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.

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 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.

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.

The investigation of antibacterial properties of peptides and protein hydrolysates derived from serum of Asian water monitor (Varanus salvator).

It is well known that the Asian water monitors or Varanus salvator are both scavengers and predators. They can live and survive in the place that exposed to harmful microorganisms. Most people believe that they have some protected mechanisms to confront those infections. The aim of this study is to determine the antibacterial activities of crude peptides and protein hydrolysates extracted from serum of the Varanus salvator. Ten types of bacteria were cultured with crude peptides and protein hydrolysates which were isolated from 21 Varanus salvator's serum. The crude peptides showed some interested inhibition percentages against Enterobacter aerogenes ATCC13048 = 25.6%, Acinetobacter baumannii ATCC19606 = 33.4%, Burkholderia cepacia ATCC25416 = 35.3% and Pseudomonas aeruginosa ATCC27853 = 25.8%, whereas the protein hydrolysates had some inhibition potential on Burkholderia cepacia ATCC25416 = 24.3%. For the rest results of other tests were below 20% of inhibition. In addition, the evidences show that crude peptides have better antibacterial performances significantly than protein hydrolysates on most tested bacteria. Furthermore, antimicrobial peptides prediction shows about 10 percent hit (41/432 sequences). The interpretation shows that the best hit sequence is highly hydrophobic. It may destroy outer membrane of Gram-negative hence prevents the invasion of those bacteria. Altogether, bioinformatics and experiments show similar trends of antimicrobial peptide efficacy from Varanus salvator. Further studies need to be conducted on peptide purification and antimicrobial peptide candidate should be identified.

Pinostrobin induces acute leukemia cell apoptosis via the regulation of miR-410-5p and SFRP5.

AIMS: This study attempted to explore the mechanisms involved in pinostrobin (PN)-mediated acute leukemia cell apoptosis regulated by miR-410-5p. MATERIAL AND METHODS: NB4 and MOLT-4 cells were cultured and treated with PN at the IC50 concentration. Apoptosis was examined by Annexin V-FITC/PI staining. RT-qPCR was used to measure the expression of caspase-3, BAK, BCL-W, and MCL-1. The target protein of PN was identified using LC-MS/MS followed by bioinformatic analysis. TargetScan, DIANA, and miRDB were used for the prediction of miRNAs involved in the PN-induced apoptosis mechanism. miRNA mimic transfection, RT-qPCR, and western blot analysis were performed to evaluate the regulatory effect of miRNA on its target and the involvement of miRNA in apoptosis induction by PN. In addition, the synergistic effect of PN and daunorubicin (DNR) were investigated by using the MTT assay. KEY FINDINGS: The results showed that PN reduced cell viability and induced apoptosis in both leukemia cell lines. From the LC-MS/MS and bioinformatics analysis, SFRP5 and miR-410-5p were selected as a potential PN target protein and miRNA, respectively. After miRNA mimic transfection, miR-410-5p, which is an onco-miRNA, was decreased and led to increased apoptosis in both cell lines, indicating that this miRNA is involved in PN-mediated apoptosis mechanisms. Moreover, PN demonstrated a synergistic effect with DNR, suggesting that PN may be used in combination with conventional chemotherapy drugs. SIGNIFICANCE: PN regulates the expression of miR-410-5p and SFRP5 to promote apoptosis in acute leukemia cells. It could be developed as an alternative treatment for leukemia in the future.

Palmelloid Formation and Cell Aggregation Are Essential Mechanisms for High Light Tolerance in a Natural Strain of Chlamydomonas reinhardtii.

Photosynthetic organisms, such as higher plants and algae, require light to survive. However, an excessive amount of light can be harmful due to the production of reactive oxygen species (ROS), which cause cell damage and, if it is not effectively regulated, cell death. The study of plants' responses to light can aid in the development of methods to improve plants' growth and productivity. Due to the multicellular nature of plants, there may be variations in the results based on plant age and tissue type. Chlamydomonas reinhardtii, a unicellular green alga, has also been used as a model organism to study photosynthesis and photoprotection. Nonetheless, the majority of the research has been conducted with strains that have been consistently utilized in laboratories and originated from the same source. Despite the availability of many field isolates of this species, very few studies have compared the light responses of field isolates. This study examined the responses of two field isolates of Chlamydomonas to high light stress. The light-tolerant strain, CC-4414, managed reactive oxygen species (ROS) slightly better than the sensitive strain, CC-2344, did. The proteomic data of cells subjected to high light revealed cellular modifications of the light-tolerant strain toward membrane proteins. The morphology of cells under light stress revealed that this strain utilized the formation of palmelloid structures and cell aggregation to shield cells from excessive light. As indicated by proteome data, morphological modifications occur simultaneously with the increase in protein degradation and autophagy. By protecting cells from stress, cells are able to continue to upregulate ROS management mechanisms and prevent cell death. This is the first report of palmelloid formation in Chlamydomonas under high light stress.

Echocardiography and MALDI-TOF Identification of Myosin-Binding Protein C3 A74T Gene Mutations Involved Healthy and Mutated Bengal Cats.

