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.

Urinary D-amino acid profiles in cats with chronic kidney disease.

Chronic kidney disease (CKD) is highly prevalent in domestic cats. This study aimed to compare urinary D-amino acid levels between control and CKD-afflicted cats as a novel noninvasive method for assessing CKD. Cats were divided into control and CKD stage II groups in accordance with the International Renal Interest Society guidelines. The urinary DL-amino acid levels of the cats were analyzed using chiral tandem liquid chromatography-tandem mass spectrometry, and their medical records were investigated. The CKD group had considerably lower urinary D-amino acid concentrations and enantiomeric ratios than the control group. The total urinary D-amino acid contents significantly correlated with blood parameters (creatinine and urea nitrogen). These findings may contribute towards the detection of CKD stage II in domestic cats.

Salivary metabolomic identification of biomarker candidates for oral melanoma and oral squamous cell carcinoma in dogs.

BACKGROUND: Oral melanoma (OM) and oral squamous cell carcinoma (OSCC) are frequently diagnosed in dogs, presenting a challenge in distinguishing them from benign oral tumors (BN). Salivary metabolomic biomarkers offer a practical solution because of saliva's direct contact with tumors and the noninvasive nature of collection. OBJECTIVE: Assess the diversity and abundance of the salivary metabolome in dogs with BN, OM, and OSCC using amine/phenol submetabolome analysis and high-performance chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC-MS). ANIMALS: Study included 11 BN, 24 OM, 10 OSCC, and 20 healthy control dogs. METHODS: Case-control cross-sectional study was conducted to assess salivary submetabolic profiles in dogs with BN, OM, and OSCC and healthy dogs. Samples were labeled with 12C-dansyl chloride and analyzed using CIL LC-MS targeted to amine- and phenol-containing metabolites for amine/phenol submetabolome analysis. RESULTS: Distinct clusters and significant differences in metabolite concentrations were observed among the oral cancer, BN, and control groups. A total of 154 and 66 metabolites showed significantly altered concentrations, particularly in OM and OSCC, respectively, when compared with BN (Padj < .05). Potential metabolic biomarkers were identified for each cancer, including decreased concentrations of seryl-arginine and sarcosine in OSCC. Moreover, high-confidence putative metabolites were identified, including an increase in tryptophyl-threonine and a decrease in 1,2-dihydroxynapthalene-6-sulfonic acid and hydroxyprolyl-hydroxyproline for OM. CONCLUSIONS AND CLINICAL IMPORTANCE: We identified high coverage of the amine/phenol submetabolome, including seryl-arginine, and sarcosine, in OSCC. Our findings emphasize the potential of these biomarkers for distinguishing between oral OSCC and BN in dogs.

Urinary steroid profiling using liquid chromatography-tandem mass spectrometry for the diagnosis of canine Cushing's syndrome.

Serum cortisol measurements by chemiluminescence enzyme immunoassay (CLEIA) are widely used to diagnose hypercortisolism (HC) or Cushing's syndrome in dogs. However, they are associated with problems such as the need for multiple blood collections under stressful conditions or cross-reactivity between hormones. Therefore, a less invasive and more accurate diagnostic method is required. This study aimed to develop a urinary steroid profile analysis method using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and to evaluate its clinical usefulness. Sixty-five healthy dogs and 38 dogs with suspected HC were included in the study. Using LC/MS/MS, the levels of 11 steroid hormones in the urine were determined. We established the upper limit of the reference interval for each urinary steroid-to-creatinine ratio and evaluated their diagnostic performances. The levels of the five steroid hormones were significantly higher in the 14 dogs with HC than in the 24 dogs with mimicking HC and 65 healthy dogs. The urinary corticosterone-to-creatinine ratio showed the highest diagnostic accuracy (area under the curve, 0.96). A significant correlation was seen between urinary cortisol concentrations measured by LC/MS/MS and CLEIA (rs = 0.88, P <0.001), although the CLEIA measurements were significantly higher than the LC/MS/MS measurements (P <0.001). LC/MS/MS-based urinary steroid profiles are a promising tool for diagnosing canine HC.

