Comparative Proteomic Profiling Research Articles

Cronobacter sakazakii lysozyme inhibitor LprI mediated by HmsP and c-di-GMP is essential for biofilm formation and virulence

ABSTRACT Cronobacter sakazakii poses a significant threat, particularly to neonates and infants. Despite its strong pathogenicity, understanding of C. sakazakii biofilms and their role in infections remains limited. This study investigates the roles of HmsP and c-di-GMP in biofilm formation and identifies key genetic and proteomic elements involved. Gene knockout experiments reveal that HmsP and c-di-GMP are linked to biofilm formation in C. sakazakii . Comparative proteomic profiling identifies the lysozyme inhibitor protein LprI, which is downregulated in hmsP knockouts and upregulated in c-di-GMP knockouts, as a potential biofilm formation factor. Further investigation of the lprI knockout strain shows significantly reduced biofilm formation and decreased virulence in a rat infection model. Additionally, LprI is demonstrated to bind extracellular DNA, suggesting a role in anchoring C. sakazakii within the biofilm matrix. These findings enhance our understanding of the molecular mechanisms underlying biofilm formation and virulence in C. sakazakii , offering potential targets for therapeutic intervention and food production settings. IMPORTANCE Cronobacter sakazakii is a bacterium that poses a severe threat to neonates and infants. This research elucidates the role of the lysozyme inhibitor LprI, modulated by HmsP and c-di-GMP, and uncovers a key factor in biofilm formation and virulence. The findings offer crucial insights into the molecular interactions that enable C. sakazakii to form resilient biofilms and persist in hostile environments, such as those found in food production facilities. These insights not only enhance our understanding of C. sakazakii pathogenesis but also identify potential targets for novel therapeutic interventions to prevent or mitigate infections. This work is particularly relevant to public health and the food industry, where controlling C. sakazakii contamination in powdered infant formula is vital for safeguarding vulnerable populations.

Comparative Proteomic Identification of Ram Sperm before and after In Vitro Capacitation.

Ram sperm undergo a sequence of physiological and biochemical changes collectively termed as capacitation to perform oocyte fertilization. However, the protein changes induced by capacitation remain in need of further exploration. Thus, the present study investigated the comparative proteomic profiling in ram spermatozoa under non-capacitating (NC) and capacitating (CAP) conditions in vitro using a liquid chromatography-tandem mass spectrometry combined with tandem mass tag labeling strategy. As a results, 2050 proteins were identified and quantified; 348 of them were differentially abundant, with 280 of the proteins upregulated and 68 of the proteins downregulated between the CAP and NC spermatozoa, respectively. Functional enrichment analysis indicated that the differentially abundant proteins Prune Exopolyphosphatase 1, Galactose-1-Phosphate Uridylyltransferase, and ATP Citrate Lyase were strictly related to energy production and conversion, and Phosphoglycolate phosphatase, Glucosamine-6-Phosphate Deaminase 1 and 2 were related to metabolism, RNA processing, and vesicular transport pathways. Furthermore, the networks of protein-protein interaction indicated a strong interaction among these differential proteins in annotated pathways such as ubiquitin and transport metabolism. Our findings indicate that capacitation progress might be regulated through different pathways, providing insights into mechanisms involved in ram sperm capacitation and fertility.

Comparative Cross-Kingdom DDA- and DIA-PASEF Proteomic Profiling Reveals Novel Determinants of Fungal Virulence and a Putative Druggable Target.

Accurate and reliable detection of fungal pathogens presents an important hurdle to manage infections, especially considering that fungal pathogens, including the globally important human pathogen, Cryptococcus neoformans, have adapted diverse mechanisms to survive the hostile host environment and moderate virulence determinant production during coinfections. These pathogen adaptations present an opportunity for improvements (e.g., technological and computational) to better understand the interplay between a host and a pathogen during disease to uncover new strategies to overcome infection. In this study, we performed comparative proteomic profiling of an in vitro coinfection model across a range of fungal and bacterial burden loads in macrophages. Comparing data-dependent acquisition and data-independent acquisition enabled with parallel accumulation serial fragmentation technology, we quantified changes in dual-perspective proteome remodeling. We report enhanced and novel detection of pathogen proteins with data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF), especially for fungal proteins during single and dual infection of macrophages. Further characterization of a fungal protein detected only with DIA-PASEF uncovered a novel determinant of fungal virulence, including altered capsule and melanin production, thermotolerance, and macrophage infectivity, supporting proteomics advances for the discovery of a novel putative druggable target to suppress C. neoformans pathogenicity.

