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.

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.

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.

Feline infectious peritonitis: A comprehensive evaluation of clinical manifestations, laboratory diagnosis, and therapeutic approaches.

Objective: This study aimed to investigate the clinical and laboratory characteristics of naturally occurring feline infectious peritonitis (FIP) and estimate the median survival time of FIP cats treated with prednisolone to guide further therapeutic planning. Materials and Methods: In this retrospective study, data from a total of 116 cats with effusion were fully recorded. Forty-five FIP-diagnosed cats were enrolled for analysis. Results: The study findings indicate that FIP was a disease affecting cats aged 1-2 years and was highly prevalent among male cats. Clinical manifestations of FIP affected the digestive (60%), hematological (53.3%), respiratory (33.3%), neurological (6.7%), and ocular (4.4%) systems. Blood profiles revealed mild anemia, lymphopenia, thrombocytopenia, hypoalbuminemia, hyperglobulinemia, and an albumin to globulin ratio of 0.4. Fluid analysis and cytology of FIP cats demonstrated a transparent yellow fluid with a protein content of 6 gm/dl and a total nucleated cell count of approximately 5,000-10,000 cells. During the observation period, FIP cats treated with prednisolone exhibited a median survival time of 31 days. Conclusion: Confirming FIP cases can be challenging; therefore, a tentative diagnosis of FIP must be made with care. This study provided practical diagnostic tools to diagnose FIP based on clinical signs and multiple abnormalities, which allowed for more efficient and rapid detection.

Exploring the Model of Cefazolin Released from Jellyfish Gelatin-Based Hydrogels as Affected by Glutaraldehyde.

Due to its excellent biocompatibility and ease of biodegradation, jellyfish gelatin has gained attention as a hydrogel. However, hydrogel produced from jellyfish gelatin has not yet been sufficiently characterized. Therefore, this research aims to produce a jellyfish gelatin-based hydrogel. The gelatin produced from desalted jellyfish by-products varied with the part of the specimen and extraction time. Hydrogels with gelatin: glutaraldehyde ratios of 10:0.25, 10:0.50, and 10:1.00 (v/v) were characterized, and their cefazolin release ability was determined. The optimal conditions for gelatin extraction and chosen for the development of jellyfish hydrogels (JGel) included the use of the umbrella part of desalted jellyfish by-products extracted for 24 h (WU24), which yielded the highest gel strength (460.02 g), viscosity (24.45 cP), gelling temperature (12.70 °C), and melting temperature (22.48 °C). The quantities of collagen alpha-1(XXVIII) chain A, collagen alpha-1(XXI) chain, and collagen alpha-2(IX) chain in WU24 may influence its gel properties. Increasing the glutaraldehyde content in JGel increased the gel fraction by decreasing the space between the protein chains and gel swelling, as glutaraldehyde binds with lateral amino acid residues and produces a stronger network. At 8 h, more than 80% of the cefazolin in JGel (10:0.25) was released, which was higher than that released from bovine hydrogel (52.81%) and fish hydrogel (54.04%). This research is the first report focused on the production of JGel using glutaraldehyde as a cross-linking agent.

A 2D-proteomic analysis identifies proteins differentially regulated by two different dengue virus serotypes.

The mosquito transmitted dengue virus (DENV) is a major public health problem in many tropical and sub-tropical countries around the world. Both vaccine development and drug development are complex as the species Dengue virus consist of four distinct viruses (DENV 1 to DENV 4) each of which is composed of multiple lineages and strains. To understand the interaction of DENV with the host cell machinery, several studies have undertaken in vitro proteomic analysis of different cell lines infected with DENV. Invariably, these studies have utilized DENV 2. In this study we sought to define proteins that are differentially regulated by two different DENVs, DENV 2 and DENV 4. A 2-dimensional proteomic analysis identified some 300 protein spots, of which only 11 showed differential expression by both DENVs. Of these, only six were coordinately regulated. One protein, prohibitin 1 (PHB1) was downregulated by infection with both DENVs. Overexpression of PHB1 increased DENV protein expression, level of infection and genome copy number. DENV E protein colocalized with PHB, and there was a direct interaction between DENV 2 E protein and PHB1, but not between DENV 4 E protein and PHB1. The low number of proteins showing coordinate regulation after infection by different DENVs is a cause for concern, particularly in determining new druggable targets, and suggests that studies should routinely investigate multiple DENVs.