Heat stress alters the ovarian proteome in prepubertal gilts.

Heat stress (HS) occurs when exogenous and metabolic heat accumulation exceeds heat dissipation; a thermal imbalance that compromises female reproduction. This study investigated the hypothesis that HS alters the ovarian proteome and negatively impacts proteins engaged with insulin signaling, inflammation, and ovarian function. Prepubertal gilts (n = 19) were assigned to one of three environmental groups: thermal neutral with ad libitum feed intake (TN; n = 6), thermal neutral pair-fed (PF; n = 6), or HS (n = 7). For 7 d, HS gilts were exposed to 12-h cyclic temperatures of 35.0 ±â€…0.2 °C and 32.2 ±â€…0.1 °C, while TN and PF gilts were housed at 21.0 ±â€…0.1 °C. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on ovarian protein homogenates. Relative to TN gilts, 178 proteins were altered (P ≤ 0.05, log2foldchange ≥ 1) by HS, with 76 increased and 102 decreased. STRING gene ontology classified and identified 45 biological processes including those associated with chaperone protein refolding, cytoplasmic translational initiation, and immune activation; with a protein-protein interaction web network of 158 nodes and 563 edges connected based on protein function (FDR ≤ 0.05). Relative to PF, HS altered 330 proteins (P ≤ 0.05, log2foldchange ≥ 1), with 151 increased and 179 decreased. Fifty-seven biological pathways associated with protein function and assembly, RNA processing, and metabolic processes were identified, with a protein-protein interaction network of 303 nodes and 1,606 edges. Comparing HS with both the TN and PF treatments, 72 ovarian proteins were consistently altered by HS with 68 nodes and 104 edges, with biological pathways associated with translation and gene expression. This indicates that HS alters the ovarian proteome and multiple biological pathways and systems in prepubertal gilts; changes that potentially contribute to female infertility.

Heat stress impairs female fertility, yet the mechanisms underlying reduced fecundity remain unclear. This study investigated the ovarian proteomic changes resultant from heat stress in prepubertal gilts and discovered changes related to several important biological processes that could be responsible for reduced female fertility.

Condensed tannin-induced variations in the rumen metabolome and the correlation with fermentation characteristics in goats.

In this study, we characterized the effects of CT dietary inclusion at 2% (wt/wt) dry matter on the goat rumen metabolome and fermentation characteristics. Barley (BA) and corn (CN) were separately used as basal grain for the control rations, and rations supplemented with CT were BACT and CNCT, respectively. The rations were tested using eight Japanese Shiba × Saanen goats in a replicated 4 × 4 Latin square arrangement (28 days for each period). Ruminal fluid was obtained on day 25 of each period, and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis was performed. Metabolites from BACT against BA and CNCT against CN were mostly associated with purine metabolism. Moreover, BACT against BA showed intensified biosynthesis of unsaturated fatty acids, and CNCT against CN resulted in strengthened amino acid metabolism. Furthermore, strong correlations were observed between rumen NH3 -N and the copy number of total bacteria with most of the differential metabolites. The present paper provides a better understanding of the relationship between the rumen metabolome and fermentation characteristics and supports a shift in concern about using CT as a strategy to manipulate rumen metabolism.

Assessment of some chemical residues in Egyptian raw milk and traditional cheese.

