Search Results (267)

Background/Objectives: Donkey milk (DM) has been considered a valuable alternative to human and bovine counterparts as well as to infant formulas. Milk extracellular vesicles (EVs) have been proposed to influence key biological processes. The purpose of this study is to provide a comprehensive characterization of the protein composition of extracellular vesicles (EVs) by extending quantitative proteomic comparisons to EVs derived from donkey colostrum (DC) and mature donkey milk (MDM). Methods: The EVs were isolated from DC and MDM samples, characterized, and subjected to proteomic analysis using the tandem mass tag-based quantitative approach. Results: In addition to typical milk proteins and EV markers, EVs from DC and MDM both contain components associated with the immune system, immune response, or promoting tissue repair, and assisting with communication between the infant and their environment. The EVs from DC were enriched in proteins associated with protein turnover, specific defense functions, and regenerative processes. Conclusions: Overall, the results can contribute to the broader characterization of the overall protein composition of DC and MDM and might help to predict the beneficial effects of the corresponding EVs on various mammalian cells. They may also provide valuable insights for the development of novel DM-based products for food, pharmaceutical, and biotechnological applications. Full article

Klebsiella pneumoniae strains that are resistant to carbapenems are of great concern. Exposure to low concentrations of antibiotics may influence tolerance to antibiotics. Novel antibiotics and treatment options are thus needed, and this need is exacerbated by the rapid and global spread of antibiotic resistance. In this study, we determined the global proteome changes in a K. pneumoniae strain (CCUG 70747) carrying carbapenem resistance genes when exposed to low concentrations of ertapenem. Quantitative proteomics was achieved by the tandem mass tag labeling of peptides generated by trypsin proteolysis and mass spectrometry analysis. Bioinformatics analyses were used to observe changes in protein abundance, as well as the gene ontology (GO) terms and pathways associated with the differentially expressed proteins. The number of proteins detected with significant differential abundance were 87 at the highest concentration applied and 61 in the lowest concentration, all compared with the strain cultured without any antibiotics present. Several of these proteins, as well as the GO terms and pathways associated with the proteins, were linked to mechanisms of antibiotic resistance. However, this strain encodes a carbapenemase and other beta-lactamases, and thus, as expected, presented a reasonably modest adaptation in the global proteome upon exposure to the low concentrations of ertapenem applied. Nevertheless, our study identifies pathways that may lead to adaptation under sublethal concentrations of antibiotics leading to strains with higher tolerance. Full article

Background/Objectives: Non-Hodgkin lymphoma (NHL) is a group of blood cancers that arise in the lymphatic nodes and other tissues after an injury to the DNA of B/T lineage and NK lymphocytes. Recently, we reported that incomptine A (IA) has in vivo antilymphoma properties. This research aimed to evaluate the effects of IA in the treatment of NHL using antilymphoma activity, Tandem Mass Tag (TMT), and bioinformatics approaches. Methods: The antilymphoma activity of IA was tested on male Balb/c mice inoculated with U-937 cells. Also, TMT, gene ontology enrichment, Reactome pathway, Kyoto Encyclopedia of Gene and Genomes pathway, molecular docking, toxicoinformatic, and pharmaceutical analyses were performed. Results: By TMT analysis of the altered levels of proteins present in the lymph nodes of Balb/c mice with NHL and treated with IA, we identified 106 significantly differentially expressed proteins (DEPs), including Il1rap, Ifi44, Timd4, Apoa4, and Fabp3 as well as Myh3, Eno 2, and H4c11. Among these, the Fhl1 result was the most important cluster altered and a potential core target of IA for the treatment of NHL. Network pharmacology studies have revealed that DEPs are associated with processes such as muscle contraction, glycolysis, hemostasis, epigenetic regulation of gene expression, transport of small molecules, neutrophil extracellular trap formation, adrenergic signaling in cardiomyocytes, systemic lupus erythematosus, alcoholism, and platelet activation, signaling, and aggregation. Computational studies revealed strong binding affinities with six proteins associated with cancer, positive pharmacokinetic properties, and no toxicity. Conclusions: Our contribution suggests that IA may be a compound with potential therapeutic effects against NHL. Full article

