Proteomic insights into the regulatory function of ARID1A in colon cancer cells.

The AT-rich interacting domain-containing protein 1A (ARID1A) is a tumor suppressor gene that has been implicated in several cancers, including colorectal cancer (CRC). The present study used a proteomic approach to elucidate the molecular mechanisms of ARID1A in CRC carcinogenesis. Stable ARID1A-overexpressing SW48 colon cancer cells were established using lentivirus transduction and the successful overexpression of ARID1A was confirmed by western blotting. Label-free quantitative proteomic analysis using liquid chromatography-tandem mass spectrometry identified 705 differentially altered proteins in the ARID1A-overexpressing cells, with 310 proteins significantly increased and 395 significantly decreased compared with empty vector control cells. Gene Ontology enrichment analysis highlighted the involvement of the altered proteins mainly in the Wnt signaling pathway. Western blotting supported these findings, as a decreased protein expression of Wnt target genes, including c-Myc, transcription factor T cell factor-1/7 and cyclin D1, were observed in ARID1A-overexpressing cells. Among the altered proteins involved in the Wnt signaling pathway, the interaction network analysis revealed that ARID1A exhibited a direct interaction with E3 ubiquitin-protein ligase zinc and ring finger 3 (ZNRF3), a negative regulator of the Wnt signaling pathway. Further analyses using the The Cancer Genome Atlas colon adenocarcinoma public dataset revealed that ZNRF3 expression significantly impacted the overall survival of patients with CRC and was positively correlated with ARID1A expression. Finally, an increased level of ZNRF3 in ARID1A-overexpressing cells was confirmed by western blotting. In conclusion, the findings of the present study suggest that ARID1A negatively regulates the Wnt signaling pathway through ZNRF3, which may contribute to CRC carcinogenesis.

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.

Menthol induces extracellular vesicle regulation of apoptosis via ATG3 and caspase-3 in acute leukemic cells.

Leukemia is one of the most deadly cancers in Thailand. Natural compounds have been developed for cancer treatment. Menthol, a peppermint compound, has shown pharmacological properties such as anti-cancer activity. However, the mechanism of menthol inducing extracellular vesicles in leukemic cells is not yet understood. In this study, we investigated the effects of menthol on leukemic extracellular vesicles and their role in apoptosis. NB4 and Molt-4 leukemic cells were cultured with menthol in various concentrations and times. Bioinformatic analysis was used to investigate target proteins of extracellular vesicle and apoptosis, followed by mRNA and protein expression by RT‒PCR and western blotting, respectively. Our findings indicate that menthol inhibits leukemic cell proliferation and increases extracellular vesicles. Furthermore, menthol treated leukemic extracellular vesicles induce apoptosis and upregulate the expression of ATG3 and caspase-3 in both mRNA and protein levels. These results suggest that menthol has an antileukemic effect through ATG3 and caspase-3 in apoptosis of leukemic extracellular vesicles.

Proteomics profile in encapsulated follicular patterned thyroid neoplasms.

Diagnosing encapsulated follicular-patterned thyroid tumors like Invasive Encapsulated Follicular Variant of Papillary Thyroid Carcinoma (IEFVPTC), Non-invasive Follicular Thyroid Neoplasm with Papillary-like Nuclear Features (NIFTP), and Well-Differentiated Tumor of Uncertain Malignant Potential (WDT-UMP) remains challenging due to their morphological and molecular similarities. This study aimed to investigate the protein distinctions among these three thyroid tumors and discover biological tumorigenesis through proteomic analysis. We employed total shotgun proteome analysis allowing to discover the quantitative expression of over 1398 proteins from 12 normal thyroid tissues, 13 IEFVPTC, 11 NIFTP, and 10 WDT-UMP. Principal component analysis revealed a distinct separation of IEFVPTC and normal tissue samples, distinguishing them from the low-risk tumor group (NIFTP and WDT-UMP). IEFVPTC exhibited the highest number of differentially expressed proteins (DEPs) compared to the other tumors. No discriminatory proteins between NIFTP and WDT-UMP were identified. Moreover, DEPs in IEFVPTC were significantly associated with thyroid tumor progression pathways. Certain hub genes linked to the response of immune checkpoint inhibitor therapy, revealing the potential predictor of prognosis. In conclusion, the proteomic profile of IEFVPTC differs from that of low-risk tumors. These findings may provide valuable insights into tumor biology and offer a basis for developing novel therapeutic strategies for follicular-patterned thyroid neoplasms.