Background: The assessment of risks related to food safety is becoming a challenge in developing countries with its consequent health hazards. Chemical risk assessment in dairy products is important to maintain consumer health locally and internationally. Since milk and dairy products are essential foods for a wide range of customers, mostly children, patients, and pregnant women, it is very important to estimate the risks of some chemical residues, such as pesticides, some heavy metals, and aflatoxins. Aim: This work aims to determine the levels of chemical contamination in milk and traditional Egyptian cheese. Methods: Heavy metals were determined in samples by atomic absorption spectrometry. GC-mass spectrometry (MS)/MS and LC-MS/MS were also used for measuring pesticide residues. The Aflatoxin M1 was determined by enzyme-linked immune-sorbent assay. Results: Raw milk samples were tested and showed elevated concentrations of lead and cadmium, (46% and 4%, respectively). The heavy metals detected in the Egyptian cheese samples were variable depending on the type of cheese. Moreover, p.p.-DDE phenofose was present in 45% and 29% of raw milk and Ras cheese samples, respectively. For Aflatoxin M1, only 7% of milk samples and 2.9% of Ras cheese samples exceeded the acceptable limits. Conclusion: More surveying and risk assessment of chemical residues in milk and milk products are essential for controlling health risks to consumers.

Quantitative tyramine analysis method for Apis mellifera larvae infected with Melissococcus plutonius, the causative agent of European foulbrood.

Tyramine, a trace monoamine produced from tyrosine by decarboxylation and found naturally in foods, plants, and animals, is a suspected virulence factor of Melissococcus plutonius that causes European foulbrood in honey bee brood. In the present study, we developed a method for quantitative analysis of tyramine in culture medium and honey bee larvae with a limit of quantitation of 3 ng/mL and a recovery rate of >97% using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry and deuterium-labeled tyramine, demonstrating for the first time that a highly virulent M. plutonius strain actually produces tyramine in infected larvae. This method will be an indispensable tool to elucidate the role of tyramine in European foulbrood pathogenesis in combination with exposure bioassays using artificially reared bee larvae.

Serum vitamin D3 (25-OH vit D3) concentrations and hemogram changes in calves with enzootic pneumonia.

BACKGROUND: Enzootic pneumonia is an important disease complex associated with insufficient colostrum intake after birth, adverse environmental conditions, and stress. Vitamin D deficiency may be an important predisposing factor for this disease. OBJECTIVE: This study aimed to investigate in calves with enzootic pneumonia. METHODS: A total of 30 calves, aged 3-5 months, under the same care and feeding conditions were used. Groups were formed according to Clinical Respiratory Scoring as the group with mild/moderate enzootic pneumonia (n = 10), the group with severe enzootic pneumonia (n = 10), and the healthy control group (n = 10) without any disease. Blood samples were collected from the jugular vein of animals in all groups on Day 0; a complete blood count was performed, and serum vitamin D levels were measured using the Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method. RESULTS: Although no statistical differences were observed in total leukocyte, lymphocyte, eosinophil, basophil, hemoglobin, and hematocrit levels between groups, statistically significant differences in blood neutrophil, monocyte, and erythrocyte counts were found between the groups. Monocyte counts were statistically decreased in the mild/moderate group compared with the control group. Neutrophil counts were significantly higher in the mild/moderate and severe groups than in the control group. Erythrocyte counts were increased in the mild/moderate and severe groups compared with the control group. Vitamin D concentrations were statistically lower in the mild/moderate and severe groups than in the control group. However, no statistical differences in Vitamin D concentrations were observed between the mild/moderate and severe groups. There was a negative and significant correlation between erythrocyte counts and vitamin D concentrations (r = -0.64, P < .0001). While erythrocyte counts increased in the severe group compared with the mild/moderate group, vitamin D concentrations decreased. Also, a negative and significant correlation was observed between platelet counts and vitamin D concentrations (r = -0.74, P < .0001). CONCLUSIONS: The results of this study determined that serum vitamin D concentrations in calves with pneumonia were lower than those in healthy calves. Detailed studies on the etiologic and prognostic importance of low vitamin D levels in calves with enzootic pneumonia may provide valuable data for prevention and treatment.

Identification and characterization of the receptors of a microneme adhesive repeat domain of Eimeria maxima microneme protein 3 in chicken intestine epithelial cells.

Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.

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.

Serum symmetric dimethylarginine in older dogs: Reference interval and comparison of a gold standard method with the ELISA.