Hypovirus infection is known to reduce the pathogenicity of Cryphonectria parasitica, the causative agent of chestnut blight. Isoforms derived from a viral protein p48 have been discovered in host mitochondria and vesicles, which may contribute to virulence attenuation, as reported in earlier work using two-dimensional electrophoresis (2-DE). In this study, a total of 1739 fungal proteins were identified in fungal vesicles through Tandem Mass Tag (TMT)-based quantitative proteomics. The infection of CHV1-EP713 was associated with 75 up-regulated and 201 down-regulated proteins, predominantly involved in vesicular transport process and related cellular functions, including protein folding, membrane fusion, retrograde transport, autophagy, and ER stress responses. The down-regulation of calnexin, COPI, ArfGAP, importin-β, and Atg8 is consistent with impairments in protein folding, retrograde transport, and autophagy. Meanwhile, the up-regulation of clathrin, dynamin, Vps10p, HSP70, and t-SNAREs indicated enhanced trafficking to vacuoles and increased stress response activity. Overall, our findings indicate that hypoviral infection is associated with extensive alterations in the vesicular transport system of C. parasitica, likely mediated through changes in the abundance of multiple key protein regulators. These alterations may underlie attenuation of virulence by impacting crucial cellular processes. Full article

There is a present need to develop alternative biotherapeutic drugs to mitigate the exacerbated inflammatory immune responses characteristic of sepsis. The potent endotoxin lipopolysaccharide (LPS), a major component of Gram-negative bacterial outer membrane, activates the immune system via Toll-like receptor 4 (TLR4), triggering macrophages and a persistent cascade of inflammatory mediators. Our previous studies have demonstrated that Fh15, a recombinant member of the Fasciola hepatica fatty acid binding protein family, can significantly increase the survival rate by suppressing many inflammatory mediators induced by LPS in a septic shock mouse model. Although Fh15 has been proposed as a TLR4 antagonist, the specific mechanisms underlying its immunomodulatory effect remained unclear. In the present study, we employed a quantitative proteomics approach using tandem mass tag (TMT) followed by LC-MS/MS analysis to identify and quantify differentially expressed proteins that participate in signaling pathways downstream TLR4 of macrophages, which can be dysregulated by Fh15. Data are available via ProteomeXchange with identifier PXD065520. Based on significant fold change (FC) cut-off of 1.5 and p-value ≤ 0.05 criteria, we focused our attention to 114 proteins that were upregulated by LPS and downregulated by Fh15. From these proteins, TNFα, IL-1α, Lck, NOS2, SOD2 and CD36 were selected for validation by Western blot on murine bone marrow-derived macrophages due to their relevant roles in the NF-κB, iNOS, oxidative stress, and phagosome signaling pathways, which are closely associated with sepsis pathogenesis. These results suggest that Fh15 exerts a broad spectrum of action by simultaneously targeting multiple downstream pathways activated by TLR4, thereby modulating various aspects of the inflammatory responses during sepsis. Full article

Background: Cochlear implantation is widely used to provide auditory rehabilitation to individuals with severe-to-profound sensorineural hearing loss. However, electrode insertion during cochlear implantation leads to inner ear trauma, damage to sensory structures, and consequently, loss of residual hearing. There is very limited information regarding the target proteins involved in electrode insertion trauma (EIT) following cochlear implantation. Methods: The aim of our study was to identify target proteins and host molecular pathways involved in cochlear damage following EIT utilizing the iTRAQ™ (isobaric tags for relative and absolute quantification) technique using our ex vivo model. The organ of Corti (OC) explants were dissected from postnatal day 3 rats and subjected to EIT or left untreated (control). The proteins were extracted, labelled, and subjected to ultra-high performance liquid chromatography–tandem mass spectrometry. Results: We identified distinct molecular pathways involved in EIT-induced cochlear damage. Confocal microscopy confirmed the expression of these identified proteins in OC explants subjected to EIT. By separating the apical, middle, and basal cochlear turns, we deciphered a topographic array of host molecular pathways that extend from the base to the apex of the cochlea, which are activated post-trauma following cochlear implantation. Conclusions: The identification of target proteins involved in cochlear damage will provide novel therapeutic targets for the development of effective treatment modalities for the preservation of residual hearing in implanted individuals. Full article