Deciphering the Intracellular Action of the Antimicrobial Peptide A11 via an In-Depth Analysis of Its Effect on the Global Proteome of Acinetobacter baumannii.

The potential antimicrobial activity and low propensity to induce the development of bacterial resistance have rendered antimicrobial peptides (AMPs) as novel and ideal candidate therapeutic agents for the treatment of infections caused by drug-resistant pathogenic bacteria. The targeting of bacterial membranes by AMPs has been typically considered their sole mode of action; however, increasing evidence supports the existence of multiple and complementary functions of AMPs that result in bacterial death. An in-depth characterization of their mechanism of action could facilitate further research and development of AMPs with higher potency. The current study employs biophysics and proteomics approaches to unveil the mechanisms underlying the antibacterial activity of A11, a potential candidate AMP, against Acinetobacter baumannii, a leading cause of hospital-acquired infections (HAIs) and consequently, a serious global threat. A11 peptide was found to induce membrane depolarization to a high extent, as revealed by flow cytometry and electron microscopy analyses. The prompt intracellular penetration of A11 peptide, observed using confocal microscopy, was found to occur concomitantly with a very low degree of membrane lysis, suggesting that its mode of action predominantly involves a nonlytic killing mechanism. Quantitative proteomics analysis employed for obtaining insights into the mechanisms underlying the antimicrobial activity of A11 peptide revealed that it disrupted energy metabolism, interfered with protein homeostasis, and inhibited fatty acid synthesis that is essential for cell membrane integrity; all these impacted the cellular functions of A. baumannii. A11 treatment also impacted signal transduction associated with the regulation of biofilm formation, hindered the stress response, and influenced DNA repair processes; these are all crucial survival mechanisms of A. baumannii. Additionally, robust antibacterial activity was exhibited by A11 peptide against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of A. baumannii; moreover, A11 peptide exhibited synergy with levofloxacin and minocycline as well as low propensity for inducing resistance. Taken together, the findings emphasize the therapeutic potential of A11 peptide as an antibacterial agent against drug-resistant A. baumannii and underscore the need for further investigation.

Comparison of proteomic profiles and biological properties for corneal wound healing of canine amniotic membrane and its extracts.

OBJECTIVE: The application of canine amniotic membrane (cAM) for corneal reconstruction is widely used in the veterinary field. However, the information on biological properties and alternative forms of cAM for corneal wound healing is limited. This study aimed to investigate the proteomic profiles and corneal wound healing properties of cAM, cAM extract (cAME), and lyophilized cAM extract (cAMX). ANIMAL STUDIED: A total number of 14 cAMs were sterilely harvested from healthy full-term puppies and randomly divided into three different forms: cAM (n = 14), cAME (n = 14), and cAMX (n = 14). PROCEDURES: Each form of cAMs was subjected to proteomic analysis using label-free liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), followed by bioinformatic analysis. The proteins were classified into properties by comparing them with the literature search on human amniotic membrane (hAM) properties and the effect on corneal wound healing when given topically. RESULTS: The analyses identified 8136 proteins in cAM, 8211 proteins in cAME, and 7093 proteins in cAMX. A total number of 100 proteins were matched with proteins in hAM properties and were classified into anti-inflammatory, anti-fibrotic, anti-microbial, anti-angiogenic, promotion of epithelialization, analgesic, and support cell adhesion and growth properties. Furthermore, proteins with corneal wound healing effects were identified in cAME and cAMX. CONCLUSIONS: cAM and its extracts contain numerous proteins, including proteins related to corneal wound healing properties. Additionally, cAME and cAMX showed proteins involved in corneal wound healing and their potential benefits for topical use in ophthalmology.

Egg white hydrolysate peptides act as antimicrobial and anti-inflammatory agents for acne.