BACKGROUND: Serum symmetric dimethylarginine (SDMA) is used to screen for renal dysfunction in dogs. The gold standard technique for measuring SDMA, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is not widely available. Age-specific reference intervals for SDMA in older dogs are lacking. OBJECTIVES: Prospective study in older dogs to validate a commercially available LC-MS/MS method for SDMA, compare SDMA concentrations with concentrations measured using ELISA and obtain a reference interval (RI) for older dogs using both methods. ANIMALS: Client-owned older dogs undergoing health screening. METHODS: The LC-MS/MS method was analytically validated (limit of detection, precision, and linearity). Serum was sent cooled overnight for ELISA or was frozen at -80°C until batch analysis using LC-MS/MS. Results of LC-MS/MS and ELISA were compared and RIs for older dogs were calculated according to international guidelines. RESULTS: The LC-MS/MS method showed good linearity (r2 = .99) and precision (coefficient of variation <10%), with a laboratory RI between 8.0 and 14.0 µg/dL. Paired measurements were available from 118 different dogs. Median SDMA concentration were 9.4 (range, 5.0-21.2) using LC-MS/MS and 12.0 (range, 5.0-22.0) µg/dL using ELISA. Both methods significantly differed with a mean difference of 2.2 µg/dL. The RI for older dogs for LC-MS/MS was 4.4-15.0 µg/dL, and for ELISA was 6.4-17.4 µg/dL. CONCLUSIONS AND CLINICAL IMPORTANCE: The ELISA provided significantly higher SDMA concentrations compared to the validated LC-MS/MS method, indicating the need for device- or assay-specific RI. The obtained age-specific RI for SDMA is considerably higher in older dogs compared to the general laboratory RI.

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.

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.

Pollen beetle (Astylus atromaculatus)-associated gastroenteric disease in cattle: report of 6 natural outbreaks.

Astylus atromaculatus is a pollen beetle native to South America, commonly found in crop flowers. Experimental intoxication of sheep and guinea pigs by this beetle resulting in fibrinonecrotizing enteritis has been reported. We describe here 6 natural outbreaks of intoxication in cattle associated with consumption of alfalfa (5 of 6) and mixed native (1 of 6) pastures heavily contaminated with A. atromaculatus. The outbreaks occurred during the summer (January-February) of 2023 in Argentina (n = 4) and Uruguay (n = 2), in beef cattle under extensive or semi-extensive rearing systems, with overall cumulative incidence and mortality of 22.3% and 17.8%, respectively. The main clinical signs included acute onset of anorexia, lethargy, hyperthermia, hindlimb weakness, reluctance to move, and diarrhea, for up to 15 d. In 2 outbreaks, sudden death was observed. Eight Hereford, Angus, and/or crossbreed heifers, cows, steers, and/or calves were autopsied. Gross and microscopic findings included multifocal necrosis with fibrinous pseudomembranes in the forestomachs and/or small and large intestines. Fragments or whole specimens of A. atromaculatus were identified in the ruminal content of all animals. Testing for multiple gastroenteric pathogens was negative as was testing of A. atromaculatus for cantharidin and batrachotoxin. GC-MS and LC-MS/MS performed on the beetles did not identify any known toxic compounds. Based on the exposure to A. atromaculatus-contaminated pasture, gross and microscopic lesions, and negative results of all testing for multiple gastroenteric pathogens, a diagnosis of intoxication by A. atromaculatus is proposed. Disease caused by A. atromaculatus consumption has not been reported previously in cattle, to our knowledge.

Research Note: Changes in chicken egg yolk metabolome during its formation.

The process of egg yolk formation involves the transport and uptake of a large number of small molecule metabolites. A qualitative and relative quantitative analysis of metabolites in the 3 formation periods of egg yolk was performed by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical workflow. A total of 398 metabolites were identified, of which "amino acids and their metabolites", "lipid", and "organic acids and their derivatives" were the dominant egg yolk metabolite categories with the most metabolite species. The findings suggested that a number of amino acids, organic acids, nucleotides and their metabolites were deposited during follicular development to provide material support for later embryonic development. At the same time, some vitamins and carbohydrates were consumed during follicular development to support the normal development process. In addition, the small hierarchical follicle (SF) period may be a critical period for the regulation of the transport and deposition of some active ingredients. These results contribute to a comprehensive understanding of the nutrient deposition pattern and nutritional properties of egg yolk.

