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.

Comprehensive genome assembly reveals genetic diversity and carcass consumption insights in critically endangered Asian king vultures.

The Asian king vulture (AKV), a vital forest scavenger, is facing globally critical endangerment. This study aimed to construct a reference genome to unveil the mechanisms underlying its scavenger abilities and to assess the genetic relatedness of the captive population in Thailand. A reference genome of a female AKV was assembled from sequencing reads obtained from both PacBio long-read and MGI short-read sequencing platforms. Comparative genomics with New World vultures (NWVs) and other birds in the Family Accipitridae revealed unique gene families in AKV associated with retroviral genome integration and feather keratin, contrasting with NWVs' genes related to olfactory reception. Expanded gene families in AKV were linked to inflammatory response, iron regulation and spermatogenesis. Positively selected genes included those associated with anti-apoptosis, immune response and muscle cell development, shedding light on adaptations for carcass consumption and high-altitude soaring. Using restriction site-associated DNA sequencing (RADseq)-based genome-wide single nucleotide polymorphisms (SNPs), genetic relatedness and inbreeding status of five captive AKVs were determined, revealing high genomic inbreeding in two females. In conclusion, the AKV reference genome was established, providing insights into its unique characteristics. Additionally, the potential of RADseq-based genome-wide SNPs for selecting AKV breeders was demonstrated.

Comparative E-cadherin and syndecan-1 protein expression in human and canine oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a prevalent form of oral cancer in humans and dogs. The altered expression of cell adhesion molecules, including E-cadherin (CDH1) and syndecan-1 (SDC1), is involved in cancer progression. This study aimed to investigate the protein expression of CDH1 and SDC1 in early and late clinical stages of human and canine OSCC (hOSCC and cOSCC, respectively), using immunohistochemistry. Formalin-fixed and paraffin embedded tissue blocks were obtained from 21 hOSCC, 8 human normal gingiva, 26 cOSCC, and 13 canine normal gingiva. Clinical stages and histological subtypes of samples were evaluated. The results indicated that both human and canine OSCC exhibited reduced levels of CDH1 and SDC1 expression at the cell membrane regardless of clinical stage or histological subtype. Additionally, decreased levels of total SDC1 expression were observed in hOSCC compared with normal controls. In conclusion, this study demonstrates a similarity in the immunohistochemical expression of CDH1 and SDC1 between humans and dogs with OSCC, lending support to the potential use of dogs as a model for studying human head and neck squamous cell carcinoma.

Comparative genomics and genome-wide SNPs of endangered Eld's deer provide breeder selection for inbreeding avoidance.

Eld's deer, a conserved wildlife species of Thailand, is facing inbreeding depression, particularly in the captive Siamese Eld's deer (SED) subspecies. In this study, we constructed genomes of a male SED and a male Burmese Eld's deer (BED), and used genome-wide single nucleotide polymorphisms to evaluate the genetic purity and the inbreeding status of 35 SED and 49 BED with limited pedigree information. The results show that these subspecies diverged approximately 1.26 million years ago. All SED were found to be purebred. A low proportion of admixed SED genetic material was observed in some BED individuals. Six potential breeders from male SED with no genetic relation to any female SED and three purebred male BED with no relation to more than 10 purebred female BED were identified. This study provides valuable insights about Eld's deer populations and appropriate breeder selection in efforts to repopulate this endangered species while avoiding inbreeding.

Tissue transcriptome profiling and pathway analyses revealed novel potential biomarkers in the tumor progression of canine oral melanoma.

