acid (2,3-BPG). CONCLUSIONS: These results provide insights into the metabolic alterations in BMECs upon IFN-γ induction and indicate potential theoretical basis for clarifying IFN-γ-induced diseases in mammary gland.

Purification and characteristics of a novel milk-clotting metalloprotease from Bacillus velezensis DB219.

Milk-clotting enzyme (MCE) is the essential active agents in dairy processing. The traditional MCE is mainly obtained from animal sources, in which calf rennet is the most widely used in cheese industry. Traditional MCE substitute is becoming necessary due to its limited production and increased cheese consumption. A novel traditional MCE substitute was produced from Bacillus velezensis DB219 in this study. The DB219 MCE exhibited a notable specific activity of 6,110 Soxhlet units/mg and 3.16-fold purification yield with 28.87% recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow. The purified DB219 MCE was a metalloprotease with a molecular weight of 36 kDa. The DB219 MCE was weak acid resistance and stable at pH 6.0 to 10.0 and temperature <45°C. The highest milk-clotting activity was observed in substrate at pH 5.5 added with 20 to 30 mM CaCl2. The Michaelis constant and maximal velocity for casein were 0.31 g/L and 14.22 µmol/min. The DB219 MCE preferred to hydrolyze ß-casein instead of α-casein. The DB219 MCE hydrolyzed α-casein, ß-casein, and κ-casein to generate significantly different peptides in comparison with calf rennet and ES6023 MCE (fungal MCE) through SDS-PAGE and reversed-phase HPLC analysis. The DB219 MCE mainly cleaved Thr124-Ile125 and Ser104-Phe105 bonds in κ-casein and had unique casein cleavage sites and peptide composition through LC-MS/MS analysis. The DB219 MCE was potential to be a new milk coagulant and enriched kinds of traditional MCE substitute.

Pregnancy monitoring in mares: Ultrasonographic and endocrine approaches.

Methods to diagnose and monitor equine pregnancy continue to advance with improved instrumentation enabling the development of novel, non-invasive approaches to assess fetal well-being and viability using ultrasound and endocrine testing. From early embryonic loss to placentitis, that is typically encountered later in gestation, fetal viability and development as well as placental function can be evaluated using two fundamentally different, structural and functional, approaches. Ultrasound provides structural information on embryonic and fetal growth using such parameters as combined thickness of the uterus and placenta (CTUP), visual assessment of fetal fluids, activity, heart rate and multiple biometrics involving the fetal head and eyes, limbs and joints among many others, depending on the stage of gestation. Endocrine profiles that include progesterone and 5α-dihydroprogesterone, other metabolites, androgens and estrogens can be evaluated simultaneously using liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing more functional information on fetal and placental competence and development. Endocrine information can be used in making clinical decisions including the need for progestin supplementation or when it can cease, and even estimating gestational stage in mares that cannot be easily palpated or scanned, as with mini-breeds or rancorous animals most notably. When used together, monitoring gestation by ultrasound and hormonal analysis provides unusual insight into feto-placental well-being and the progress of pregnancy, helping to identify problems needing therapeutic intervention.

Seminal plasma lipid profiles of differential cryotolerance of semen in Mediterranean Buffalo bulls.

The cryotolerance of semen obtained from Mediterranean buffalo bulls usually is more likely to deteriorate during the summer. To obtain the optimal sperm for fertility, the physiological status and reproductive performance of Mediterranean buffalo bulls in the summer and spring were first analysed by assessing blood serum and seminal plasma samples; then, the lipid profiles of seminal plasma were investigated by LC-MS/MS. The T, T3 and SOD levels of serum and seminal plasma in the spring were significantly higher than in the summer (p < .05). The results suggest that T3 level is positively correlated with semen cryotolerance; sphingolipids are potential markers for semen cryotolerance of Mediterranean buffalo. To our knowledge, this is the first report of targeted lipidomics in semen cryotolerance.

Novel holistic pharmacokinetic model applied to plasma and urine concentrations of 2,5-dihydroxybenzene sulphonate following administrations of calcium dobesilate and etamsylate to exercised horses.