Glycolysis is reduced in dengue virus 2 infected liver cells.

Infections with dengue virus (DENV) remain a worldwide public health problem. A number of bona fide cellular targets of DENV have been identified including liver cells. Despite the many lines of evidence confirming the involvement of hepatocytes during DENV infection, only a few studies have used proteomic analysis to understand the modulation of the cellular proteome occurring upon DENV infection. We utilized a 2D-gel electrophoresis analysis to identify proteins that were differentially regulated by DENV 2 infection of liver (Hep3B) cells at 12 h post infection (hpi) and at 48 hpi. The analysis identifies 4 proteins differentially expressed at 12 hpi, and 14 differentially regulated at 48 hpi. One candidate protein identified as downregulated at 48 hpi in the proteomic analysis (GAPDH) was validated in western blotting in Hep3B cells, and subsequently in induced pluripotent stem cell (iPSC) derived human hepatocytes. The reduced expression of GAPDH was coupled with an increase in NADH, and a significantly reduced NAD + /NADH ratio, strongly suggesting that glycolysis is down regulated in response to DENV 2 infection. Metformin, a well characterized drug used in the treatment of diabetes mellitus, is an inhibitor of hepatic gluconeogenesis was shown to reduce the level of DENV 2 infection and new virus production. Collectively these results show that although glycolysis is reduced, glucose is still required, possibly for use by the pentose phosphate pathway to generate nucleosides required for viral replication.

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.

Role of Dietary Factors on DNA Methylation Levels of TNF-Alpha Gene and Proteome Profiles in Obese Men.

Integrated omics-based platforms from epigenomics and proteomics technologies are used to identify several important mechanisms in obesity etiology, food components, dietary intake, regulation of biological pathways, and potential new intervention targets. Therefore, this study aimed to analyze whether dietary factors involved in the methylation of tumor necrosis factor (TNF)-α are implicated in differential protein expression in people with normal weight and obesity. METHODS: The participants were classified into the non-obese (N = 100) and obese (N = 133) groups. DNA methylation levels of the TNF-alpha gene and proteomics were analyzed using the pyrosequencing method and LC-MS-MS, respectively. RESULTS: Comparison between geometric means of DNA methylation of TNF-α showed lower levels in subjects with obesity than in those without obesity (p < 0.05). There were associations between dietary factors and some metabolic syndrome components and TNF-α DNA methylation levels. Proteomic analysis showed important signaling pathways related to obesity, with 95 significantly downregulated proteins and 181 upregulated proteins in the non-obese group compared with the obese group. CONCLUSION: This study shows an association between the dietary factors involved in the methylation of TNF-α and differential protein expression related to obesity. However, a large sample size in future studies is required to confirm our results.

The pomegranate-derived peptide Pug-4 alleviates nontypeable Haemophilus influenzae-induced inflammation by suppressing NF-kB signaling and NLRP3 inflammasome activation.