Canine oral melanoma (COM) is an aggressive oral malignancy in dogs, mostly with metastasis. However, the understanding of total gene expression of oral melanoma (OM) at different clinical stages has been limited. The objective of this study was to identify novel mRNA biomarkers of early-stage OM (EOM) and late-stage OM (LOM). Transcriptome sequencing of 3 EOM, 5 LOM and 4 normal gingival tissues (controls) was performed. Selected transcriptome results were validated by quantitative reverse transcription-PCR (qRT-PCR) using 12 LOM and 10 controls. We found 534 differentially expressed in EOM compared with controls, whereas 696 genes in LOM were differentially expressed compared with controls (P < 0.05). Moreover, 27 genes were differentially expressed in LOM compared with EOM (P < 0.05). The genes expressed in COM were involved in the molecular mechanism of cancer and melanocyte development pathways, promoting melanoma progression. qRT-PCR confirmed an increased expression of genes encoding an important protein in chemotherapy resistance (dopachrome tautomerase, DCT) and tumor progression (forkhead box M1, FOXM1), and decreased expression of a tumor suppression gene (N-myc downstream-regulated gene 2, NDRG2) in LOM, concordant with transcriptome results. In conclusion, this study revealed the comprehensive transcriptome from COM tissues, and increased DCT and FOXM1 and decreased NDRG2 gene expression indicated the potential candidate biomarkers in COM progression.

Metaproteomic Analysis of Gut Resistome in the Cecal Microbiota of Fattening Pigs Raised without Antibiotics.

Improper use of antibiotics in swine could reduce commensal bacteria and possibly increase pathogen infections via the gut resistome. This study aimed to compare the metaproteomic profiles of the gut resistome and related metabolism in the cecal microbiota of fattening pigs raised under antibiotic-free (ABF) conditions with those of ordinary industrial pigs (controls [CTRL]). The top three relatively abundant microbes in both groups were Escherichia coli, Ruminococcus, and Lactobacillus, followed by Bacteroides and Bifidobacterium. E. coli, Lactobacillus, and Bacteroides were found to be increased in the CTRL group, whereas Ruminococcus and Clostridium were greater in the ABF group. The highest abundances of antibiotic resistance proteins (log2 expression levels [ELs] of >10) were found to be for tetracycline resistance (Tetr) and aminoglycoside resistance (AMGr) proteins found in Bacteroides, with a significant increase in the CTRL group. High Tetr (ELs of 5.32) was found in Ruminococcus in the CTRL group, although pigs in both groups had never received tetracycline, possibly reflecting the influence of environments in farms. In E. coli, AMGr and ß-lactamase family proteins were observed in both groups (ELs of 3 to 6), whereas multidrug resistance protein MdtL was significantly expressed in the CTRL group (ELs of around 3). In the ABF group, CRISPR-associated endonucleases Cas1 and Cas9, which function to defend against viruses, were markedly observed in Ruminococcus and Lactobacillus, respectively, with ELs of 8.6 and 4.15, respectively. In conclusion, this study demonstrated that CRISPR-associated endonucleases were markedly observed in the ABF group, whereas higher levels of Tetr, AMGr, and multidrug resistance protein MdtL was markedly observed in dominant bacterial species in the CTRL group. IMPORTANCE In order to control and reduce antibiotic use in animals, the Department of Livestock Development, Thailand, has launched a campaign for antibiotic-free livestock production. The present study has shown for the first time that CRISPR-associated endonucleases Cas1 and Cas9, which function to defend against viruses, were markedly observed in Ruminococcus and Lactobacillus, respectively, in ceca of pigs raised without antibiotics (ABF). The highest abundances of antibiotic resistance proteins were for tetracycline (Tetr) and aminoglycoside resistance (AMGr) proteins found in Bacteroides, with a significant increase in the controls. In E. coli, the microbe with the highest relative abundance, AMGr and ß-lactamase family proteins were observed in both groups, whereas multidrug resistance protein MdtL was significantly expressed in the controls. Pigs in both ABF and control groups had never received tetracycline, possibly reflecting the influence of farm environments. We suggest that pigs raised without antibiotics may have more beneficial microorganisms for the gut than pigs raised with antibiotics.

Utilizing MALDI-TOF MS and LC-MS/MS to access serum peptidome-based biomarkers in canine oral tumors.