Comparative Proteomic Profiling of Blood Plasma Revealed Marker Proteins Involved in Temporal Lobe Epilepsy.

Temporal lobe epilepsy has various origins, involving or not involving structural changes in brain tissue. The mechanisms of epileptogenesis are associated with cell regulation and signaling disruptions expressed in varied levels of proteins. The blood plasma proteomic profiling of temporal lobe epilepsy patients (including magnetic resonance imaging (MRI)-positive and MRI-negative ones) and healthy volunteers using mass spectrometry and label-free quantification revealed a list of differently expressed proteins. Several apolipoproteins (APOA1, APOD, and APOA4), serpin protease inhibitors (SERPINA3, SERPINF1, etc.), complement components (C9, C8, and C1R), and a total of 42 proteins were found to be significantly upregulated in the temporal lobe epilepsy group. A classification analysis of these proteins according to their biological functions, as well as a review of the published sources, disclosed the predominant involvement of the processes mostly affected during epilepsy such as neuroinflammation, intracellular signaling, lipid metabolism, and oxidative stress. The presence of several proteins related to the corresponding compensatory mechanisms has been noted. After further validation, the newly identified temporal lobe epilepsy biomarker candidates may be used as epilepsy diagnostic tools, in addition to other less specific methods such as electroencephalography or MRI.

Comparison of the Proteome of Huh7 Cells Transfected with Hepatitis B Virus Subgenotype A1, with or without G1862T.

HBeAg is a non-structural, secreted protein of hepatitis B virus (HBV). Its p25 precursor is post-translationally modified in the endoplasmic reticulum. The G1862T precore mutation leads to the accumulation of P25 in the endoplasmic reticulum and activation of unfolded protein response. Using mass spectrometry, comparative proteome profiling of Huh-7 cells transfected with wildtype (WT) or G1862T revealed significantly differentially expressed proteins resulting in 12 dysregulated pathways unique to WT-transfected cells and 7 shared between cells transfected with either WT or G1862T. Except for the p38 MAPK signalling pathway, WT showed a higher number of DEPs than G1862T-transfected cells in all remaining six shared pathways. Two signalling pathways: oxidative stress and cell cycle signalling were differentially expressed only in cells transfected with G1862T. Fifteen pathways were dysregulated in G1862T-transfected cells compared to WT. The 15 dysregulated pathways were involved in the following processes: MAPK signalling, DNA synthesis and methylation, and extracellular matrix organization. Moreover, proteins involved in DNA synthesis signalling (replication protein A (RPA) and DNA primase (PRIM2)) were significantly upregulated in G1862T compared to WT. This upregulation was confirmed by mRNA quantification of both genes and immunofluorescent confocal microscopy for RPA only. The dysregulation of the pathways involved in these processes may lead to immune evasion, persistence, and uncontrolled proliferation, which are hallmarks of cancer.

Comparative proteomic profiling of the ovine and human PBMC inflammatory response

Understanding the cellular and molecular mechanisms of inflammation requires robust animal models. Sheep are commonly used in immune-related studies, yet the validity of sheep as animal models for immune and inflammatory diseases remains to be established. This cross-species comparative study analyzed the in vitro inflammatory response of ovine (oPBMCs) and human PBMCs (hPBMCs) using mass spectrometry, profiling the proteome of the secretome and whole cell lysate. Of the entire cell lysate proteome (oPBMCs: 4217, hPBMCs: 4574 proteins) 47.8% and in the secretome proteome (oPBMCs: 1913, hPBMCs: 1375 proteins) 32.8% were orthologous between species, among them 32 orthologous CD antigens, indicating the presence of six immune cell subsets. Following inflammatory stimulation, 71 proteins in oPBMCs and 176 in hPBMCs showed differential abundance, with only 7 overlapping. Network and Gene Ontology analyses identified 16 shared inflammatory-related terms and 17 canonical pathways with similar activation/inhibition patterns in both species, demonstrating significant conservation in specific immune and inflammatory responses. However, ovine PMBCs also contained a unique WC1+γδ T-cell subset, not detected in hPBMCs. Furthermore, differences in the activation/inhibition trends of seven canonical pathways and the sets of DAPs between sheep and humans, emphasize the need to consider interspecies differences in translational studies and inflammation research.

