Use Of Proteomics Research Articles

Use of proteomics to elucidate characteristics of Cronobacter sakazakii under mild heat stress

Cronobacter sakazakii is an opportunistic pathogen known for causing severe diseases. Mild heat treatment is commonly used in food processing, however, the pathogenic characteristics and underlying mechanisms of Cronobacter sakazakii strains remain poorly understood. In this study, we found that mild heat stress (MHS) at 52 °C can induce several deleterious effects in Cronobacter sakazakii, including damage to the cell wall, genomic DNA breakage, and misfolding of cytoplasmic proteins. These conditions lead to a decreased survival ability under acid, desiccation, and osmotic stress; a reduction in biofilm formation; and diminished motility. Notably, surviving C. sakazakii cells retain their pathogenicity, causing significant intestinal damage in newborn mice. This damage is characterized by epithelial sloughing and disruption of the intestinal structure. Tandem mass tag (TMT)-based proteomics identified 736 proteins with differential abundance across C. sakazakii strains subjected to mild heat stress, highlighting adaptations in biofilm formation, motility, and stress tolerance. Key regulatory changes were observed in phospholipid metabolism and protein synthesis, which underpin this complex stress response. This data illustrates a sophisticated balance between environmental adaptability and pathogenic potential. The metabolic and pathogenic responses of C. sakazakii to mild heat stress are closely linked to its phospholipid metabolism and the production of secretory proteins, both crucial for its virulence and reliant on membrane transport. This complex interplay emphasizes the need to understand these mechanisms to develop effective control strategies.

Proteomics Study of Benzene Metabolite Hydroquinone Induced Hematotoxicity in K562 Cells

ObjectiveHydroquinone (HQ), one of the phenolic metabolites of benzene, is widely recognized as an important participant in benzene-induced hematotoxicity. However, there are few relevant proteomics in HQ-induced hematotoxicity and the mechanism hasn't been fully understood yet. MethodsIn this study, we treated K562 cells with 40 μmol/L HQ for 72 h, examined and validated protein expression changes by Label-free proteomic analysis and Parallel reaction monitoring (PRM), and performed bioinformatics analysis to identify interaction networks. ResultsOne hundred and eighty-seven upregulated differentially expressed proteins (DEPs) and 279 downregulated DEPs were identified in HQ-exposed K562 cells, which were involved in neutrophil-mediated immunity, blood microparticle, and other GO terms, as well as the lysosome, metabolic, cell cycle, and cellular senescence-related pathways. Focusing on the 23 DEGs and 5 DEPs in erythroid differentiation-related pathways, we constructed the network of protein interactions and determined 6 DEPs (STAT1, STAT3, CASP3, KIT, STAT5B, and VEGFA) as main hub proteins with the most interactions, among which STATs made a central impact and may be potential biomarkers of HQ-induced hematotoxicity. ConclusionOur work reinforced the use of proteomics and bioinformatic approaches to advance knowledge on molecular mechanisms of HQ-induced hematotoxicity at the protein level and provide a valuable basis for further clarification.

Scientometrics Evaluation of Published Scientific Papers on the Use of Proteomics Technologies in Mastitis Research in Ruminants.