Tumors frequently found in dogs include canine oral tumors, either cancerous or noncancerous. The bloodstream is an important route for tumor metastasis, particularly for late-stage oral melanoma (LOM) and late-stage oral squamous cell carcinoma (LOSCC). The present study aimed to investigate serum peptidome-based biomarkers of dogs with early-stage oral melanoma, LOM, LOSCC, benign oral tumors, chronic periodontitis and healthy controls, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography tandem mass spectrometry. A principal component analysis plot showed distinct clusters among all groups. Four peptides were identified, including peptidyl-prolyl cis-trans isomerase FKBP4 isoform X2 (FKBP4), steroid hormone receptor ERR1 (ESRRA or ERRA), immunoglobulin superfamily member 10 (IGSF10) and ATP-binding cassette subfamily B member 5 (ABCB5). FKBP4, ESRRA and ABCB5 were found to be overexpressed in both LOM and LOSCC, whereas IGSF10 expression was markedly increased in LOSCC only. These four proteins also played a crucial role in numerous pathways of cancer metastasis and showed a strong relationship with chemotherapy drugs. In conclusion, this study showed rapid screening of canine oral tumors using serum and MALDI-TOF MS. In addition, potential serum peptidome-based biomarker candidates for LOM and LOSCC were identified.

Biological Compositions of Canine Amniotic Membrane and Its Extracts and the Investigation of Corneal Wound Healing Efficacy In Vitro.

The usage of canine amniotic membrane (cAM) is mainly of interest in veterinary ophthalmology. Topical formulations of cAM could deliver the beneficial properties of cAM without the need for surgical intervention. The present study aimed to investigate biological compositions of cAM and its extracts, including their corneal wound healing efficacy. In this study, canine amniotic membrane extract (cAME) and lyophilized canine amniotic membrane extract (cAMX) were developed. Bioactive molecules related to corneal wound healing, including hepatocyte growth factor, tissue inhibitor of metalloproteinase-1 and -2, Thrombospondin-1 and Interleukin-1 receptor antagonist were studied at both gene and protein expression levels. Cell viability and wound healing assays were investigated for the possibility of cAME and cAMX as topical applications. The results demonstrated that all of the relevant genes and proteins were detected in cAM, cAME and cAMX. Both cAME and cAMX showed wound healing properties in vitro and cAME at 1.0 mg/mL concentration appeared to have the best healing efficacy. In conclusion, cAME and cAMX generated for topical use provided promising results in the healing of corneal defects.

Dynamics of the fecal microbiome and antimicrobial resistome in commercial piglets during the weaning period.

This study aimed to characterize the alteration of the fecal microbiome and antimicrobial resistance (AMR) determinants in 24 piglets at day 3 pre-weaning (D. - 3), weaning day (D.0), days 3 (D.3) and 8 post-weaning (D.8), using whole-genome shotgun sequencing. Distinct clusters of microbiomes and AMR determinants were observed at D.8 when Prevotella (20.9%) was the major genus, whereas at D. - 3-D.3, Alistipes (6.9-12.7%) and Bacteroides (5.2-8.5%) were the major genera. Lactobacillus and Escherichia were notably observed at D. - 3 (1.2%) and D. - 3-D.3 (0.2-0.4%), respectively. For AMR, a distinct cluster of AMR determinants was observed at D.8, mainly conferring resistance to macrolide-lincosamide-streptogramin (mefA), ß-lactam (cfxA6 and aci1) and phenicol (rlmN). In contrast, at D. - 3-D.3, a high abundance of determinants with aminoglycoside (AMG) (sat, aac(6')-aph(2''), aadA and acrF), ß-lactam (fus-1, cepA and mrdA), multidrug resistance (MDR) (gadW, mdtE, emrA, evgS, tolC and mdtB), phenicol (catB4 and cmlA4), and sulfonamide patterns (sul3) was observed. Canonical correlation analysis (CCA) plot associated Escherichia coli with aac(6')-aph(2''), emrA, mdtB, catB4 and cmlA4 at D. - 3, D.0 and/or D.3 whereas at D.8 associations between Prevotella and mefA, cfxA6 and aci1 were identified. The weaning age and diet factor played an important role in the microbial community composition.