One shot-proteomic profiling, allergenicity assessment and varietal discrimination of lentils seeds by discovery high resolution tandem mass spectrometry

Lentils (Lens culinaris) represent important food staple replacing meat products in human diet with various applications of foods and feeds due to the high protein content. This work reported on the comparative proteomic profiling of four lentils commercial varieties Crimson, Eston, Laird and Black, providing novel knowledge on the differential expression of specific protein accessions with specific focus placed on the major protein families also investigating their allergenicity risk. The electrophoretic profile of lentil extracts confirmed that most of the proteomic profile was conserved across the investigated varieties, with only few differences highlighted for convicillin, vicilin and legumin subunits. A more in-depth analysis was carried out by one-shot discovery proteomics and provided the unequivocal identification and label-free quantification of 365 protein entries ranked into the main protein families. Statistically significant differences were disclosed for most of these protein groups and/or subgroups, cupins being the major contributors to the protein profile for all lentil samples. Noteworthy, the 7 S/11 S ratio describing seed nutritional quality highlighted clear differences among the four varieties with highest values reported for Eston and Laird samples. However, such higher nutritional quality was offset by a higher risk of allergenicity for sensitized individuals; indeed, the in-silico allergenicity prediction reported that most of the identified proteins presented either strong or weak evidence of immunogenicity with highest abundance in the Eston and Laird lentil samples. In perspective, the differential expression of specific protein accessions suggested the possibility to identify protein markers for varietal discrimination: a preliminary multivariate statistical analysis was accomplished to this aim.

Comparative Seed Proteome Profile Reveals No Alternation of Major Allergens in High-Yielding Mung Bean Cultivars.

Mung bean contains up to 32.6% protein and is one of the great sources of plant-based protein. Because many allergens also function as defense-related proteins, it is important to determine their abundance levels in the high-yielding, disease-resistant cultivars. In this study, for the first time, we compared the seed proteome of high-yielding mung bean cultivars developed by a conventional breeding approach. Using a label-free quantitative proteomic platform, we successfully identified and quantified a total of 1373 proteins. Comparative analysis between the high-yielding disease-resistant cultivar (MC5) and the other three cultivars showed that a total of 69 common proteins were significantly altered in their abundances across all cultivars. Bioinformatic analysis of these altered proteins demonstrated that PDF1 (a defensin-like protein) exhibited high sequence similarity and epitope matching with the established peanut allergens, indicating a potential mung bean allergen that showed a cultivar-specific response. Conversely, known mung bean allergen proteins such as PR-2/PR-10 (Vig r 1), Vig r 2, Vig r 4, LTP1, β-conglycinin, and glycinin G4 showed no alternation in the MC5 compared to other cultivars. Taken together, our findings suggest that the known allergen profiles may not be impacted by the conventional plant breeding method to develop improved mung bean cultivars.

Comparative Proteome Profiling of Extracellular Vesicles from Three Growth Phases of Haematococcus pluvialis under High Light and Sodium Acetate Stresses.

Extracellular vesicles (EVs) are nano-sized particles involved in intercellular communications that intrinsically possess many attributes as a modern drug delivery platform. Haematococcus pluvialis-derived EVs (HpEVs) can be potentially exploited as a high-value-added bioproduct during astaxanthin production. The encapsulation of HpEV cargo is a crucial key for the determination of their biological functions and therapeutic potentials. However, little is known about the composition of HpEVs, limiting insights into their biological properties and application characteristics. This study examined the protein composition of HpEVs from three growth phases of H. pluvialis grown under high light (350 µmol·m-2·s-1) and sodium acetate (45 mM) stresses. A total of 2038 proteins were identified, the majority of which were associated with biological processes including signal transduction, cell proliferation, cell metabolism, and the cell response to stress. Comparative analysis indicated that H. pluvialis cells sort variant proteins into HpEVs at different physiological states. It was revealed that HpEVs from the early growth stage of H. pluvialis contain more proteins associated with cellular functions involved in primary metabolite, cell division, and cellular energy metabolism, while HpEVs from the late growth stage of H. pluvialis were enriched in proteins involved in cell wall synthesis and secondary metabolism. This is the first study to report and compare the protein composition of HpEVs from different growth stages of H. pluvialis, providing important information on the development and production of functional microalgal-derived EVs.