A simple method to generate antibacterial peptides by alkaline hydrolysis of hen egg whites is reported. The method reproducibly generates short peptides with molecular weight of less than 14.4 kDa that exhibit low to no cytotoxicity on RAW 264.7 macrophage cells, but do inhibit the bacterial growth of Cutibacterium acnes (C. acnes), Staphylococcus aureus (S. aureus) and antibiotic-resistant S. aureus (MRSA), while also reducing nitric oxide production from heat-killed C. acnes-treated RAW 264.7 cells. Peptidomics revealed at least thirty peptides within the complex mixture, of which eight were evaluated individually. Three peptides (PK8, EE9 and RP8) were potent anti-inflammation and antibacterial agents, but notably the complex egg white hydrolysate (EWH) was more effective than the individual peptides. Electron microscopy suggests the antibacterial mechanism of both the hydrolysate and the selected peptides is through disruption of the cell membrane of C. acnes. These findings suggest that EWH and EWH-derived peptides are promising candidates for infection and inflammation treatment, particularly in managing acne and combating antibiotic-resistant bacteria like MRSA.

Investigation of coagulation and proteomics profiles in symptomatic feline hypertrophic cardiomyopathy and healthy control cats.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a crucial heart disease in cats. The clinical manifestations of HCM comprise pulmonary edema, dyspnea, syncope, arterial thromboembolism (ATE), and sudden cardiac death. D-dimer and prothrombin time (PT) are powerful biomarkers used to assess coagulation function. Dysregulation in these two biomarkers may be associated with HCM in cats. This study aims to assess D-dimer levels, PT, and proteomic profiling in healthy cats in comparison to cats with symptomatic HCM. RESULTS: Twenty-nine client-owned cats with HCM were enrolled, including 15 healthy control and 14 symptomatic HCM cats. The D-dimer concentration and PT were examined. Proteomic analysis was conducted by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In symptomatic cats, D-dimer levels were statistically significantly higher (mean ± SEM: 372.19 ng/ml ± 58.28) than in healthy cats (mean ± SEM: 208.54 ng/ml ± 10.92) with P-value of less than 0.01, while PT was statistically significantly lower in symptomatic cats (mean ± SEM: 9.8 s ± 0.15) compared to healthy cats (mean ± SEM: 11.08 s ± 0.23) with P-value of less than 0.0001. The proteomics analysis revealed upregulation of integrin subunit alpha M (ITGAM), elongin B (ELOB), and fibrillin 2 (FBN2) and downregulation of zinc finger protein 316 (ZNF316) and ectonucleoside triphosphate diphosphohydrolase 8 (ENTPD8) in symptomatic HCM cats. In addition, protein-drug interaction analysis identified the Ras signaling pathway and PI3K-Akt signaling pathway. CONCLUSIONS: Cats with symptomatic HCM have higher D-dimer and lower PT than healthy cats. Proteomic profiles may be used as potential biomarkers for the detection and management of HCM in cats. The use of D-dimer as a biomarker for HCM detection and the use of proteomic profiling for a better understanding of disease mechanisms remain to be further studied in cats.

Secretomic changes of amyloid beta peptides on Alzheimer's disease related proteins in differentiated human SH-SY5Y neuroblastoma cells.

Alzheimer's disease (AD) is a neurodegenerative disease that causes physical damage to neuronal connections, leading to brain atrophy. This disruption of synaptic connections results in mild to severe cognitive impairments. Unfortunately, no effective treatment is currently known to prevent or reverse the symptoms of AD. The aim of this study was to investigate the effects of three synthetic peptides, i.e., KLVFF, RGKLVFFGR and RIIGL, on an AD in vitro model represented by differentiated SH-SY5Y neuroblastoma cells exposed to retinoic acid (RA) and brain-derived neurotrophic factor (BDNF). The results demonstrated that RIIGL peptide had the least significant cytotoxic activity to normal SH-SY5Y while exerting high cytotoxicity against the differentiated cells. The mechanism of RIIGL peptide in the differentiated SH-SY5Y was investigated based on changes in secretory proteins compared to another two peptides. A total of 380 proteins were identified, and five of them were significantly detected after treatment with RIIGL peptide. These secretory proteins were found to be related to microtubule-associated protein tau (MAPT) and amyloid-beta precursor protein (APP). RIIGL peptide acts on differentiated SH-SY5Y by regulating amyloid-beta formation, neuron apoptotic process, ceramide catabolic process, and oxidative phosphorylation and thus has the potentials to treat AD.