Salivary proteomics in monitoring the therapeutic response of canine oral melanoma.

Saliva biomarkers are suitable for monitoring the therapeutic response of canine oral melanoma (COM), because saliva directly contacts the tumor, and saliva collection is non-invasive, convenient and cost effective. The present study aimed to investigate novel biomarkers from the salivary proteome of COM treated with surgery and a chemotherapy drug, carboplatin, 1-6 times, using a liquid chromatography-tandem mass spectrometry approach. The expression of a potential salivary biomarker, ubiquitin D (UBD), was observed and verified by western blot analysis. A significantly increased ratio of free UBD (fUBD) to conjugated UBD (cUBD) was shown in the pre-surgery stage (PreS) in OM dogs with short-term survival (STS) (less than 12 months after surgery) compared with that with long-term survival (more than 12 months after surgery). In dogs with STS, the ratio was also shown to be augmented in PreS compared with that after surgery, followed by treatment with carboplatin twice, 4 and 5 times [After treatment (AT)2, AT4 and AT5]. In addition, the expression of fUBD was enhanced in PreS compared with that of AT2 in the STS group. In conclusion, this study revealed that a ratio of fUBD to cUBD in PreS was plausibly shown to be a potential prognostic biomarker for survival in dogs with OM.

Salivary Proteomic Analysis of Canine Oral Melanoma by MALDI-TOF Mass Spectrometry and LC-Mass Spectrometry/Mass Spectrometry.

Canine oral melanoma (COM) is a common oral cancer with aggressiveness and high metastasis. A tumor in a dog's mouth makes it difficult to be observed at the early-clinical stage. Salivary biomarkers may be useful for early detection, prognosis, and monitoring of therapies. In addition, salivary collection is a simple and non-invasive technique. The present study describes how to identify salivary biomarkers in COM using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) approaches. Western blot analysis has been used to confirm the protein expression. The sequence of expressed protein from western blot has been verified by LC-MS/MS.

Metagenomics of antimicrobial and heavy metal resistance in the cecal microbiome of fattening pigs raised without antibiotics.

This study aimed to detect the cecal microbiome, antimicrobial resistance (AMR) and heavy metal resistance genes (MRGs) in fattening pigs raised under antibiotic-free (ABF) conditions compared with ordinary industrial pigs (control, C) using whole-genome shotgun sequencing. ABF pigs showed the enrichment of Prevotella (33%) and Lactobacillus (13%), whereas Escherichia coli (40%), Fusobacterium and Bacteroides (each at 4%) were notably observed in the C group. Distinct clusters of cecal microbiota of ABF and C pigs were revealed; however, microbiota of some C pigs (C1) appeared in the same cluster as ABF and were totally separated from the remaining C pigs (C2). For AMR genes, the highest abundance tet(Q) (35.7%) and mef(A) (12.7%) were markedly observed in the ABF group whereas tet(Q) (26.2%) and tet(W) (10.4%) were shown in the C group. tet(Q) was positively correlated to Prevotella in ABF and C1 samples. In the C2 group, the prominent tet(W) was positively correlated to Fusobacterium and Bacteroides Pigs have never received tetracycline but pregnant sows used chlortetracycline once 7 d before parturition. Chromosomal Cu and Zn resistance genes were also shown in both groups regardless the received Cu and Zn feed additives. A higher abundance of multi-metal resistance genes was observed in the C group (44%) compared with the ABF group (41%). In conclusion, the microbiome clusters in some C pigs were similar to that in ABF pigs. High abundant tetracycline resistance genes interrelated to major bacteria were observed in both ABF and C pigs. MRGs were also observed.IMPORTANCE: Owing to the increased problem of AMR in farm animals, raising farm animals without antibiotics is one method that could solve this problem. Our study showed that only some tetracycline and macrolide resistance genes, tet(Q), tet(W) and mef(A), were markedly abundant in ABF and C groups. The tet(Q) and tet(W) genes interrelated to different predominant bacteria in each group, showing the potential role of major bacteria as reservoirs of AMR genes. In addition, chromosomal Cu and Zn resistance genes were also observed in both pig groups, not depending on the use of Cu and Zn additives in both farms. The association of MRGs and AMR genotypes and phenotypes together with the method to re-sensitize bacteria to antibiotics should be studied further to unveil the cause of high resistance genes and solve the problems.

Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches.

BACKGROUND: Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1-2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1-2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography-tandem mass spectrometry. RESULTS: A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1-2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. CONCLUSIONS: The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

In-gel digestion coupled with mass spectrometry (GeLC-MS/MS)-based salivary proteomic profiling of canine oral tumors.

BACKGROUND: Various types of oral tumors, either benign or malignant, are commonly found in dogs. Since saliva directly contacts the tumors and saliva collection is non-invasive, easily accessible and cost effective, salivary biomarkers are practical to be used for the diagnosis and/or prognosis of these diseases. However, there is limited knowledge of protein expression in saliva for canine oral tumors. The present study aimed to investigate novel biomarkers from the salivary proteome of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP), using an in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). The relationships between protein candidates and chemotherapy drugs were explored and the expression of potential biomarkers in saliva and tissues was verified by western blot analysis. RESULTS: For saliva samples, increased expression of protein tyrosine phosphatase non-receptor type 5 (PTPN5) was shown in all tumor groups compared with the CP group. Marked expression of PTPN5 was also observed in LOM and OSCC compared with that in BN and EOM. In addition, tumor protein p53 (p53), which appeared in the PTPN5-drug interactions, was exhibited to be expressed in all tumor groups compared with that in the CP group. For tissue samples, increased expression of p53 was shown in LOM compared with the control group. CONCLUSION: PTPN5 and p53 were proposed to be potential salivary biomarkers of canine oral tumors.

Effect of glycerol concentrations and temperatures on epidermal growth factor protein expression in preserved canine amniotic membrane.

Amniotic membrane has been widely applied as a biological graft in both medical and veterinary practice. In ophthalmology, epidermal growth factor (EGF) in human amniotic membrane (HAM) promotes corneal epithelial cell proliferation and migration, thus it facilitates corneal wound healing. In dogs, with limited cryopreserved HAM availability, different cold glycerol preserving protocols have been developed for the storage canine amniotic membrane (CAM). This study aimed to study protein expression of EGF in CAM preserved with different concentrations of glycerol and storage temperatures, using enzyme-linked immunosorbent assay. CAM preserved in 50% glycerol and 99.5% glycerol and kept at 4 and - 20 °C for 7-30 days were compared. We found that preserving membrane with 50% glycerol at - 20 °C has significantly higher EGF protein expression compared with that at 4 °C (p < 0.05). There was a trend that the storage in 50% glycerol achieved higher EGF protein expression than 99.5% glycerol at both 4 °C and - 20 °C. In conclusion, 50% glycerol at - 20 °C was the best condition to preserve CAM in our study. Therefore, there is likely an alternative method to maintain level of EGF protein expression in preserved CAM.

Salivary proteomics of canine oral tumors using MALDI-TOF mass spectrometry and LC-tandem mass spectrometry.