This study aimed to identify the potential peptide candidates and expected proteins associated with MYBPC3-A74T gene mutations in Bengal cats and determine if peptidome profiles differ between healthy controls and cats with MYBPC3-A74T gene mutations. All animals were evaluated using echocardiography. DNA was isolated and followed by the screening test of MYBPC3 gene mutation. The MALDI-TOF mass spectrometry was conducted for analyzing the targeted peptide and protein patterns. The expected protein candidates were searched for within the NCBI database. Our results demonstrated that the MYBPC3-A74T gene mutation was dominant in Bengal cats but not in domestic shorthair cats. Correlations between baseline characteristics and echocardiographic parameters were discovered in Bengal cats. Mass spectrometry profiles of the candidate proteins were suspected to accompany the cat with the MYBPC3-A74T gene mutation, involving integral protein-membrane, organization of nucleus, DNA replication, and ATP-binding protein. Therefore, MYBPC3-A74T gene mutations occur frequently in Bengal cat populations. The high incidence of homozygotes for the mutation supports the causal nature of the MYBPC3-A74T mutation. In addition, peptidomics analysis was established for the first time under this condition to promise a complementary technique for the future clinical diagnosis of the MYBPC3-A74T mutation associated with physiological variables and cardiac morphology in cats.

Discovery of potential protein biomarkers associated with sugarcane white leaf disease susceptibility using a comparative proteomic approach.

Sugarcane white leaf disease (SCWLD) is caused by phytoplasma, a serious sugarcane phytoplasma pathogen, which causes significant decreases in crop yield and sugar quality. The identification of proteins involved in the defense mechanism against SCWLD phytoplasma may help towards the development of varieties resistant to SCWLD. We investigated the proteomes of four sugarcane varieties with different levels of susceptibility to SCWLD phytoplasma infection, namely K88-92 and K95-84 (high), KK3 (moderate), and UT1 (low) by quantitative label-free nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). A total of 248 proteins were identified and compared among the four sugarcane varieties. Two potential candidate protein biomarkers for reduced susceptibility to SCWLD phytoplasma were identified as proteins detected only in UT1. The functions of these proteins are associated with protein folding, metal ion binding, and oxidoreductase. The candidate biomarkers could be useful for further study of the sugarcane defense mechanism against SCWLD phytoplasma, and in molecular and conventional breeding strategies for variety improvement.

Analysis of the Serum Peptidomics Profile for Cats With Sarcomeric Gene Mutation and Hypertrophic Cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) has a complex phenotype that is partly explained by genetic variants related to this disease. The serum peptidome profile is a promising approach to define clinically relevant biomarkers. This study aimed to classify peptide patterns in serum samples between cats with sarcomeric gene mutations and normal cats. Materials and Methods: In the total serum samples from 31 cats, several essential proteins were identified by peptidomics analysis. The 5,946 peptides were differentially expressed in cats with sarcomeric gene mutations compared with cats without mutations. Results: Our results demonstrated characteristic protein expression in control cats, Maine Coon cats, and Maine Coon cats with gene mutations. In cats with gene mutations, peptide expression profiling showed an association with three peptides, Cytochrome 3a132 (CYP3A132), forkhead box O1 (FOXO1), and ArfGAP, with GTPase domains, ankyrin repeats, and PH domain 2 (AGAP2). Discussion: The serum peptidome of cats with mutations might provide supporting evidence for the dysregulation of metabolic and structural proteins. Genetic and peptidomics investigations may help elucidate the phenotypic variability of HCM and treatment targets to reduce morbidity and mortality of HCM in cats.

Fungicidal Activity of Recombinant Javanicin against Cryptococcus neoformans Is Associated with Intracellular Target(s) Involved in Carbohydrate and Energy Metabolic Processes.

The occurrence of Cryptococcus neoformans, the human fungal pathogen that primarily infects immunocompromised individuals, has been progressing at an alarming rate. The increased incidence of infection of C. neoformans with antifungal drugs resistance has become a global concern. Potential antifungal agents with extremely low toxicity are urgently needed. Herein, the biological activities of recombinant javanicin (r-javanicin) against C. neoformans were evaluated. A time-killing assay was performed and both concentration- and time-dependent antifungal activity of r-javanicin were indicated. The inhibitory effect of the peptide was initially observed at 4 h post-treatment and ultimately eradicated within 36 to 48 h. Fungal outer surface alteration was characterized by the scanning electron microscope (SEM) whereas a negligible change with slight shrinkage of external morphology was observed in r-javanicin treated cells. Confocal laser scanning microscopic analysis implied that the target(s) of r-javanicin is conceivably resided in the cell thereby allowing the peptide to penetrate across the membrane and accumulate throughout the fungal body. Finally, cryptococcal cells coped with r-javanicin were preliminarily investigated using label-free mass spectrometry-based proteomics. Combined with microscopic and proteomics analysis, it was clearly elucidated the peptide localized in the intracellular compartment where carbohydrate metabolism and energy production associated with glycolysis pathway and mitochondrial respiration, respectively, were principally interfered. Overall, r-javanicin would be an alternative candidate for further development of antifungal agents.