Quantitative proteomics analysis reveals possible anticancer mechanisms of 5'-deoxy-5'-methylthioadenosine in cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is an aggressive cancer originating from bile duct epithelium, particularly prevalent in Asian countries with liver fluke infections. Current chemotherapy for CCA often fails due to drug resistance, necessitating novel anticancer agents. This study investigates the potential of 5'-deoxy-5'-methylthioadenosine (MTA), a naturally occurring nucleoside, against CCA. While MTA has shown promise against various cancers, its effects on CCA remain unexplored. We evaluated MTA's anticancer activity in CCA cell lines and drug-resistant sub-lines, assessing cell viability, migration, invasion, and apoptosis. The potential anticancer mechanisms of MTA were explored through proteomic analysis using LC-MS/MS and bioinformatic analysis. The results show a dose-dependent reduction in CCA cell viability, with enhanced effects on cancer cells compared to normal cells. Moreover, MTA inhibits growth, induces apoptosis, and suppresses cell migration and invasion. Additionally, MTA enhanced the anticancer effects of gemcitabine on drug-resistant CCA cells. Proteomics revealed the down-regulation of multiple proteins by MTA, affecting various molecular functions, biological processes, and cellular components. Network analysis highlighted MTA's role in inhibiting proteins related to mitochondrial function and energy derivation, crucial for cell growth and survival. Additionally, MTA suppressed proteins involved in cell morphology and cytoskeleton organization, important for cancer cell motility and metastasis. Six candidate genes, including ZNF860, KLC1, GRAMD1C, MAMSTR, TANC1, and TTC13, were selected from the top 10 most down-regulated proteins identified in the proteomics results and were subsequently verified through RT-qPCR. Further, KLC1 protein suppression by MTA treatment was confirmed through Western blotting. Additionally, based on TCGA data, KLC1 mRNA was found to be upregulated in the tissue of CCA patients compared to that of normal adjacent tissues. In summary, MTA shows promising anticancer potential against CCA by inhibiting growth, inducing apoptosis, and suppressing migration and invasion, while enhancing gemcitabine's effects. Proteomic analysis elucidates possible molecular mechanisms underlying MTA's anticancer activity, laying the groundwork for future research and development of MTA as a treatment for advanced CCA.

Proteomic Analysis of Butyrate-Resistant Colorectal Cancer-Derived Exosomes Reveals Potential Resistance to Anti-Cancer Drugs.

BACKGROUND: Butyrate-resistant (BR) cells play an important role in acquiring chemoresistance in colorectal cancer (CRC). Our previous study demonstrated that BR CRC cells showed cross-resistance to chemotherapy drugs, including 5-fluorouracil and oxaliplatin, in both monolayer and spheroid cultures. The mechanisms underlying drug resistance were also elucidated. However, the link between parental (PT) and BR cells remains unclear. Extracellular vesicles (EVs) are key cell-cell communications that transport various molecules, including DNA, RNA, and proteins, between the donor and target cells. EVs contribute to drug resistance in cancers, such as melanoma and lung cancer. Recently, we focused on the correlation of proteomic profiles of EVs from different cell types. METHODS: In this study, we analyzed the proteomic profiles of EVs derived from PT and BR cells to investigate the mechanisms underlying the butyrate- and chemo-resistant phenotypes. EVs were isolated from PT and BR cells using ultracentrifugation. The characteristics of the EVs were evaluated using western blot and transmission electron microscopy. The EV proteomic data were further analyzed using liquid chromatography-mass spectrometry. RESULTS: We identified a unique protein expressed in BR cells related to the chemoresistant phenotype. Functional enrichment analysis showed that BR cells had higher protein catalytic activity, binding, and transcription activity. The STITCH database showed a greater correlation between protein-drug interactions in BR cells than in PT cells. Moreover, our findings support the hypothesis that EVs promote tumor progression and metastasis and affect the tumor microenvironment. CONCLUSIONS: Proteomic analysis of EVs from BR CRC cells reveals insights into drug resistance mechanisms, including protein-mediated carcinogenesis and reduced drug uptake, offering potential strategies to overcome resistance in clinical practice.