The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) is the most common cause of exacerbation of chronic obstructive pulmonary disease (COPD), of which an excessive inflammatory response is a hallmark. With the limited success of current medicines there is an urgent need for the development of novel therapeutics that are both safe and effective. In this study, we explored the regulatory potential of pomegranate-derived peptides Pug-1, Pug-2, Pug-3, and Pug-4 on NTHi-induced inflammation. Our results clearly showed that to varying degrees the Pug peptides inhibited NTHi-induced production of IL-1ß, a pivotal cytokine in COPD, and showed that these effects were not related to cytotoxicity. Pug-4 peptide exhibited the most potent inhibitory activity. This was demonstrated in all studied cell types including murine (RAW264.7) and human (differentiated THP-1) macrophages as well as human lung epithelial cells (A549). Substantial reduction by Pug-4 of TNF-α, NO and PGE2 in NTHi-infected A549 cells was also observed. In addition, Pug-4 strongly inhibited the expression of nuclear-NF-κB p65 protein and the NF-κB target genes (determined by IL-1ß, TNF-α, iNOS and COX-2 mRNA expression) in NTHi-infected A549 cells. Pug-4 suppressed the expression of NLRP3 and pro-IL-1ß proteins and inhibited NTHi-mediated cleavage of caspase-1 and mature IL-1ß. These results demonstrated that Pug-4 inhibited NTHi-induced inflammation through the NF-κB signaling and NLRP3 inflammasome activation. Our findings herein highlight the significant anti-inflammatory activity of Pug-4, a newly identified peptide from pomegranate, against NTHi-induced inflammation. We therefore strongly suggest the potential of the Pug-4 peptide as an anti-inflammatory medicine candidate for treatment of NTHi-mediated inflammation.

Cardiac endothelial ischemia/reperfusion injury-derived protein damage-associated molecular patterns disrupt the integrity of the endothelial barrier.

Human cardiac microvascular endothelial cells (HCMECs) are sensitive to ischemia and vulnerable to damage during reperfusion. The release of damage-associated molecular patterns (DAMPs) during reperfusion induces additional tissue damage. The current study aimed to identify early protein DAMPs in human cardiac microvascular endothelial cells subjected to ischemia-reperfusion injury (IRI) using a proteomic approach and their effect on endothelial cell injury. HCMECs were subjected to 60 min of simulated ischemia and 6 h of reperfusion, which can cause lethal damage. DAMPs in the culture media were subjected to liquid chromatography-tandem mass spectrometry proteomic analysis. The cells were treated with endothelial IRI-derived DAMP medium for 24 h. Endothelial injury was assessed by measuring lactate dehydrogenase activity, morphological features, and the expression of endothelial cadherin, nitric oxide synthase (eNOS), and caveolin-1. The top two upregulated proteins, DNAJ homolog subfamily B member 11 and pyrroline-5-carboxylate reductase 2, are promising and sensitive predictors of cardiac microvascular endothelial damage. HCMECs expose to endothelial IRI-derived DAMP, the lactate dehydrogenase activity was significantly increased compared with the control group (10.15 ± 1.03 vs 17.67 ± 1.19, respectively). Following treatment with endothelial IRI-derived DAMPs, actin-filament dysregulation, and downregulation of vascular endothelial cadherin, caveolin-1, and eNOS expressions were observed, along with cell death. In conclusion, the early protein DAMPs released during cardiac microvascular endothelial IRI could serve as novel candidate biomarkers for acute myocardial IRI. Distinct features of impaired plasma membrane integrity can help identify therapeutic targets to mitigate the detrimental consequences mediated of endothelial IRI-derived DAMPs.

Proteomic analysis of crocodile white blood cells reveals insights into the mechanism of the innate immune system.

Crocodiles have a particularly powerful innate immune system because their blood contains high levels of antimicrobial peptides. They can survive injuries that would be fatal to other animals, and they are rarely afflicted with diseases. To better understand the crocodile's innate immune response, proteomic analysis was performed on the white blood cells (WBC) of an Aeromonas hydrophila-infected crocodile. Levels of WBC and red blood cells (RBC) rapidly increased within 1 h. In WBC, there were 109 up-regulated differentially expressed proteins (DEP) that were up-regulated. Fifty-nine DEPs dramatically increased expression from 1 h after inoculation, whereas 50 up-regulated DEPs rose after 24 h. The most abundant DEPs mainly had two biological functions, 1) gene expression regulators, for example, zinc finger proteins and histone H1 family, and 2) cell mechanical forces such as actin cytoskeleton proteins and microtubule-binding proteins. This finding illustrates the characteristic effective innate immune response mechanism of crocodiles that might occur via boosted transcription machinery proteins to accelerate cytoskeletal protein production for induction of phagocytosis, along with the increment of trafficking proteins to transport essential molecules for combating pathogens. The findings of this study provide new insights into the mechanisms of the crocodile's innate immune system.