The objective of this study was the presentation of quantitative characteristics regarding the scientific content and bibliometric details of the relevant publications. In total, 156 papers were considered. Most papers presented original studies (n = 135), and fewer were reviews (n = 21). Most original articles (n = 101) referred to work involving cattle. Most original articles described work related to the diagnosis (n = 72) or pathogenesis (n = 62) of mastitis. Most original articles included field work (n = 75), whilst fewer included experimental (n = 31) or laboratory (n = 30) work. The tissue assessed most frequently in the studies was milk (n = 59). Milk was assessed more frequently in studies on the diagnosis (61.1% of relevant studies) or pathogenesis (30.6%) of the infection, but mammary tissue was assessed more frequently in studies on the treatment (31.0%). In total, 47 pathogens were included in the studies described; most were Gram-positive bacteria (n = 34). The three bacteria most frequently included in the studies were Staphylococcus aureus (n = 55 articles), Escherichia coli (n = 31) and Streptococcus uberis (n = 19). The proteomics technology employed more often in the respective studies was liquid chromatography-tandem mass spectrometry (LC-MS/MS), either on its own (n = 56) or in combination with other technologies (n = 40). The median year of publication of articles involving bioinformatics or LC-MS/MS and bioinformatics was the most recent: 2022. The 156 papers were published in 78 different journals, most frequently in the Journal of Proteomics (n = 16 papers) and the Journal of Dairy Science (n = 12). The median number of cited references in the papers was 48. In the papers, there were 1143 co-authors (mean: 7.3 ± 0.3 co-authors per paper, median: 7, min.-max.: 1-19) and 742 individual authors. Among them, 15 authors had published at least seven papers (max.: 10). Further, there were 218 individual authors who were the first or last authors in the papers. Most papers were submitted for open access (n = 79). The median number of citations received by the 156 papers was 12 (min.-max.: 0-339), and the median yearly number of citations was 2.0 (min.-max.: 0.0-29.5). The h-index of the papers was 33, and the m-index was 2. The increased number of cited references in papers and international collaboration in the respective study were the variables associated with most citations to published papers. This is the first ever scientometrics evaluation of proteomics studies, the results of which highlighted the characteristics of published papers on mastitis and proteomics. The use of proteomics in mastitis research has focused on the elucidation of pathogenesis and diagnosis of the infection; LC-MS/MS has been established as the most frequently used proteomics technology, although the use of bioinformatics has also emerged recently as a useful tool.

Research Trends in Proteomic Studies Using Serum from COVID-19 Patients: A Bibliometric Analysis.

The COVID-19 pandemic significantly impacted the global populace, resulting in a staggering number of deaths across the globe. New approaches and biomarkers to evaluate disease progression are crucial for improving disease management. In this context, serum proteomics has emerged as a promising tool for identifying molecular alterations related to COVID-19. This work carried out a bibliometric evaluation of the current status and trends of studies applying serum proteomics to COVID-19 subjects. The search was performed using Web of Science and Scopus databases, and the results were analyzed in VOSviewer software. The investigation was limited to articles published between January 2020 and February 2023. The analysis found 48 articles, primarily experimental studies. China is the most influential country in this field, followed by the USA. The co-occurrence analysis performed by VOSviewer showed 170 keywords, of which 9 reached the occurrence threshold and were divided into two groups. The most cited words were related to biomarker identification and the use of proteomics for diagnosing and treating COVID-19. The most cited proteins include those classically associated with the immune system (IgG, IgM, interleukins, CXCL, CCL, MCP, CRP) and SAA1, SAA1, ApoA-1, TTR (prealbumin), SerpinA and ITIH4. Other studies have validated the predictive value of these serum markers and have the potential to improve the management of COVID-19 patients. The findings highlighted in this bibliometric study can help the researchers design new projects to enhance our understanding of the complex interplay between SARS-CoV-2 and host immunity.

Key Proteomics Tools for Fundamental and Applied Microalgal Research.

Microscopic, photosynthetic prokaryotes and eukaryotes, collectively referred to as microalgae, are widely studied to improve our understanding of key metabolic pathways (e.g., photosynthesis) and for the development of biotechnological applications. Omics technologies, which are now common tools in biological research, have been shown to be critical in microalgal research. In the past decade, significant technological advancements have allowed omics technologies to become more affordable and efficient, with huge datasets being generated. In particular, where studies focused on a single or few proteins decades ago, it is now possible to study the whole proteome of a microalgae. The development of mass spectrometry-based methods has provided this leap forward with the high-throughput identification and quantification of proteins. This review specifically provides an overview of the use of proteomics in fundamental (e.g., photosynthesis) and applied (e.g., lipid production for biofuel) microalgal research, and presents future research directions in this field.