Calcium dobesilate (CD) is a synthetic venoactive drug used in veterinary medicine to treat equine navicular disease. Etamsylate is a haemostatic agent used in horses for the treatment of exercise-induced pulmonary haemorrhage. Both etamsylate and CD dissociate in the circulatory system with 2,5-HBSA as the active drug. The aim of the research was to be able to provide detection time (DT) advice from pharmacokinetic (PK) studies in Thoroughbred horses to better inform trainers, and their veterinary surgeons, prescribing these substances for treatment of Thoroughbred racehorses. Two (pilot study) and six (final study) horses were given 28 and 9 repeated dose of CD (3 mg/kg BID) respectively. Two horses were each given a single intravenous (IV) dose of etamsylate (10 mg/kg). Plasma and urine 2,5-HBSA concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CD pilot study revealed that steady state could be reached with a few days and that 2,5-HBSA in plasma and urine shows instability during storage at -20°C but appears stable at -80°C. A novel holistic non-linear mixed-effects three-compartmental PK model was developed that described both plasma and urine concentrations of 2,5-HBSA, from either CD or etamsylate administration. Typical values for 2,5-HBSA clearance and bioavailability were 2.0 mL/min/kg and 28% respectively. Using the parameters obtained from this PK model, in conjunction with methodology developed by Toutain, afforded a possible screening limit (SL) that can regulate for a DT of 3 days in urine; however, a corresponding SL in plasma would be below current levels of detection. However, it is the responsibility of the individual racing authorities to apply their own risk management with regard to SLs and DTs.

Pharmacokinetics of aminocaproic acid in thoroughbred horses.

The antifibrinolytic agent aminocaproic acid (ACA) is occasionally used prior to episodes of intense training in racehorses suffering from exercise-induced pulmonary hemorrhage. Although a previous study indicated that the drug is cleared rapidly in horses, some racetrack practitioners claim that recent adverse analytical findings for ACA in postrace samples were from ACA administrations 5-7 days before the race. The purpose of this study was to re-examine the pharmacokinetics of ACA in horses to address this apparent conundrum. Eight exercise-conditioned thoroughbred horses were administered 5 g of ACA IV, and blood and urine samples were collected at pre-determined time points prior to drug administration and for up to 168 h after dosing. Concentrations of ACA in the serum and urine samples were determined by LC-MS/MS. The pharmacokinetics of ACA in serum were best described by a three-compartment model with a terminal elimination half-life of 24.2 ± 2.9 h. After dosing, ACA was above the lower limit of detection (1 ng/mL for serum and 10 ng/mL for urine) in all serum and urine samples at all time points. In a similar manner, ACA was above the lower limit of quantification (LLOQ; 10 ng/mL for serum and 100 ng/mL for urine) in all serum and urine samples collected from all horses from 0.5 to 120 h post dosing. In six of the eight horses, ACA was above the LLOQ 168 h after dosing in serum and urine samples. LC-MS/MS methodology is the industry standard for testing of samples collected from racehorses with the purpose of controlling the use of medications and performance altering substances. The improved sensitivity of the analytical procedure used in the present study allowed the detection of a prolonged terminal elimination phase of ACA in horses that had not previously been described. Currently, most racing jurisdictions have not adopted a permitted concentration or threshold for ACA in postrace samples, and therefore veterinarians need to allow for an extended withdrawal time of a minimum of 11 days after the administration of ACA to racehorses to substantially reduce the risk of adverse analytical findings of ACA in postrace samples.

Pharmacokinetic, sedative, and physiological effects of oral compounded formulations of trazodone alone or in combination with gabapentin in male cats.

Trazodone and gabapentin are common oral sedatives in cats, used alone or combined, but no pharmacokinetic studies exist for trazodone in this species. The objective of this study was to determine the pharmacokinetics of oral trazodone (T) alone, or in combination with gabapentin (G) in healthy cats. Cats (n = 6) were randomly allocated to receive T (3 mg/kg) intravenously (IV), T (5 mg/kg) orally (PO), or T (5 mg/kg) and G (10 mg/kg) PO with a 1-week washout period between treatments. Heart rate, respiratory rate, indirect blood pressure, and level of sedation were assessed, and venous blood samples were collected serially over 24 h. Analysis of plasma trazodone concentration was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oral T administration resulted in a bioavailability of 54.9(7-96)%, and 17.2(11-25)% when administered with G. Tmax 0.17 (0.17-0.5) and 0.17 (0.17-0.75) h; Cmax 1.67 ± 0.91 and 1.22 ± 0.54 µg/mL, AUC 5.23 (2.0-18.76) and 2.37 (1.17-7.80) h*µg/mL; T1/2 5.12 ± 2.56 and 4.71 ± 1.07 h; for T and TG, respectively. Sedation was significant when compared to baseline in all groups from 20 or 45 min to 8 h indicating some lag between peak plasma concentration and sedative effects. Physiological variables remained within normal limits. This study concludes that oral trazodone is rapidly absorbed in healthy cats. Addition of gabapentin did not result in more profound sedation, showing no clinical advantage of combining these drugs in this study population.

Rational determination of cefazolin dosage regimen in horses based on pharmacokinetics/pharmacodynamics principles and Monte Carlo simulations.