Proteomic analysis of pulmonary arteries and lung tissues from dogs affected with pulmonary hypertension secondary to degenerative mitral valve disease.

In dogs with degenerative mitral valve disease (DMVD), pulmonary hypertension (PH) is a common complication characterized by abnormally elevated pulmonary arterial pressure (PAP). Pulmonary arterial remodeling is the histopathological changes of pulmonary artery that has been recognized in PH. The underlying mechanisms that cause this arterial remodeling are poorly understood. This study aimed to perform shotgun proteomics to investigate changes in protein expression in pulmonary arteries and lung tissues of DMVD dogs with PH compared to normal control dogs and DMVD dogs without PH. Tissue samples were collected from the carcasses of 22 small-sized breed dogs and divided into three groups: control (n = 7), DMVD (n = 7) and DMVD+PH groups (n = 8). Differentially expressed proteins were identified, and top three upregulated and downregulated proteins in the pulmonary arteries of DMVD dogs with PH including SIK family kinase 3 (SIK3), Collagen type I alpha 1 chain (COL1A1), Transforming growth factor alpha (TGF-α), Apoptosis associated tyrosine kinase (AATYK), Hepatocyte growth factor activator (HGFA) and Tyrosine-protein phosphatase non-receptor type 13 (PTPN13) were chosen. Results showed that some of the identified proteins may play a role in the pathogenesis of pulmonary arterial remodeling. This study concluded shotgun proteomics has potential as a tool for exploring candidate proteins associated with the pathogenesis of PH secondary to DMVD in dogs.

Ceftriaxone exerts antitumor effects in MYCN-driven retinoblastoma and neuroblastoma by targeting DDX3X for translation repression.

MYCN proto-oncogene, bHLH transcription factor (MYCN) amplification is associated with aggressive retinoblastoma (RB) and neuroblastoma (NB) cancer recurrence that is resistant to chemotherapies. Therefore, there is an urgent need to identify new therapeutic tools. This study aimed to evaluate the potential repurposing of ceftriaxone for the treatment of MYCN-amplified RB and NB, based on the clinical observations that the drug was serendipitously found to decrease the volume of the MYCN-driven RB subtype. Using patient-derived tumor organoids and tumor cell lines, we demonstrated that ceftriaxone is a potent and selective growth inhibitor targeting MYCN-driven RB and NB cells. Profiling of drug-induced transcriptomic changes, cell-cycle progression, and apoptotic death indicated cell-cycle arrest and death of drug-treated MYCN-amplified tumor cells. Drug target identification, using an affinity-based proteomic and molecular docking approach, and functional studies of the target proteins revealed that ceftriaxone targeted DEAD-box helicase 3 X-linked (DDX3X), thereby inhibiting translation in MYCN-amplified tumors but not in MYCN-nonamplified cells. The data suggest the feasibility of repurposing ceftriaxone as an anticancer drug and provide insights into the mechanism of drug action, highlighting DDX3X as a potential target for treating MYCN-driven tumors.

Comparative Serum Proteome Profiling of Canine Benign Prostatic Hyperplasia before and after Castration.