Canine oral tumors are relatively common neoplasms in dogs. For disease monitoring and early diagnosis, salivary biomarkers are appropriate because saliva collection is non-invasive and requires no professional skills. In the era of omics, matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) coupled with liquid chromatography-tandem MS (LC-MS/MS) are suitable to identify potential disease-associated peptides and proteins. The present study aimed to use MALDI-TOF MS and LC-MS/MS to search for particular peptide mass fingerprints (PMFs) and conceivable biomarkers in saliva of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP). Pooled saliva samples in each group were used to be representative of population change. Unique PMFs were obtained and specific peptide fragments were sequenced by LC-MS/MS and BLAST-searched with mammalian protein databases. Seven peptide fragments appeared in the tumor groups (EOM, LOM, OSCC and BN) at 1096, 1208, 1322, 1794, 1864, 2354 and 2483 Da, two peptide fragments appeared in the LOM and OSCC groups at 2450 and 3492 Da, and in the CP controls at 2544 and 3026 Da. Also, protein-chemotherapy drug interaction networks were exhibited. Using western blot analysis, the expression of sentrin-specific protease 7 (SENP7), a peptide fragment at 1096 Da, in OSCC was significantly increased, as was the expression of TLR4, a peptide fragment at 3492 Da, in LOM and OSCC, compared with the CP group. The expression of nuclear factor kappa B (NF-κB), a TLR4 partner, was notably increased in OSCC compared with CP, BN and EOM. The expression was also enhanced in LOM compared with EOM. Expressed protein sequences from western blots were verified by LC-MS/MS. Western blots were then performed with individual samples in each group. The results showed the elevated expression of TLR4 in LOM and OSCC, compared with that in CP and BN, the increased expression of NF-κB in LOM and OSCC, compared with CP and in LOM compared with BN, and the enhanced expression of SENP7 in LOM and OSCC, compared with that in CP and BN. In conclusion, discrete clusters of EOM, LOM, OSCC, BN and CP groups and potential protein candidates associated with the diseases were demonstrated by salivary proteomics. Western blot analysis verified SENP7, TLR4 and NF-κB as potential salivary biomarkers of canine oral tumors.

Alteration of androgen receptor expression, apoptosis and cell proliferation in cryptorchid suckling, nursery and growing-finishing pigs.

Cryptorchidism, a condition of one or two undescended testicles, is a common congenital disease in pigs, causing loss in the pig industry. One of the major factors affecting testicular descent is the androgen receptor (AR), which binds to androgen and then regulates the expression of androgen-responsive genes in the inguinoscrotal phase of testicular descent. AR expression has been reported to regulate apoptosis in testicular stem cells. The present study aimed to immunohistochemically examine AR and Ki-67 protein expression and apoptosis detection in unilateral undescended testicles (UDT) and descended testicles in cryptorchid pigs (DT) of suckling (aged 1-2 weeks), nursery (aged 6 weeks) and growing-finishing pigs (aged 12, 15 and 20 weeks) and in normal testicles (NT) at 1-2 and 12 weeks of age. At 1-2 weeks, decreased expression of AR was observed in UDT and DT compared with NT and was lower than that at 6-20 weeks. The expression of Ki-67, a marker of cell proliferation, in UDT and DT at 12 weeks was lower than that in NT at the same age. In addition, Ki-67 expression in UDT at 6 and 12 weeks was lower than that in UDT at 1-2 and 15-20 weeks. More testicular apoptosis was revealed in UDT at 1-2 weeks than in DT and NT at the same age. At 15-20 weeks, more apoptosis was detected in UDT than in DT. Positive correlation of AR expression in DT at 6 and 12 weeks was also noted, in addition to the association of the expression of AR and Ki-67 in NT at 12 weeks. Taken together, this study unveiled the low expression of AR and high apoptosis detection in UDT, whereas low expression of AR and low apoptosis detection were noted in DT in suckling piglets. Diminished cell proliferation was shown in UDT at 6-12 weeks, whereas high apoptosis was observed in UDT at 15-20 weeks. High expression of AR was shown only in nursery pigs. Distinct expression of AR in DT and NT at 1-2 and 12 weeks indicated that both conditions were not interchangeable.

Proteomic analysis of canine oral tumor tissues using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC MS/MS) approaches.