Paeoniflorin (PF), a monomeric compound extracted from the dry roots of Paeonia lactiflora, has been widely used in the treatment of nervous system diseases, marking it as a critical formula in Parkinson’s disease (PD). However, the action of PF against PD and its molecular mechanism are still unclear. In this study, tandem mass tags quantitative proteomics was performed to systematically clarify the underlying mechanism of action of PF against PD and to confirm it using in vivo and in vitro studies. The results showed that PF notably enhanced the viability of PC12 cells and mitigated MPP⁺-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Tandem mass tag-based quantitative proteome analysis revealed the identification of 6405 proteins, of which 92 were downregulated and 190 were upregulated. Among them, the levels of PI3K, AKT, CREB, and BDNF in the MPP⁺-induced PC12 cell and MPTP mice were considerably lower than in the control group, indicating the role of the BDNF/CREB pathway in the pathogenesis of neuroprotection. The related DEP (PI3K, AKT, CREB, and BDNF) expression levels were verified by Western blot. PF effectively restored the altered expression of the four DEPs induced by MPP⁺ and MPTP. Summarily, PF exerted remarkable neuroprotective effects in MPP⁺-induced PC12 cell and MPTP-induced mice. Further, our research may provide proteomics insights that contribute to the further exploration of PF as a potential treatment for PD. Full article

Background/Objectives: Canine intervertebral disc herniation (IVDH) is an important musculoskeletal pathology. Unlike in humans, IVDH mechanisms in dogs are underinvestigated from a system-level integrative omics point of view. The aim of this study was to identify key serum molecular players in canine IVDH. Methods: An integrative multi-omics approach combining high-resolution LC-MS-based untargeted metabolomics and tandem mass tag (TMT)-based proteomics was applied. Additionally, serum zinc concentration was determined by spectrophotometry. Results: Nineteen serum metabolites were differentially abundant in IVDH dogs. Metabolite analysis highlighted dysregulation in lipoic acid and branched-chain amino acid (BCAA) metabolism, with elevated levels of valine, leucine, and isoleucine in IVDH. These findings suggest disrupted energy, nitrogen, and neurotransmitter metabolism, potentially contributing to the IVDH pathophysiology. Additionally, lower serum uridine, possibly influenced by BCAA accumulation, was observed, indicating altered neuroinflammatory responses. ELISA validation confirmed elevated serum levels of zinc-α2-glycoprotein (ZAG), alpha-1-microglobulin/bikunin precursor (AMBP), and vitronectin (VTN) in IVDH, supporting immune modulation and neuroprotective mechanisms. Serum prekallikrein (KLKB1) and Protein C inhibitor (SERPINA5), involved in fibrin cloth formation, were found to be lowered in IVDH patients. Pathway enrichment revealed disturbances in aromatic amino acid biosynthesis, with elevated phenylalanine, tyrosine, and tryptophan influencing neurotransmission and inflammation. In addition, elevated serum Zn concentration emphasized its antioxidant importance in immune response, wound healing, and neuropathic pain signaling. Conclusions: Integration with our prior CSF multi-omics data reinforced the relevance of identified molecules in IVDH-associated neurodegeneration, inflammation, and repair processes. This study offers insight into potential diagnostic biomarkers and therapeutic targets for canine IVDH through serum-based molecular profiling. Full article