BPH is the most prevalent prostatic condition in aging dogs. Nevertheless, clinical diagnosis and management remain inconsistent. This study employed in-solution digestion coupled with nano-liquid chromatography tandem mass spectrometry to assess serum proteome profiling of dogs with BPH and those dogs after castration. Male dogs were divided into two groups; control and BPH groups. In the BPH group, each dog was evaluated at two time points: Day 0 (BF subgroup) and Day 30 after castration (AT subgroup). In the BF subgroup, three proteins were significantly upregulated and associated with dihydrotestosterone: solute carrier family 5 member 5, tyrosine-protein kinase, and FRAT regulator of WNT signaling pathway 1. Additionally, the overexpression of polymeric immunoglobulin receptors in the BF subgroup hints at its potential as a novel protein linked to the BPH development process. Conversely, alpha-1-B glycoprotein (A1BG) displayed significant downregulation in the BF subgroup, suggesting A1BG's potential as a predictive protein for canine BPH. Finasteride was associated with increased proteins in the AT subgroup, including apolipoprotein C-I, apolipoprotein E, apolipoprotein A-II, TAO kinase 1, DnaJ homolog subfamily C member 16, PH domain and leucine-rich repeat protein phosphatase 1, neuregulin 1, and pseudopodium enriched atypical kinase 1. In conclusion, this pilot study highlighted alterations in various serum proteins in canine BPH, reflecting different pathological changes occurring in this condition. These proteins could be a source of potential non-invasive biomarkers for diagnosing this disease.

Proteomic analysis of small extracellular vesicles unique to cervical cancer.

Background: Cervical cancer (CC) is the fourth most common cancer in females worldwide. Existing biomarkers for CC, such as squamous cell carcinoma antigens, show low specificity. Hence, a novel biomarker for the diagnosis of CC is required. Through proteomic analysis, this study aimed to distinguish between the small extracellular vesicle (sEV) protein profiles of healthy controls (HC) and CC sera and to identify potential sEV proteins that can serve as biomarkers for CC diagnosis. Methods: The number and size distribution of sEVs in HC and CC sera were measured using nanoparticle tracking analysis. Differential ultracentrifugation combined with size-exclusion chromatography was used to isolate and purify sEVs. Liquid chromatography-tandem mass spectrometry was used to identify and compare the protein profiles between patients with CC and HC. Differentially expressed extracellular vesicle (EV) proteins were validated using The Cancer Genome Atlas database. Results: The EV particle concentration in patients with CC was marginally higher than that in HC. Proteomic and functional protein analyses revealed a difference in the EV protein profiles between HC and CC and identified proteins that can serve as biomarkers for CC. Conclusions: This study provides insights into the potential of sEVs as less invasive biomarkers for CC diagnosis. Validation with a well-designed cohort should be performed to determine the clinical diagnostic value of specific protein markers for CC.

The Dual Functions of Andrographolide in the Epstein-Barr Virus-Positive Head-and-Neck Cancer Cells: The Inhibition of Lytic Reactivation of the Epstein-Barr Virus and the Induction of Cell Death.

Andrographolide, a medicinal compound, exhibits several pharmacological activities, including antiviral and anticancer properties. Previously, we reported that andrographolide inhibits Epstein-Barr virus (EBV) lytic reactivation, which is associated with viral transmission and oncogenesis in epithelial cancers, including head-and-neck cancer (HNC) cells. However, the underlying mechanism through which andrographolide inhibits EBV lytic reactivation and affects HNC cells is poorly understood. Therefore, we investigated these mechanisms using EBV-positive HNC cells and the molecular modeling and docking simulation of protein. Based on the results, the expression of EBV lytic genes and viral production were significantly inhibited in andrographolide-treated EBV-positive HNC cells. Concurrently, there was a reduction in transcription factors (TFs), myocyte enhancer factor-2D (MEF2D), specificity protein (SP) 1, and SP3, which was significantly associated with a combination of andrographolide and sodium butyrate (NaB) treatment. Surprisingly, andrographolide treatment also significantly induced the expression of DNA Methyltransferase (DNMT) 1, DNMT3B, and histone deacetylase (HDAC) 5 in EBV-positive cells. Molecular modeling and docking simulation suggested that HDAC5 could directly interact with MEF2D, SP1, and SP3. In our in vitro study, andrographolide exhibited a stronger cytotoxic effect on EBV-positive cells than EBV-negative cells by inducing cell death. Interestingly, the proteome analysis revealed that the expression of RIPK1, RIPK3, and MLKL, the key molecules for necroptosis, was significantly greater in andrographolide-treated cells. Taken together, it seems that andrographolide exhibits concurrent activities in HNC cells; it inhibits EBV lytic reactivation by interrupting the expression of TFs and induces cell death, probably via necroptosis.