A pharmacokinetics/pharmacodynamics (PK/PD) approach was used to determine the best empirical dosage regimen of cefazolin (CEZ) after intramuscular (IM) administration of CEZ in horses. Seven horses received a single IM or intravenous (IV) administration of CEZ of 5 mg/kg bodyweight (BW) according to a crossover design. CEZ plasma concentrations were measured using LC-MS/MS. The plasma concentrations in these seven horses and those of six other horses obtained in a previous study with an IV CEZ dose of 10 mg/kg were modelled simultaneously using NonLinear Mixed-Effect modelling followed by Monte Carlo simulations to establish a rational dosage regimen. A 90% Probability of Target Attainment (PTA) for a PK/PD target of a free plasma concentration exceeding MIC90 (fT > MIC ) for 40% of the dosing interval was set for selecting an effective dosing regimen. The typical half-life of absorption and bioavailability after IM administration were 1.25 h and 96.8%, respectively. A CEZ dosage regimen of 5 mg/kg BW q12h IM administration achieved therapeutic concentrations to control both Streptococcus zooepidemicus and Staphylococcus aureus. For the same dose, the fT > MIC after IM administration was significantly longer than after IV administration, and the IM route should be favoured by clinicians for its efficiency and convenience.

Pharmacokinetics of intramuscular maropitant in pigs (Sus scrofa domesticus).

Pigs are at risk of vomiting from medical conditions as well as the emetic side effects of drugs administered for peri-operative manipulations, but there is a lack of pharmacokinetic data for potential anti-emetic therapies, such as maropitant, in this species. The main objective of this study was to estimate plasma pharmacokinetic parameters for maropitant in pigs after a single intramuscular (IM) administration dosed at 1.0 mg/kg. A secondary objective was to estimate pilot pharmacokinetic parameters in pigs after oral (PO) administration at 2.0 mg/kg. Maropitant was administered to six commercial pigs at a dose of 1.0 mg/kg IM. Plasma samples were collected over 72 h. After a 7-day washout period, two pigs were administered maropitant at a dose of 2.0 mg/kg PO. Maropitant concentrations were measured via liquid chromatography/mass spectrometry (LC-MS/MS). A non-compartmental analysis was used to derive pharmacokinetics parameters. No adverse events were noted in any of the study pigs after administration. Following single IM administration, maximum plasma concentration was estimated at 412.7 ± 132.0 ng/mL and time to maximum concentration ranged from 0.083 to 1.0 h. Elimination half-life was estimated at 6.7 ± 1.28 h, and mean residence time was 6.1 ± 1.2 h. Volume of distribution after IM administration was 15.9 L/kg. Area under the curve was 1336 ± 132.0 h*ng/mL. The relative bioavailability of PO administration was noted to be 15.5% and 27.2% in the two pilot pigs. The maximum systemic concentration observed in the study pigs after IM administration was higher than what was observed after subcutaneous administration in dogs, cats, or rabbits. The achieved maximum concentration exceeded the concentrations for anti-emetic purposes in dogs and cats; however, a specific anti-emetic concentration is currently not known for pigs. Further research is needed into the pharmacodynamics of maropitant in pigs to determine specific therapeutic strategies for this drug.

METTL21C mediates lysine trimethylation of IGF2BP1 to regulate chicken myoblast proliferation.

1. Methyltransferase-like 21C (METTL21C) and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) play important roles in the proliferation of chicken myoblasts. However, it remains unclear whether there is protein-protein interaction between METTL21C and IGF2BP1 to regulate proliferation of chicken myoblasts.2. In this study, the Igf2bp1 gene was amplified from cDNA of liver tissue of Lueyang black-bone chicken to construct the overexpression vector HA-Igf2bp1. The HA-Igf2bp1 and Flag-Mettl21c vectors were individually transfected and co-transfected into HEK293T, respectively. Co-immunoprecipitation (Co-IP) assay indicated a protein-protein interaction between METTL21C and IGF2BP1.3. Using the Western blotting and LC-MS/MS, it was found that METTL21C could mediate the lysine methylation modification of IGF2BP1. Furthermore, the His-tagged overexpression vector HA-Igf2bp1-His was constructed, transfected and co-transfected with Flag-Mettl21c into HEK293T. His-tagged IGF2BP1 was purified by nickel ion affinity chromatography. Western blotting revealed that IGF2BP1 was successfully purified, and the trimethylation modification level of co-transfection group was significantly elevated compared with the single-transfection Igf2bp1 group.4. Mettl21c and Igf2bp1 overexpression vectors were transfected and co-transfected into primary chicken myoblasts, respectively. The results of 5-ethynyl-2'-deoxyuridine assay and the expression level of Pax7 and MyoD indicated that overexpression of Igf2bp1 alone inhibited the chicken myoblast proliferation, whereas co-expression of Mettl21c and Igf2bp1 eliminated the inhibitory effects of Igf2bp1, thereby favouring cell proliferation and differentiation.5. The results, for the first time, revealed that METTL21C mediated the lysine trimethylation modification of IGF2BP1 to regulate the proliferation of chicken myoblasts, which provided a new insight into in-depth analysis of the molecular mechanism of METTL21C methylation involved in regulating the growth and development of skeletal muscle in Lueyang black-bone chicken.