Proteins regulate various cellular processes and are of great biological interest. The protein search engine is a crucial tool in proteomics research, used to analyze high-throughput tandem mass spectrometry data and to identify protein sequence information. A core step in protein search engines is constructing sequence tag indexes and performing the rapid retrieval of protein databases. However, as the scale of protein sequence data continues to grow, traditional protein search engines face the dual challenges of the high storage cost of sequence tag indexes and low retrieval efficiency. To address these issues, we propose a sequence tag index scheme named STIP, which is based on an inverted index and compression techniques. Based on STIP, we design a peptide retrieval algorithm named STIP-Search. This algorithm utilizes the sequence tag index constructed by STIP for peptide sequence retrieval. STIP uses the greedy algorithm to partition the tag index into blocks; in this way, STIP can generate tag indexes for very large protein databases, such as NCBI-nr. Compared to the current four mainstream tag index generation algorithms used in Open-pFind, MODplus, TIIP and PIPI2, STIP has the lowest storage and time consumption. It utilizes delta encoding, index reduction, and dynamic bit width encoding to compress the tag index, reducing the storage cost by 76.2%. Compared to TIIP, which is currently the algorithm with the lowest time complexity, the time cost of the peptide sequence retrieval of STIP-Search is reduced by 8.94% to 23.31%. Full article

Infections caused by dengue virus (DENV) result in significant morbidity and mortality. A proportion of infected individuals develop dengue haemorrhagic fever (DHF) characterized by circulatory collapse and multiorgan failure. Early detection of individuals likely to develop DHF could lead to improved outcomes for patients and help us use healthcare resources more efficiently. We identified proteins that are differentially regulated during early disease in peripheral blood mononuclear cells (PBMCs) of patients who subsequently developed DHF. Four dengue fever (DF), four DHF and two healthy control PBMCs were subjected to tandem mass tag mass spectrometry. Differentially regulated proteins were used to identify up- or down-regulated Gene Ontology pathways. One hundred and sixty proteins were differentially expressed in DENV-infected samples compared to healthy controls. PBMCs from DHF patients differentially expressed 90 proteins compared to DF; these were involved in down-regulation of platelet activation and aggregation, cell adhesion, and cytoskeleton arrangement pathways. Proteins involved in oxidative stress and p38 MAPK signalling were upregulated in DHF samples during early infection compared to DF. This study has identified 90 proteins differentially regulated in PBMCs that could potentially serve as biomarkers to identify patients at risk of developing DHF at an early disease stage. Full article

Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental disorder, with lead (Pb) exposure increasingly linked to its risk. However, the molecular mechanisms linking Pb to ASD remain poorly understood. This study established a postnatal Pb-exposed mouse model and employed the three-chamber social test and the marble-burying test to assess ASD-like behavioral phenotypes. The Pb levels in both blood and the hippocampus were quantified, and hippocampal neurons were assessed for morphological alterations. Moreover, a Tandem Mass Tag (TMT)-based quantitative proteomics approach was applied to elucidate the underlying mechanisms. Neurobehavioral experiments revealed Pb-exposed C57BL/6 offspring exhibited reduced social interaction and novelty preference along with increased repetitive marble-burying behavior. The Pb levels in both the blood and hippocampus of Pb-treated mice were significantly elevated compared with those of control animals. Postnatal Pb exposure resulted in a reduction in the neuronal numbers and disorganized neuronal arrangement in the hippocampus. A total of 66 proteins were identified as being differentially expressed after postnatal Pb exposure. Among them, 34 differentially expressed proteins were common in both Pb exposure groups, with 33 downregulated and 1 upregulated. Bioinformatic analysis revealed multi-pathway regulation involved in Pb-induced neurodevelopmental disorders, including dysregulation of synaptic signaling, abnormal activation of neuron apoptosis, and neuroinflammation. Notably, the SYT10/IGF-1 signaling pathway may play a potential key role. These findings enhance understanding of Pb-induced autism-like behaviors, providing novel proteomic insights into the etiology of ASD. Full article