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.

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.

Oral cytarabine ocfosfate pharmacokinetics and assessment of leukocyte biomarkers in normal dogs.

BACKGROUND: Cytosine arabinoside (Ara-C) is a nucleoside analog prodrug utilized for immunomodulatory effects mediated by its active metabolite Ara-CTP. Optimal dosing protocols for immunomodulation in dogs have not been defined. Cytarabine ocfosfate (CO) is a lipophilic prodrug of Ara-C that can be administered PO and provides prolonged serum concentrations of Ara-C. OBJECTIVES: Provide pharmacokinetic data for orally administered CO and determine accumulation and functional consequences of Ara-CTP within peripheral blood leukocytes. ANIMALS: Three healthy female hound dogs and 1 healthy male Beagle. METHODS: Prospective study. Dogs received 200 mg/m2 of CO PO q24h for 7 doses. Serum and cerebrospinal fluid (CSF) CO and Ara-C concentrations were measured by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Complete blood counts, flow cytometry, and leukocyte activation assays were done up to 21 days. Incorporation of Ara-CTP within leukocyte DNA was determined by LC-MS/MS. RESULTS: Maximum serum concentration (Cmax ) for Ara-C was 456.1-724.0 ng/mL (1.88-2.98 µM) and terminal half-life was 23.3 to 29.4 hours. Cerebrospinal fluid: serum Ara-C ratios ranged from 0.54 to 1.2. Peripheral blood lymphocyte concentrations remained within the reference range, but proliferation rates poststimulation were decreased at 6 days. Incorporation of Ara-CTP was not saturated and remained >25% of peak concentration at 13 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Oral CO may produce prolonged serum Ara-C half-lives at concentrations sufficient to induce functional changes in peripheral leukocytes and is associated with prolonged retention of DNA-incorporated Ara-CTP. Application of functional and active metabolite assessment is feasible and may provide more relevant data to determine optimal dosing regimens for Ara-C-based treatments.

Proteomic analysis of high and low-motility frozen-thawed spermatozoa in yak provides important insights into the molecular mechanisms underlying sperm cryodamage.

Sperm cryodamage caused by cryopreservation limits the use of frozen yak spermatozoa in artificial insemination (AI). However, the proteomic changes involved in the cryodamage of yak spermatozoa have not been investigated to date. Therefore, this study aimed to identify proteins related to freezing tolerance. Tandem mass tag (TMT) were used in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for identifying differentially expressed proteins (DEPs) between high-motility (HM) and low-motility (LM) frozen-thawed yak spermatozoa. A total of 116 DEPs were identified (>1.5-fold, P < 0.05); of which, 104 proteins were upregulated in HM spermatozoa and 12 proteins were upregulated in LM spermatozoa. The results of functional annotation analysis revealed that the DEPs were mainly involved in metabolic processes. A total of 20 DEPs that were abundantly expressed in HM spermatozoa were strongly associated with carbohydrate metabolism. The results of KEGG analysis revealed that the DEPs were enriched in glycolysis/gluconeogenesis, PPAR signaling pathway, and Ras signaling pathway. In addition, many antioxidant enzymes such as superoxide dismutase (SOD1), peroxiredoxin-6 (PRDX6), and Parkinson disease protein 7 (PARK7) were upregulated in HM spermatozoa, suggesting that these enzymes affect the motility of spermatozoa by regulating the levels of reactive oxygen species (ROS) in frozen-thawed spermatozoa. Altogether, the findings of this study elucidate the mechanisms through which cryopreservation affects the movement of yak spermatozoa and offer a novel basis for refining freezing techniques and modifying cryopreservation extender components.