Mitomycin C and its analog trigger cytotoxicity in MCF-7 and K562 cancer cells through the regulation of RAS and MAPK/ERK pathways

Mitomycin C (MC) is an anti-cancer drug which functions by forming interstrand crosslinks (ICLs) between opposing DNA strands. MC analog, 10-decarbamoyl mitomycin C (DMC), unlike MC, has stronger cytotoxic effects on cancer cells with TP53 mutation. We previously demonstrated that MC/DMC could activate p21WAF1/CIP1 in MCF-7 (TP53-proficient) and K562 (TP53 deficient) cells in a TP53-independent mode. We also found that MC/DMC regulate AKT activation in a TP53-dependent manner and that AKT deactivation is not associated with the activation of p21WAF1/CIP1 in response to MC/DMC treatment. RAS proteins are known players in the upstream mediated signaling of p21WAF1/CIP1 activation that leads to control of cell proliferation and cell death. Thus, this prompted us to investigate the effect of both drugs on the expression of RAS proteins and regulation of the MAPK/ERK signaling pathways in MCF-7 and K562 cancer cells. To accomplish this goal, we performed comparative label free proteomics profiling coupled to bioinformatics/complementary phosphoprotein arrays and Western blot validations of key signaling molecules. The MAPK/ERK pathway exhibited an overall downregulation upon MC/DMC treatment in MCF-7 cells but only DMC exhibited a mild downregulation of that same pathway in TP53 mutant K562 cells. Furthermore, treatment of MCF-7 and K562 cell lines with oligonucleotides containing the interstrand crosslinks (ICLs) formed by MC or DMC shows that both ICLs had a stronger effect on the downregulation of RAS protein expression in mutant TP53 K562 cells. We discuss the implication of this regulation of the MAPK/ERK pathway in relation to cellular TP53 status.

Urine proteome profile of firefighters with exposure to emergency fire-induced smoke: A pilot study to identify potential carcinogenic effects

Firefighters are frequently exposed to a variety of chemicals formed from smoke, which pose a risk for numerous diseases, including cancer. Comparative urine proteome profiling could significantly improve our understanding of the early detection of potential cancer biomarkers. In this study, for the first time, we conducted a comparative protein profile analysis of 20 urine samples collected from ten real-life firefighters prior to and following emergency fire-induced smoke. Using a label-free quantitative proteomics platform, we identified and quantified 1325 unique protein groups, of which 45 proteins showed differential expressions in abundance in response to fire-smoke exposure (post) compared to the control (pre). Pathway analysis showed proteins associated with epithelium development (e.g., RHCG, HEG1, ADAMTSL2) and Alzheimer's disease (SORL1) were significantly increased in response to smoke exposure samples. A protein-protein-network study showed a possible link between these differentially abundant proteins and the known cancer gene (TP53). Moreover, a cross-comparison analysis revealed that seven proteins—ALDH1A1, APCS, POMC, COL2A1, RDX, DDAH2, and SDC4 overlapped with the previously published urine cancer proteome datasets, suggesting a potential cancer risk. Our findings demonstrated that the discovery proteomic platform is a promising analytical technique for identifying potential non-invasive biomarkers associated with fire-smoke exposure in firefighters that may be related to cancer.

Proteomic profiling of Arabidopsis G-protein β subunit AGB1 mutant under salt stress.

Salt stress is a limiting environmental factor that inhibits plant growth in most ecological environments. The functioning of G-proteins and activated downstream signaling during salt stress is well established and different G-protein subunits and a few downstream effectors have been identified. Arabidopsis G-protein β-subunit (AGB1) regulates the movement of Na+ from roots to shoots along with a significant role in controlling Na+ fluxes in roots, however, the molecular mechanism of AGB1 mediated salt stress regulation is not well understood. Here, we report the comparative proteome profiles of Arabidopsis AGB1 null mutant agb1-2 to investigate how the absence of AGB1 modulates the protein repertoire in response to salt stress. High-resolution two-dimensional gel electrophoresis (2-DE) showed 27 protein spots that were differentially modulated between the control and NaCl treated agb1-2 seedlings of which seven were identified by mass spectrometry. Functional annotation and interactome analysis indicated that the salt-responsive proteins were majorly associated with cellulose synthesis, structural maintenance of chromosomes, DNA replication/repair, organellar RNA editingand indole glucosinolate biosynthesis. Further exploration of the functioning of these proteins could serve as a potential stepping stone for dissection of molecular mechanism of AGB1 functions during salt stress and in long run could be extrapolated to crop plants for salinity stress management.