Using Electronic Health Record Data to Determine the Safety of Aqueous Humor Liquid Biopsies for Molecular Analyses

PurposeKnowing the surgical safety of anterior chamber liquid biopsies will support the increased use of proteomics and other molecular analyses to better understand disease mechanisms and therapeutic responses in patients and clinical trials. Manual review of operative notes from different surgeons and procedures in electronic health records (EHR) is cumbersome, but free-text software tools could facilitate efficient searches. DesignRetrospective case series. Participants1,418 aqueous humor (AH) liquid biopsies from patients undergoing intraocular surgery. MethodsFree-text EHR searches were performed using the Stanford Research Repository (STARR) cohort discovery tool to identify complications associated with anterior chamber paracentesis and subsequent endophthalmitis. Complications of the surgery unrelated to the biopsy were not reviewed. Main Outcome MeasuresBiopsy associated intraoperative complications and endophthalmitis. Results1,418 AH liquid biopsies were performed by 17 experienced surgeons. EHR free-text searches were 100% error-free for surgical complications, >99% for endophthalmitis (<1% false positive), and >93.6% for anesthesia type, requiring manual review for only a limited number of cases. More than 85% of cases were performed under local anesthesia without ocular muscle akinesia. Although the most common indication was cataract (50.1%), other diagnoses included glaucoma, diabetic retinopathy, uveitis, age-related macular degeneration, endophthalmitis, retinitis pigmentosa, and uveal melanoma. A 50-100μL sample was collected in all cases using either a 30-gauge needle or a blunt cannula via a paracentesis. The median follow-up was more than seven months. There was only one minor complication (0.07%) identified: a case of a small tear in Descemet’s membrane without long-term sequelae. No other complications occurred, including other corneal injuries, lens or iris trauma, hyphema, or suprachoroidal hemorrhage. There was no case of postoperative endophthalmitis. ConclusionsAnterior chamber liquid biopsy during intraocular surgery is a safe procedure and may be considered for large-scale collection of AH samples for molecular analyses. Free-text EHR searches are an efficient approach to reviewing intraoperative procedures.

Harnessing the power of proteomics in precision diabetes medicine.

Precision diabetes medicine (PDM) aims to reduce errors in prevention programmes, diagnosis thresholds, prognosis prediction and treatment strategies. However, its advancement and implementation are difficult due to the heterogeneity of complex molecular processes and environmental exposures that influence an individual's disease trajectory. To address this challenge, it is imperative to develop robust screening methods for all areas of PDM. Innovative proteomic technologies, alongside genomics, have proven effective in precision cancer medicine and are showing promise in diabetes research for potential translation. This narrative review highlights how proteomics is well-positioned to help improve PDM. Specifically, a critical assessment of widely adopted affinity-based proteomic technologies in large-scale clinical studies and evidence of the benefits and feasibility of using MS-based plasma proteomics is presented. We also present a case for the use of proteomics to identify predictive protein panels for type 2 diabetes subtyping and the development of clinical prediction models for prevention, diagnosis, prognosis and treatment strategies. Lastly, we discuss the importance of plasma and tissue proteomics and its integration with genomics (proteogenomics) for identifying unique type 2 diabetes intra- and inter-subtype aetiology. We conclude with a call for action formed on advancing proteomics technologies, benchmarking their performance and standardisation across sites, with an emphasis on data sharing and the inclusion of diverse ancestries in large cohort studies. These efforts should foster collaboration with key stakeholders and align with ongoing academic programmes such as the Precision Medicine in Diabetes Initiative consortium.

Potential urinary biomarkers in preeclampsia: a narrative review.

Preeclampsia (PE) is a highly relevant pregnancy-related disorder. An early and accurate diagnosis is crucial to prevent major maternal and neonatal complications and mortality. Due to the association of kidney dysfunction with the pathophysiology of the disease, urine samples have the potential to provide biomarkers for PE prediction, being minimally invasive and easy to perform. Therefore, searching for novel biomarkers may improve outcomes. This narrative review aimed to summarize the scientific literature about the traditional and potential urinary biomarkers in PE and to investigate their applicability to screen and diagnose the disorder. A non-systematic search was performed in PubMed/MEDLINE, Scopus, and SciELO databases. There is significant divergence in the literature regarding traditionally used serum markers creatinine, cystatin C, and albuminuria, accuracy in PE prediction. As for the potential renal biomarkers investigated, including vascular epithelial growth factor (VEGF), placental growth factor (PlGF), and soluble fms-like tyrosine kinase (sFlt-1), urinary levels of PlGF and sFtl-1/PlGF ratio in urine seem to be the most promising as screening tests. The assessment of the global load of misfolded proteins through urinary congophilia, podocyturia, and nephrinuria has also shown potential for screening and diagnosis. Studies regarding the use of proteomics and metabolomics have shown good accuracy, sensitivity, and specificity for predicting the development and severity of PE. However, there are still many divergences in the literature, which requires future and more conclusive research to confirm the predictive role of urinary biomarkers in pregnant women with PE.