UPLC-MS-based plasma metabolomics for identifying energy metabolism biomarkers of maintenance in growing pigs.

The purpose of this study is to explore the potential plasma metabolism biomarkers reflecting the maintenance status of growing pigs. The repeated measurement design was used in this experiment, and six barrows (28.6 ± 0.5 kg BW) were selected and kept in metabolism crates. The feeding level in growing pigs close to ad libitum was 2400 kJ ME/kg BW0.6 ·day-1 during Day 1 to Day 7, while a feeding level of 782 kJ ME/kg BW0.6 ·day-1 was provided as energy requirement for maintenance during Day 8 to Day 14. Plasma samples of each pig were collected from the anterior vena cava on the morning of Day 8 and Day 15. The metabolites of plasma were determined by high-resolution mass spectrometry using a metabolomics approach. Results showed that metabolomics analysis between ad libitum-fed state and maintained status revealed differences in 16 compounds. Identified compounds were enriched in metabolic pathways related to linoleic acid metabolism, tryptophan metabolism, and alanine, aspartate and glutamate metabolism. In conclusion, linoleic acid metabolism, tryptophan metabolism, alanine, aspartate and glutamic acid metabolism pathways played a major regulatory role in the maintenance status of growing pigs. The potential metabolism biomarkers of maintenance in growing pigs were linoleic acid, glutamine and tyrosine.

Identification of excretory and secretory proteins from Haemonchus contortus inducing a Th9 immune response in goats.

Th9 cells have been shown to play crucial roles in anti-parasite immunity, pathogenic microbe infection, and allergy. Previous studies have demonstrated that Haemonchus contortus excretory and secretory proteins (HcESPs) induce the proliferation of Th9 cells and alter the transcriptional level of IL-9 as well as its related pathways in the Th9 immune response after infection. However, the exact molecule(s) in HcESPs inducing the Th9 immune response is not yet known. In this study, flow cytometry, co-immunoprecipitation (Co-IP) and shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) were used, and a total of 218 proteins from HcESPs that might interact with goat Th9 cells were identified. By in vitro culture of Th9 cells with HcESPs, 40 binding proteins were identified. In vivo, 38, 47, 42 and 142 binding proteins were identified at 7, 15, 35 and 50 days post-infection (dpi), respectively. Furthermore, 2 of the 218 HcESPs, named DNA/RNA helicase domain containing protein (HcDR) and GATA transcription factor (HcGATA), were confirmed to induce the proliferation of Th9 cells and promote the expression of IL-9 when incubated with goat peripheral blood mononuclear cells (PBMCs). This study represents a proteomics-guided investigation of the interactions between Th9 cells and HcESPs. It provides a new way to explore immunostimulatory antigens among HcESPs and identifies candidates for immune-mediated prevention of H. contortus infection.

Proteomic profiling of aspergillus flavus endophthalmitis derived extracellular vesicles in an in-vivo murine model.

Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell communication, and are involved in potential pathological and physiological cellular processes. The aim of this study was to understand the proteomic cargo of these vesicles, in a murine model of Aspergillus flavus (AF) endophthalmitis. EVs were isolated from A. flavus infected C57BL/6 mice eyes by differential ultracentrifugation at 24 h post infection (p.i) and isolated EVs were characterized by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), Exocet assay, and western blot. Proteomic profiling of EVs was then evaluated by mass spectrometry (LC-MS/MS) and compared it with control uninfected mice. The average size of the EVs were 180-280 nm by DLS and the number of EVs increased to 1.55 × 1010 in infected mice in comparison to EVs from uninfected eye (1.24 × 109). Western blot was positive for CD9, CD63, and CD81 confirming the presence of EVs. LC-MS/MS analysis, identified 81 differentially expressed proteins, of these 22 were up-regulated and 59 were down-regulated. Gene Ontology (GO) analysis revealed enrichment of lipid metabolism, protein complex binding, and transferase activity, and the proteins associated were Aquaporin-5, CD177 antigen, Solute carrier family-25, and Calcium/calmodulin-dependent protein kinase. Additionally, KEGG pathway analysis indicated that glucagon signalling, metabolic, and PPAR signalling pathway were significantly associated with EVs from A. flavus infected mice eyes. The protein cargo in EVs from A. flavus endophthalmitis provides new insights into the pathogenesis of fungal endophthalmitis and validation of these proteins can serve as diagnostic and/or prognostic biomarkers for patients with a clinical suspicion of fungal endophthalmitis. LAY SUMMARY: EVs play an important role in cell communication. In our study proteomic profiling of EVs isolated from A. flavus infected mice provided new insights into the understanding of the pathobiology of A. flavus endophthalmitis and validation of these proteins can serve as biomarkers.