Metabolic Profiling Reveals the Importance of Arginine Metabolism via the Arginine Deiminase Pathway in Vancomycin-intermediate S. aureus

Gene mutations located in VraSR and GraSR, two-component regulatory systems have been reported to cause vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). Subsequent comparative proteomic profiling between vancomycin-susceptible S. aureus (VSSA) and VISAs of the Mu50 genetic lineage revealed up-regulated level of catabolic ornithine carbamoyltransferase (ArcB) in the latter, suggesting a role of arginine catabolism in VISA development. This study aimed to further investigate metabolic pathways associated with VISA development using liquid chromatography mass spectrometry (LCMS)-based untargeted metabolomic profiling. Metabolite profiles were compared among 3 isogenic strains of the Mu50 lineage: Mu50Ω (VSSA), Mu50Ω-vraSm (VISA) and Mu50Ω-vraSm-graRm (VISA). Intracellular levels of α-hydroxyglutaric acid, choline, lysine, malic acid, N-acetylornithine, nicotinamide, and cystathionine were found to be significantly different between VISAs compared to VSSA. VISA cells were found to have lower levels of intracellular citrulline and extracellular arginine, with higher extracellular level of ornithine compared to VSSA. These differences in metabolite levels between VISA and VSSA suggested the importance of arginine metabolism. Activation of the arginine deiminase pathway (ADI) in VISAs of the Mu50 lineage could be further explored as a target for antibiotic development.

Proteomic analysis of extracellular vesicle cargoes mirror the cardioprotective effects of rivaroxaban in patients with venous thromboembolism.

Venous thromboembolism (VTE) remains a significant cause of morbidity and mortality worldwide. Rivaroxaban, a direct oral factor Xa inhibitor, mediates anti-inflammatory and cardiovascular-protective effects besides its well-established anticoagulant properties; yet, these remain poorly characterized. Extracellular vesicles (EVs) are considered proinflammatory messengers regulating a myriad of (patho)physiological processes and may be highly relevant to the pathophysiology of VTE. The effects of Rivaroxaban on circulating EVs in VTE patients remain unknown. We have established that differential EV biosignatures are found in patients with non-valvular atrial fibrillation anticoagulated with Rivaroxaban versus warfarin. Here, we investigated whether differential proteomic profiles of circulating EVs could also be found in patients with VTE. We performed comparative label-free quantitative proteomic profiling of enriched plasma EVs from VTE patients anticoagulated with either Rivaroxaban or warfarin using a tandem mass spectrometry approach. Of the 182 quantified proteins, six were found to be either exclusive to, or enriched in, Rivaroxaban-treated patients. Intriguingly, these proteins are involved in negative feedback regulation of inflammatory and coagulation pathways, suggesting that EV proteomic signatures may reflect both Rivaroxaban's anti-coagulatory and anti-inflammatory potential. These differences suggest Rivaroxaban may have pleiotropic effects, supporting the reports of its emerging anti-inflammatory and cardiovascular-protective characteristics relative to warfarin.

Proteome profiling of Paenibacillus sonchi genomovar Riograndensis SBR5T under conventional and alternative nitrogen fixation