Background: Tomatoes are self-pollinating plants, and successful fruit set depends on the production of functional pollen within the same flower. Our previous studies have shown that the ‘Black Vernissage’ tomato variety exhibits greater resilience to heat stress in terms of pollen productivity compared to the ‘Micro-Tom’ variety. Pollen productivity is determined by meiotic activity during microsporogenesis and the development of free microspores during gametogenesis. This study focused on identifying heat stress (HS)-induced proteomes in pollen mother cells (PMCs) and microspores. Methods: Tomato plants were grown under two temperature conditions: 26 °C (non-heat-treated control) and 37 °C (heat-treated). Homogeneous cell samples of meiotic PMCs (prior to the tetrad stage) and free microspores were collected using laser capture microdissection (LCM). The heat-induced proteomes were identified using tandem mass tag (TMT)–quantitative proteomics analysis. Results: The enrichment of the meiotic cell cycle in PMCs and the pre-mitotic process in free microspores confirmed the correlation between proteome expression and developmental stage. Under HS, PMCs in both tomato varieties were enriched with heat shock proteins (HSPs). However, the ‘Black Vernissage’ variety exhibited a greater diversity of HSP species and a higher level of enrichment compared to the ‘Micro-Tom’ variety. Additionally, several proteins involved in gene expression and protein translation were downregulated in PMCs and microspores of both varieties. In the PMC proteomes, the relative abundance of proteins showed no significant differences between the two varieties under normal conditions, with very few exceptions. However, HS induced significant differential expression both within and between the varieties. More importantly, these heat-induced differentially abundant proteins (DAPs) in PMCs are directly involved in meiotic cell division, including the meiosis-specific protein ASY3 (Solyc01g079080), the cell division protein kinase 2 (Solyc11g070140), COP9 signalosome complex subunit 1 (Solyc01g091650), the kinetochore protein ndc80 (Solyc01g104570), MORC family CW-type zinc finger 3 (Solyc02g084700), and several HSPs that function in protecting the fidelity of the meiotic processes, including the DNAJ chaperone (Solyc04g009770, Solyc05g055160), chaperone protein htpG (Solyc04g081570), and class I and class II HSPs. In the microspores, most of the HS-induced DAPs were consistently observed across both varieties, with only a few proteins showing significant differences between them under heat stress. These HS-induced DAPs include proteases, antioxidant proteins, and proteins related to cell wall remodeling and the generation of pollen exine. Conclusions: HS induced more dynamic proteomic changes in meiotic PMCs compared to microspores, and the inter-varietal differences in the PMC proteomes align with the effects of HS on pollen productivity observed in the two varieties. This research highlights the importance of the cell-type-specific proteomics approach in identifying the molecular mechanisms that are critical for the pollen developmental process under elevated temperature conditions. Full article

Obesity arises from a complex interplay of genetic and environmental factors. Even among individuals with the same genetic predisposition, diet-induced obesity (DIO) exhibits varying degrees of susceptibility, which are categorized as DIO and diet-induced obesity resistance (DR). The hypothalamus plays a pivotal role in regulating energy homeostasis. This study performed a comparative hypothalamic proteomic analysis in DIO and DR rats to identify differentially expressed proteins (DEPs) associated with alterations in body weight. Male Sprague Dawley rats were fed either a standard chow diet or a high-fat diet for 12 weeks. DIO rats exhibited the most rapid weight gain compared to both the control and DR rats. Despite consuming similar caloric intake, DR rats exhibited less weight gain relative to DIO rats. Proteomic analysis revealed 31 DEPs in the hypothalamus of DR rats compared to DIO rats (with a false discovery rate (FDR) < 1%). Notably, 14 proteins were upregulated and 17 proteins were downregulated in DR rats. Gene ontology analysis revealed an enrichment of ion-binding proteins, such as those binding to Fe²⁺, Zn²⁺, Ca²⁺, and Se, as well as proteins involved in neuronal activity and function, potentially enhancing neuronal development and cognition in DR rats. The DEPs pathway analysis via the Kyoto Encyclopedia of Genes and Genomes (KEGG) implicated starch and sucrose metabolism, antigen processing and presentation, and the regulation of inflammatory mediator affecting TRP channels. Western blotting confirmed the proteomic findings for TRPV4, CaMKV, RSBN1, and BASP1, which were consistent with those obtained from Tandem Mass tag (TMT) proteomic analysis. In conclusion, our study highlights the hypothalamic proteome as a critical determinant in the susceptibility to DIO and provides novel targets for obesity prevention and treatment. Full article