High-resolution mass spectrometer-based identification of β-Casein variant (A2/A1) in the milk of Indian Holstein Friesian crossed cows

Foodomics tools are ameliorating the compositional and quality analysis part of various domains of food products. The use of proteomics in the polymorphic product investigation of beta-casein (β-Cn) is intriguing because of the fast and reliable results from the accurate mass spectrometry. This study delineates the LC-MS/MS-based sequential analysis of gel fractionated β-Cn of eighteen Holstein Friesian (HF) crossed cow milk for the identification of A2 milk. The probe of genetically induced polymorphism of β-Cn milk protein in the crossed animals is essential for the quality concern and fulfilling the demand of the consumers. Several in-vivo studies have shown that proteolytic digestion of the A1 milk secreted an opioid, i.e., β-casomorphin-7 that can cause many types of non-communicable diseases. SDS-PAGE was done on 15% resolving gel and an orbitrap mass spectrometer equipped with UniProt database search was used for the detection of the genetic variants in the HF crossed cows have shown ten animals are lactating milk with A2 β-Cn (Pro67), whereas only eight animals have A1 β-Cn (His67). The high number of HF-crossed cows are producing A2 milk. Among the molecular biology methods, top-down proteomics is the most efficient, accurate, and sustainable technique of milk protein genetic variant analysis.

Precision Medicine in Depression: The Role of Proteomics and Metabolomics in Personalized Treatment Approaches.

Depression, or major depressive disorder (MDD), is a widespread mental health condition marked by enduring feelings of sorrow and loss of interest. Treatment of depression frequently combines psychotherapy, medication, and lifestyle modifications. However, the occurrence of treatment resistance in certain individuals makes it difficult for physicians to effectively manage this disorder, calling for the implementation of alternative therapeutic strategies. Recently, precision medicine has gained increased attention in the field of mental health, paving the way for more personalized and effective therapeutic interventions in depression. Also known as personalized medicine, this approach relies on genetic composition, molecular profiles, and environmental variables to customize therapies to individual patients. In particular, precision medicine has offered novel viewpoints on depression through two specific domains: proteomics and metabolomics. On one hand, proteomics is the thorough study of proteins in a biological system, while metabolomics focuses on analyzing the complete set of metabolites in a living being. In the past few years, progress in research has led to the identification of numerous depression-related biomarkers using proteomics and metabolomics techniques, allowing for early identification, precise diagnosis, and improved clinical outcome. However, despite significant progress in these techniques, further efforts are required for advancing precision medicine in the diagnosis and treatment of depression. The overarching goal of this chapter is to provide the current state of knowledge regarding the use of proteomics and metabolomics in identifying biomarkers related to depression. It also highlights the potential of proteomics and metabolomics in elucidating the intricate processes underlying depression, opening the door for tailored therapies that could eventually enhance clinical outcome in depressed patients. This chapter finally discusses the main challenges in the use of proteomics and metabolomics and discusses potential future research directions.