Paenibacillus sonchi SBR5T is a Gram-positive, endospore-forming facultative aerobic diazotrophic bacterium that can fix nitrogen via an alternative Fe-only nitrogenase (AnfHDGK). In several bacteria, this alternative system is expressed under molybdenum (Mo)-limiting conditions when the conventional Mo-dependent nitrogenase (NifHDK) production is impaired. The regulatory mechanisms, metabolic processes, and cellular functions of N2 fixation by alternative and/or conventional systems are poorly understood in the Paenibacillus genus. We conducted a comparative proteomic profiling study of P. sonchi SBR5T grown under N2-fixing conditions with and without Mo supply through an LC-MS/MS and label-free quantification analysis to address this gap. Protein abundances revealed overrepresented processes related to anaerobiosis growth adaption, Fe-S cluster biosynthesis, ammonia assimilation, electron transfer, and sporulation under N2-fixing conditions compared to non-fixing control. Under Mo limitation, the Fe-only nitrogenase components were overrepresented together with the Mo-transporter system, while the dinitrogenase component (NifDK) of Mo‑nitrogenase was underrepresented. The dinitrogenase reductase component (NifH) and accessory proteins encoded by the nif operon had no significant differential expression, suggesting post-transcriptional regulation of nif gene products in this strain. Overall, this was the first comprehensive proteomic analysis of a diazotrophic strain from the Paenibacillaceae family, and it provided insights related to alternative N2-fixation by Fe-only nitrogenase. SignificanceIn this work, we try to understand how the alternative nitrogen fixation system, presented by some diazotrophic bacteria, works. For this, we used the SBR5 lineage of P. sonchi, which presents the alternative system in which the nitrogenase cofactor is composed only of iron. In addition, we tried to unravel the proteome of this strain in different situations of nitrogen fixation, since, for Gram-positive bacteria, these systems are little known. The results achieved, through LC-MS/MS and label-free quantitative analysis, showed an overrepresentation of proteins related to different processes involved with growth under stressful conditions in situations of nitrogen deficiency, in addition to suggesting that some encoded proteins by the nif operon may be regulated at post-transcriptional levels. Our findings represent important steps toward the elucidation of nitrogen fixation systems in Gram-positive diazotrophic bacteria.

Integrated “-omics” analysis highlights the role of brassinosteroid signaling and antioxidant machinery underlying improved rice seed longevity during artificial aging treatment

Seed longevity is a critical characteristic in agriculture, yet the specific genes/proteins responsible for this trait and the molecular mechanisms underlying reduced longevity during seed aging remain largely elusive. Here we report the comparative proteome and metabolome profiling of three rice cultivars exhibiting varying degrees of aging tolerance: Dharial, an aging-tolerant cultivar; Ilmi, an aging-sensitive cultivar; and A2, a moderately aging-tolerant cultivar developed from the crossbreeding of Dharial and Ilmi. Artificial aging treatment (AAT) markedly reduced the germination percentage and enhanced the activities of antioxidant enzymes in all the cultivars. Further, proteomics results showed a key role of the ubiquitin (Ub)-proteasome pathway in the degradation of damaged proteins during AAT while other proteases were majorly reduced. In addition, proteins associated with energy production and protein synthesis were strongly reduced in Ilmi while these were majorly increased in A2 and Dharial. These, along with metabolomics results, suggest that Ub-proteasome mediated protein degradation during AAT results in the accumulation of free amino acids in Ilmi while tolerant cultivars potentially utilize those for energy production and synthesis of stress-related proteins, especially hsp20/alpha-crystallin family protein. Additionally, both Dharial and A2 seem to activate brassinosteroid signaling and suppress jasmonate signaling which initiates a signaling cascade that allows accumulation of enzymatic and non-enzymatic antioxidants for efficient detoxification of aging-induced ROS. Taken together, these results provide an in-depth understanding of the aging-induced changes in rice seeds and highlight key pathways responsible for maintaining seed longevity during AAT.

Maltodextrin-binding protein as a key factor in Cronobacter sakazakii survival under desiccation stress

Cronobacter sakazakii (C. sakazakii) is a notorious pathogen responsible for infections in infants and newborns, often transmitted through contaminated infant formula. Despite the use of traditional pasteurization methods, which can reduce microbial contamination, there remains a significant risk of pathogenic C. sakazakii surviving due to its exceptional stress tolerance. In our study, we employed a comparative proteomic approach by comparing wild-type strains with gene knockout strains to identify the essential genes crucial for the successful survival of C. sakazakii during desiccation. Our investigation revealed the significance of envZ-ompR, recA, and flhD gene cassettes in contributing to desiccation tolerance in C. sakazakii. Furthermore, through our comparative proteomic profiling, we identified the maltodextrin-binding protein encoded by ESA_03421 as a potential factor influencing dry tolerance. This protein is regulated by EnvZ-OmpR, RecA, and FlhD. Notably, the knockout of ESA_03421 resulted in a 150% greater reduction in Log CFU compared to the wild-type C. sakazakii. Overall, our findings offer valuable insights into the mechanisms underlying C. sakazakii desiccation tolerance and provide potential targets for the development of new antimicrobial strategies aimed at reducing the risk of infections in infants and newborns.

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.