Secretome profiling of human epithelial cells exposed to cigarette smoke extract and their effect on human lung microvascular endothelial cells.

Cigarette smoke (CS) is one of the leading causes of pulmonary diseases and can induce lung secretome alteration. CS exposure-induced damages to human pulmonary epithelial cells and microvascular endothelial cells have been extensively demonstrated; however, the effects of the secretome of lung epithelial cells exposed to CS extracts (CSE) on lung microvascular endothelial cells are not fully understood. In this study, we aimed to determine the effects of the secretome of lung epithelial cells exposed to CSE on lung microvascular endothelial cells. Human lung epithelial cells, A549, were exposed to CSE, and the secretome was collected. Human lung microvascular endothelial cells, HULEC-5a, were used to evaluate the effect of the secretome of A549 exposed to CSE. Secretome profile, endothelial cell death, inflammation, and permeability markers were determined. CSE altered the secretome expression of A549 cells, and secretome derived from CSE-exposed A549 cells caused respiratory endothelial cell death, inflammation, and moderately enhanced endothelial permeability. This study demonstrates the potential role of cellular interaction between endothelial and epithelial cells during exposure to CSE and provides novel therapeutic targets or beneficial biomarkers using secretome analysis for CSE-related respiratory diseases.

Novel Serum Proteomes Expressed from Benzene Exposure Among Gasoline Station Attendants.

Background: Research on the proteomes impact of benzene exposure in fuel station employees remains sparse, underscoring the need for detailed health impact assessments focusing on biomarker evaluation. Objectives: This investigation aimed to analyze the differences in blood parameters and serum proteomes resulting from benzene exposure between gasoline station attendants (B-GSA) and a control group. Design and methods: A cross-sectional analytical study was conducted with 96 participants, comprising 54 in the B-GSA group and 42 in the control group. The methodology employed included an interview questionnaire alongside urine and blood sample collections. The urine samples were analyzed for trans,trans-muconic acid (t,t-MA) levels, while the blood samples underwent complete blood count analysis and proteome profiling. Results: Post-shift analysis indicated that the B-GSA group exhibited significantly higher levels of t,t-MA and monocytes compared to the control group (P < .05). Proteome quantification identified 1448 proteins differentially expressed between the B-GSA and control groups. Among these, 20 proteins correlated with the levels of t,t-MA in urine. Notably, 4 proteins demonstrated more than a 2-fold down-regulation in the B-GSA group: HBS1-like, non-structural maintenance of chromosomes element 1 homolog, proprotein convertase subtilisin/kexin type 4, and zinc finger protein 658. The KEGG pathway analysis revealed associations with apoptosis, cancer pathways, p53 signaling, and the TNF signaling pathway. Conclusion: The changes in these 4 significant proteins may elucidate the molecular mechanisms underlying benzene toxicity and suggest their potential as biomarkers for benzene poisoning in future assessments.

Analysis of serum proteomic profiles of endangered Siamese and Burmese Eld's deer infected with subclinical Babesia bovis in Thailand.