Serum proteomic profile of wild stump-tailed macaques (Macaca arctoides) infected with malaria parasites in Thailand.

The number of patients infected with simian malaria is gradually increasing in many countries of Southeast Asia and South America. The most important risk factor for a zoonotic spillover event of malarial infection is mostly influenced by the interaction between humans, monkeys, and vectors. In this study, we determine the protein expression profile of a wild stump-tailed macaque (Macaca arctoides) from a total of 32 blood samples collected from Prachuap Kiri Khan Province, Thailand. The malarial parasite was analyzed using nested polymerase chain reaction (PCR) assays by dividing the samples into three groups: non-infected, mono-infected, and multiple-infected. The identification and differential proteomic expression profiles were determined using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and bioinformatics tools. A total of 9,532 proteins (total proteins) were identified with the filter-based selection methods analysis, and a subset of 440 proteins were found to be different between each group. Within these proteins, the GhostKOALA functional enrichment analysis indicated that 142 important proteins were associated with either of the organismal system (28.87%), genetic information processing (23.24%), environmental information processing (16.20%), metabolism (13.38%), cellular processes (11.97%), or causing human disease (6.34%). Additionally, using interaction network analysis, nine potential reporter proteins were identified. Here, we report the first study on the protein profiles differentially expressed in the serum of wild stump-tailed macaques between non, mono, and multiple malarial infected living in a natural transmission environment. Our findings demonstrate that differentially expressed proteins implicated in host defense through lipid metabolism, involved with TGF pathway were suppressed, while those with the apoptosis pathway, such as cytokines and proinflammation signals were increased. Including the parasite's response via induced hemolysis and disruption of myeloid cells. A greater understanding of the fundamental processes involved in a malarial infection and host response can be crucial for developing diagnostic tools, medication development, and therapies to improve the health of those affected by the disease.

Proteomic Profiling of Early Secreted Proteins in Response to Lipopolysaccharide-Induced Vascular Endothelial Cell EA.hy926 Injury.

Sepsis is a crucial public health problem with a high mortality rate caused by a dysregulated host immune response to infection. Vascular endothelial cell injury is an important hallmark of sepsis, which leads to multiple organ failure and death. Early biomarkers to diagnose sepsis may provide early intervention and reduce risk of death. Damage-associated molecular patterns (DAMPs) are host nuclear or cytoplasmic molecules released from cells following tissue damage. We postulated that DAMPs could potentially be a novel sepsis biomarker. We used an in vitro model to determine suitable protein-DAMPs biomarkers for early sepsis diagnosis. Low and high lipopolysaccharide (LPS) doses were used to stimulate the human umbilical vein endothelial cell line EA.hy926 for 24, 48, and 72 h. Results showed that cell viability was reduced in both dose-dependent and time-dependent manners. Cell injury was corroborated by a significant increase in lactate dehydrogenase (LDH) activity within 24 h in cell-conditioned medium. Secreted protein-DAMPs in the supernatant, collected at different time points within 24 h, were characterized using shotgun proteomics LC-MS/MS analysis. Results showed that there were 2233 proteins. Among these, 181 proteins from the LPS-stimulated EA.hy926 at 1, 12, and 24 h were significantly different from those of the control. Twelve proteins were up-regulated at all three time points. Furthermore, a potential interaction analysis of predominant DAMPs-related proteins using STITCH 5.0 revealed the following associations with pathways: response to stress; bacterium; and LPS (GO:0080134; 0009617; 0032496). Markedly, alpha-2-HS-glycoprotein (AHSG or fetuin-A) and lactotransferrin (LTF) potentially presented since the first hour of LPS stimulation, and were highly up-regulated at 24 h. Taken together, we reported proteomic profiling of vascular endothelial cell-specific DAMPs in response to early an in vitro LPS stimulation, suggesting that these early damage-response protein candidates could be novel early biomarkers associated with sepsis.