Oral tumors, including highly invasive and metastatic oral melanoma (OM), non-tonsillar oral squamous cell carcinoma (OSCC) and benign tumors (BN), are common neoplasms in dogs. Although these tumors behave differently, limited data of their protein expression profiles have been exhibited, particularly at the proteome level. The present study aimed to i.) characterize peptide-mass fingerprints (PMFs) and identify potential protein candidates of OM, OSCC, BN and normal control subjects, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS), ii.) identify potential protein candidates associated with the diseases, using in-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS) and iii.) search for relationships between chemotherapy drugs and disease-perturbed proteins. A distinct cluster of each sample group and unique PMFs with identified protein candidates were revealed. The unique peptide fragment at 2,274 Da of sacsin molecular chaperone (SACS) was observed in early-stage OM whereas the fragment at 1,958 Da of sodium voltage-gated channel alpha subunit 10 (SCN10A) was presented in early- and late-stage OM. The peptide mass at 2,316 Da of Notch1 appeared in early-stage OM and benign oral tumors while the peptide mass at 2,505 Da of glutamate ionotropic receptor N-methyl-D-aspartate type subunit 3A (GRIN3A) was identified in all groups. Markedly expressed proteins from GeLC-MS/MS included Jumonji domain containing 1C (JMJD1C) in benign tumors, inversin (INVS) and rho guanine nucleotide exchange factor 28 (ARHGEF28) in OM, BTB domain-containing 16 (BTBD16) in OSCC, and protein tyrosine phosphatase non-receptor type 1 (PTPN1), BRCA2, DNA repair associated (BRCA2), WW domain binding protein 2 (WBP2), purinergic receptor P2Y1 and proteasome activator subunit 4 (PSME4) in all cancerous groups. The network connections between these proteins and chemotherapy drugs, cisplatin and doxorubicin, were also demonstrated. In conclusion, this study unveiled the unique PMFs and novel candidate protein markers of canine oral tumors.

Gene expression profiles of cell adhesion molecules, matrix metalloproteinases and their tissue inhibitors in canine oral tumors.

Perturbation of cell adhesion can be essential for tumor cell invasion and metastasis, but the current knowledge on the gene expression of molecules that mediate cell adhesion in canine oral tumors is limited. The present study aimed to investigate changes in the gene expression of cell adhesion molecules (E-cadherin or CDH1, syndecan 1 or SDC1, NECTIN2 and NECTIN4), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), in canine oral tumors, including benign tumors, oral melanoma (OM) and non-tonsillar oral squamous cell carcinoma (OSCC), by quantitative real-time reverse transcription PCR. When compared with the normal gingival controls, decreased CDH1, SDC1 and NECTIN4 expression levels were observed in OSCC and OM, reflecting a possible role as cell adhesion molecules and tumor suppressors in canine oral cancers in contrast to the upregulation of MMP2 expression. Downregulated MMP7 was specifically revealed in the OM group. In the late-stage OM, the positive correlation of MMP7 and CDH1 expression was noticed as well as that of SDC1 and NECTIN4. Enhanced TIMP1 expression was shown in all tumor groups with prominent expression in the benign tumors and the early-stage OM. MMP14 expression was notable in the early-stage OM. Higher MMP9 and TIMP1 expression was observed in the acanthomatous ameloblastoma. In conclusion, this study revealed that the altered expression of cell adhesion molecules, MMP7 and MMP2 was correlated with clinicopathologic features in canine oral cancers whereas TIMP1 and MMP14 expression was probably associated with early-stage tumors; therefore, these genes might serve as molecular markers for canine oral tumors.

Alterations of keratins, involucrin and filaggrin gene expression in canine atopic dermatitis.

Canine atopic dermatitis (CAD) is a common allergic skin disease in dogs, associated with a defective epidermal barrier. In this study we investigated the alterations in skin keratinocyte proliferation and differentiation in CAD by quantitative reverse transcription-polymerase chain reaction. Gene expression of keratin (KRT) markers of proliferative and differentiated keratinocytes, together with that of cornified envelope proteins, involucrin (IVL) and filaggrin (FLG), were evaluated. An upregulation of KRT5 and KRT17 in both lesional and non-lesional AD skin was observed (p<0.05) whereas KRT2e, KRT14, IVL and FLG expression were significantly increased only in lesional AD skin (p<0.05). Additionally, the expression levels of KRT5, KRT14, KRT17 and IVL in CAD were strongly correlated. In conclusion, the expression of the majority of the studied keratins, as well as IVL and FLG is increased in CAD with close correlation between the proliferative keratins. This is the first report of a correlation of KRT and IVL genes with CAD.