The endangered Eld's deer is a conserved species in Thailand, where tropical parasitic infections are endemic. Although Eld's deer with babesiosis are generally asymptomatic, they can still harbor the parasite and serve as reservoirs for ticks, spreading the infection to healthy animals within the herd. The present study aimed to investigate potential serum proteome biomarkers of Eld's deer with subclinical Babesia bovis infection. A total of 67 blood samples were collected from captive Siamese and Burmese Eld's deer showing no signs of parasitic infection. The nested polymerase chain reaction (nPCR) of a conserved spherical body protein 2 (sbp-2) gene of B. bovis was utilized to classify Eld's deer groups, with 25.37 % (17/67) testing positive for B. bovis. Additionally, the application of proteomic studies showed that six B. bovis proteins, such as Obg-like ATPase 1 (OLA1) and heat shock protein 90 (HSP90), were significantly upregulated by more than a two-fold change compared with the PCR-negative samples. Of the 55 overexpressed serum proteins in the PCR-positives, alpha 2-HS glycoprotein (AHSG) and immunoglobulin lambda variable 2-8 (IGLV2-8) were notably among the top 10 proteins with the highest area under curve (AUC) values. Hence, they were proposed as potential biomarkers for subclinical B. bovis infection in Eld's deer. Analysis of the protein interaction network revealed interactions between Eld's deer AHSG and B. bovis OLA1 and HSP90, alongside associations with other proteins such as erb-b2 receptor tyrosine kinase 2 (ERBB2) and epidermal growth factor receptor (EGFR). These interactions were involved in the immune system pathway and inflammatory responses. Our findings shed light on subclinical infection of B. bovis in Eld's deer and identify potential biomarkers, contributing to the further effective detection and monitoring of B. bovis infection in this endangered species.

Antibacterial Activities of Phenolic Compounds in Miang Extract: Growth Inhibition and Change in Protein Expression of Extensively Drug-Resistant Klebsiella pneumoniae.

The rising incidence of extensively drug-resistant (XDR) Klebsiella pneumoniae, including carbapenem- and colistin-resistant strains, leads to the limitation of available effective antibiotics. Miang, known as chewing tea, is produced from Camellia sinensis var. assamica or Assam tea leaves fermentation. Previous studies revealed that the extract of Miang contains various phenolic and flavonoid compounds with numerous biological activities including antibacterial activity. However, the antibacterial activity of Miang against XDR bacteria especially colistin-resistant strains had not been investigated. In this study, the compositions of phenolic and flavonoid compounds in fresh, steamed, and fermented Assam tea leaves were examined by HPLC, and their antibacterial activities were evaluated by the determination of the MIC and MBC. Pyrogallol was detected only in the extract from Miang and showed the highest activities with an MIC of 0.25 mg/mL and an MBC of 0.25-0.5 mg/mL against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli ATCC 25922, colistin-resistant E. coli, and colistin-resistant K. pneumoniae. The effects on morphology and proteomic changes in K. pneumoniae NH54 treated with Miang extract were characterized by SEM and label-free quantitative shotgun proteomics analysis. The results revealed that Miang extract caused the decrease in bacterial cell wall integrity and cell lysis. The up- and downregulated expression with approximately a 2 to >5-fold change in proteins involved in peptidoglycan synthesis and outer membrane, carbohydrate, and amino acid metabolism were identified. These findings suggested that Miang containing pyrogallol and other secondary metabolites from fermentation has potential as an alternative candidate with an antibacterial agent or natural active pharmaceutical ingredient against XDR bacteria including colistin-resistant bacteria.

Comparative proteomic analysis of dental pulp from supernumerary and normal permanent teeth.

OBJECTIVES: To obtain and compare the protein profiles of supernumerary and normal permanent dental pulp tissues. MATERIALS AND METHODS: Dental pulp tissues were obtained from supernumerary and normal permanent teeth. Proteins were extracted and analyzed by liquid chromatography-tandem mass spectrometry (LC/MS-MS). Protein identification and quantification from MS data was performed with MaxQuant. Statistical analysis was conducted using Metaboanalyst to identify differentially expressed proteins (DEPs) (P-value < 0.05, fold-change > 2). Gene Ontology enrichment analyses were performed with gProfiler. RESULTS: A total of 3,534 proteins were found in normal dental pulp tissue and 1,093 in supernumerary dental pulp tissue, with 174 DEPs between the two groups. This analysis revealed similar functional characteristics in terms of cellular component organization, cell differentiation, developmental process, and response to stimulus, alongside exclusive functions unique to normal permanent dental pulp tissues such as healing, vascular development and cell death. Upon examination of DEPs, these proteins were associated with the processes of wound healing and apoptosis. CONCLUSIONS: This study provides a comprehensive understanding of the protein profile of dental pulp tissue, including the first such profiling of supernumerary permanent dental pulp. There are functional differences between the proteomic profiles of supernumerary and normal permanent dental pulp tissue, despite certain biological similarities between the two groups. Differences in protein expression were identified, and the identified DEPs were linked to the healing and apoptosis processes. CLINICAL RELEVANCE: This discovery enhances our knowledge of supernumerary and normal permanent pulp tissue, and serves as a valuable reference for future studies on supernumerary teeth.