Comparative proteomic profiling represents an inhibition of protein synthesis to regulate osmotic stress in Nile tilapia (Oreochromis niloticus) embryos

Seawater (SW)-acclimated Nile tilapia, Oreochromis niloticus, can tolerate up to 30 g.L−1 SW but rarely produce offspring. The embryos of SW-acclimated O. niloticus survived equally well from 0- to 10-g.L−1 environment but not under 20-g. L−1. However, when the embryos were incubated under 10 g.L−1 during days 0–3, and then the salinity was suddenly shifted to and maintained at 20 g.L−1 during days 4–6, their survival rate was comparable to those incubated under 0 and 10 g.L−1. To elucidate a molecular adaptation of the embryos that survived different salinity environments, the proteomic profiles of the newly hatched embryos, or early larvae, hatched under 0 g.L−1, 10 g.L−1, and those being incubated at 10 g.L−1 during days 0–3 followed at 20 g.L−1 during days 4–6 were compared. Total proteins extracted from the samples were identified with a gel-free shot-gun proteomics approach using the Nile tilapia protein database. The early larvae from the three groups expressed 2295 proteins, and 279 proteins showed statistically different expressions among groups. Downregulation of the 182 proteins in the larvae hatched under 10 and 20 g.L−1 was found to include 22 proteins that are responsible for cellular responses to osmotic stress. This adaptation may be a crucial factor in reducing cellular metabolism and ion transport between the intra- and extra-cellular environment to stabilize cellular osmolality. In addition, some of these proteins suppress cellular damage from oxygen free radicals generated from the osmotic stress. Eighty-seven proteins significantly changed in the larvae hatched under 20 g.L−1 were clustered. Nineteen of the cellular stress response proteins, which were considered to be mortality induction, were described.

Unveiling the role of osteosarcoma-derived secretome in premetastatic lung remodelling

BackgroundLung metastasis is the most adverse clinical factor and remains the leading cause of osteosarcoma-related death. Deciphering the mechanisms driving metastatic spread is crucial for finding open therapeutic windows for successful organ-specific interventions that may halt or prevent lung metastasis.MethodsWe employed a mouse premetastatic lung-based multi-omics integrative approach combined with clinical features to uncover the specific changes that precede lung metastasis formation and identify novel molecular targets and biomarker of clinical utility that enable the design of novel therapeutic strategies.ResultsWe found that osteosarcoma-bearing mice or those preconditioned with the osteosarcoma cell secretome harbour profound lung structural alterations with airway damage, inflammation, neutrophil infiltration, and extracellular matrix remodelling with increased deposition of fibronectin and collagens by resident stromal activated fibroblasts, favouring the adhesion of disseminated tumour cells. Systemic-induced microenvironmental changes, supported by transcriptomic and histological data, promoted and accelerated lung metastasis formation. Comparative proteome profiling of the cell secretome and mouse plasma identified a large number of proteins involved in extracellular-matrix organization, cell-matrix adhesion, neutrophil degranulation, and cytokine-mediated signalling, consistent with the observed lung microenvironmental changes. Moreover, we identified EFEMP1, an extracellular matrix glycoprotein exclusively secreted by metastatic cells, in the plasma of mice bearing a primary tumour and in biopsy specimens from osteosarcoma patients with poorer overall survival. Depletion of EFEMP1 from the secretome prevents the formation of lung metastasis.ConclusionsIntegration of our data uncovers neutrophil infiltration and the functional contribution of stromal-activated fibroblasts in ECM remodelling for tumour cell attachment as early pro-metastatic events, which may hold therapeutic potential in preventing or slowing the metastatic spread. Moreover, we identified EFEMP1, a secreted glycoprotein, as a metastatic driver and a potential candidate prognostic biomarker for lung metastasis in osteosarcoma patients.Graphical abstractOsteosarcoma-derived secreted factors systemically reprogrammed the lung microenvironment and fostered a growth-permissive niche for incoming disseminated cells to survive and outgrow into overt metastasis.Daily administration of osteosarcoma cell secretome mimics the systemic release of tumour-secreted factors of a growing tumour in mice during PMN formation;Transcriptomic and histological analysis of premetastatic lungs revealed inflammatory-induced stromal fibroblast activation, neutrophil infiltration, and ECM remodelling as early onset pro-metastatic events;Proteome profiling identified EFEMP1, an extracellular secreted glycoprotein, as a potential predictive biomarker for lung metastasis and poor prognosis in osteosarcoma patients.Osteosarcoma patients with EFEMP1 expressing biopsies have a poorer overall survival.