Currently, developing cotton cultivation in saline–alkali soils is a vital focus for restructuring the cotton industry in China. The seedling stage, specifically the three-leaf stage, is a crucial period for assessing the salt tolerance of cotton. This research examined 430 natural populations of upland cotton, including 45 semi-wild germlines of Gossypium purpurascens. We measured the phenotypic responses of salt stress injury on seedlings as well as potassium (K), calcium (Ca), sodium (Na), and magnesium (Mg) concentrations in the roots, stems, and leaves following a 72 h exposure. The comprehensive salt tolerance index (CSTI) was determined using a membership function, principal component analysis, and cluster analysis based on 48 phenotypic traits related to salt tolerance. The results revealed significant variations in the phenotypic traits of the ion group under salt stress. Salt stress greatly affected the relative contents of Mg, K, and Ca ions in the aboveground parts of cotton, and correlations were observed among the 48 indices. The CSTI was calculated using seven principal component indexes, identifying 30 salt-tolerant, 114 weakly salt-tolerant, 39 salt-sensitive, and 4 highly sensitive materials based on cluster analysis. Among the 45 G. purpurascens cotton resources, 28 were weakly salt-tolerant, while 17 were salt-sensitive. Through TMT (Tandem Mass Tag)-based quantitative analysis, we identified 3107 unique peptides among 28,642 detected peptides, resulting in 203,869 secondary mass spectra, with 50,039 spectra successfully matched to peptides. Additionally, we identified several salt tolerance-related pathways (carbon metabolism; glutathione metabolism; the biosynthesis of amino acids, etc.) and proteins classified within the CAZy (Carbohydrate-Active EnZYme) family and expansin proteins. The results of this study concerning salt-tolerant materials provide a crucial theoretical foundation for the identification and evaluation of salt-tolerant breeding parents in cultivated cotton. Full article

Highland animals have unique hair growth mechanisms to allow them to adapt to harsh living environments. Compared with other species, their hair cycle growth is affected by more environmental factors. Yaks, as highland animals, have obvious periodic hair growth characteristics in a year; this biological process is regulated by numerous proteins, but the specific molecular regulatory mechanism is still unclear. Here we analyzed the histological characteristics of yak hair follicles (HFs) at each stage and conducted TMT proteomics research. The protein expression network of yak hair at each stage and the mechanism of the yak HF growth cycle were systematically explored, and the candidate proteins Sfrp1 and Ppard were verified. A total of 3176 proteins were quantifiable and 1142 differentially expressed proteins (DEPs) were obtained at five stages of the yak hair cycle. DEPs enriched in complement activation change, tissue development, lipid metabolism, WNT pathway, VEGF pathway, JAK-STAT pathway, and PPAR pathway may promote the growth of yak hair follicles, such as Serpinf1, Ppard, and Stat3. DEPs enriched in complement system, coagulation, cell adhesion, lipid metabolic process, proliferation of epidermal cells, and estrogen pathway may promote the degeneration of yak hair, such as Sfrp1, Eppk1, and Egfr. Using Protein-Protein Interaction (PPI) analysis, we found that core nodes of DEP networks in yak skin are significantly different at three critical time points in hair follicle development, and lipid metabolism proteins are common core DEP nodes during yak HF growth and degeneration. The expression of Sfrp1 and Ppard in yak hair follicles at different periods showed they are related to yak hair cycle control. This study showed that the protein regulatory network of the yak HF growth cycle is complex and dynamically changing and revealed key candidate proteins that may affect yak hair follicle development. These findings provided detailed data for further understanding of the plateau adaptation mechanism of the yak, which is of great significance to make better use of the yak livestock resources and enhance their economic value. Full article