Phosphoproteomics analysis of serum from dogs affected with pulmonary hypertension secondary to degenerative mitral valve disease.

Pulmonary hypertension (PH), a common complication in dogs affected by degenerative mitral valve disease (DMVD), is a progressive disorder characterized by increased pulmonary arterial pressure (PAP) and pulmonary vascular remodeling. Phosphorylation of proteins, impacting vascular function and cell proliferation, might play a role in the development and progression of PH. Unlike gene or protein studies, phosphoproteomic focuses on active proteins that function as end-target proteins within signaling cascades. Studying phosphorylated proteins can reveal active contributors to PH development. Early diagnosis of PH is crucial for effective management and improved clinical outcomes. This study aimed to identify potential serum biomarkers for diagnosing PH in dogs affected with DMVD using a phosphoproteomic approach. Serum samples were collected from healthy control dogs (n = 28), dogs with DMVD (n = 24), and dogs with DMVD and PH (n = 29). Phosphoproteins were enriched from the serum samples and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data analysis was performed to identify uniquely expressed phosphoproteins in each group and differentially expressed phosphoproteins among groups. Phosphoproteomic analysis revealed nine uniquely expressed phosphoproteins in the serum of dogs in the DMVD+PH group and 15 differentially upregulated phosphoproteins in the DMVD+PH group compared to the DMVD group. The phosphoproteins previously implicated in PH and associated with pulmonary arterial remodeling, including small nuclear ribonucleoprotein G (SNRPG), alpha-2-macroglobulin (A2M), zinc finger and BTB domain containing 42 (ZBTB42), hemopexin (HPX), serotransferrin (TRF) and complement C3 (C3), were focused on. Their unique expression and differential upregulation in the serum of DMVD dogs with PH suggest their potential as biomarkers for PH diagnosis. In conclusion, this phosphoproteomic study identified uniquely expressed and differentially upregulated phosphoproteins in the serum of DMVD dogs with PH. Further studies are warranted to validate the diagnostic utility of these phosphoproteins.

Dual-function antimicrobial-antibiofilm peptide hybrid to tackle biofilm-forming Staphylococcus epidermidis.

BACKGROUND: Due to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication. METHODS: A novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area. RESULTS: BiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area. CONCLUSIONS: Due to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis.

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.

Proteomic analysis of the serum in dogs with pulmonary hypertension secondary to myxomatous mitral valve disease: the preliminary study.

Background: Pulmonary hypertension (PH) is a common complication in dogs with myxomatous mitral valve disease (MMVD), characterized by elevated blood pressure in pulmonary artery. Echocardiography is a reliable technique for PH diagnosis in veterinary medicine. However, it is limited to use as an early detection method. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has found extensive application in the discovery of serum protein biomarkers for various diseases. The objective of this study was to identify serum proteins in healthy control dogs and MMVD dogs both with and without PH using LC-MS/MS. Materials and methods: In this research, a total of 81 small-breed dogs participated, and they were categorized into three groups: the control (n = 28), MMVD (n = 24) and MMVD+PH (n = 29) groups. Serum samples were collected and analyzed by LC-MS/MS. Results: Differentially expressed proteins were identified, and the upregulated and downregulated proteins in MMVD+PH group including Myomesin 1 (MYOM1) and Histone deacetylase 7 (HDAC7), Pleckstrin homology domain containing M3 (PLEKHM3), Diacylglycerol lipase alpha (DAGLA) and Tubulin tyrosine ligase like 6 (TTLL6) were selected as proteins of interest in MMVD dogs with PH. Conclusion: Different types of proteins have been identified in healthy dogs and MMVD dogs with and without PH. Additional studies are needed to investigate the potential of these proteins as biomarkers for PH in dogs with MMVD.