SINGLE-CELL AND SPATIAL OMICS FOR MAPPING CELLULAR SENESCENCE IN HEALTH, AGING AND DISEASE

Abstract NIH SenNet consortium aims to dissect the heterogeneity of senescent cells (SnCs) and map their impact on the microenvironment at a single cell resolution and in the spatial tissue context, which requires the implementation of an array of omics technologies to comprehensively identify, characterize, and spatially profile SnCs across tissues in humans and mice. These technologies are broadly categorized into two groups –single cell omics and spatial mapping. To achieve single cell resolution and overcome the scarcity of SnCs, high-throughput single-cell and single-nucleus transcriptomic techniques have become a mainstay tool for surveying tens of thousands of cells to identify transcriptional signatures in rare cell populations, enabling discovery of potential new SnC biomarkers. Novel single cell mass spectrometry methods are developed for unbiased discovery of proteomic signatures of SnCs. A hallmark of SnCs is the senescence-associated secretory phenotype (SASP), which requires the use of proteomics, secretomics, metabolomics and lipidomics, especially SASP-associated extracellular vesicles, for comprehensive characterization of SAPS. High resolution molecular and cellular imaging of gene expression (e.g., MERFISH) or protein markers (e.g., CODEX) is critical for the study of SnCs in the large-scale tissue context. NGS-based spatial omics sequencing is poised to bridge the gap to realize both genome scale and cellular resolution in mapping SnCs in tissue. Novel technologies such as Seq-Scope and Pixel-Seq developed within SenNet further enabled subcellular resolution. SenNet investigators also developed spatially resolved epigenome and multi-omics sequencing techniques to link transcriptional or proteomic phenotype of SnCs to epigenetic mechanism. Further integration with high-resolution imaging makes spatial omics the crucial linchpin in connecting mechanistic underpinnings and molecular signatures with morphological features and spatial distribution. All these are critical for the construction of a map of SnCs and associated niches in the native tissue environment implicated in human health, aging, and disease, which is one of the main goals of the SenNet consortium.

Application of Proteomics in Alzheimer’s Disease: A Mini Review

Alzheimer’s disease (AD) is classified as one of neurodegenerative disease caused by neuronal death. It is characterized as memory impairment, including the inability to produce new memories. Since AD has low treatment effectiveness, proteomics research opens possibilities for advancement. Proteomics is the study of proteomes produced by the disease-bearing host to identify and understand diseases. In this case, to investigate the use of protein as a reliable molecular entity and their involvement in AD. Therefore, this review focused on three main applications of proteomics; the potential use of proteomics as a diagnostic tool for AD, the use of proteomics to assess the treatment progression of AD and the advancement in AD research. The review discussed three research areas utilizing the proteomics approach: ageing, behavioural, and demographic research of AD populations. Proteomic approaches have also been shown to be effective to discover the biomarkers for infectious diseases, cancers, heart diseases, and neurological disorders. Although much work remained to be done, the proteomics approach is an interesting method to be carried out in detecting AD at an earlier stage and will be very useful for AD treatment and management in the future.

Biochemical typing and evaluation of pathogenicity in vulvovaginal isolates of Candida albicans complex

Candida albicans, C. dubliniensis, and C. africana form the Candida albicans complex. To identify the phenotypic and pathogenic characteristics of isolates of the C. albicans complex preserved in a collection. Three hundred presumptive strains of the C. albicans complex were evaluated using CHROMagarTM Candida. Germ tube production was determined by three methods, chlamydospores formation was assessed and colonies were characterized in artisanal agars (Rosmarinus officinalis and Nicotiana tabacum). MALDI-TOF was used as the gold standard identification test. To detect pathogenicity factors, we evaluated the hemolytic activity of each isolate and cocultured with Staphylococcus aureus, coagulase enzyme production, and biofilm formation. Out of the 300 isolates, 43.7% produced germ tube in the heart-brain infusion broth and 47% of the isolates produced chlamydospores. In the artisan media, 6% of the isolates produced brown colonies on rosemary agar and 5% did so on tobacco agar. None of the strains hemolyzed the blood agar alone or cocultured with S. aureus. However, 50% of the isolates hemolyzed the potato dextrose agar supplemented with blood. All strains were coagulase producers, and biofilm production was variable. For germ tube production, the human serum method showed the same positivity as the milk broth method. All isolates were identified as C. albicans by MALDI-TOF. The use of proteomics, molecular tests or a combination of methods is required for species identification.

In-depth characterization of the sarcoplasmic muscle proteome changes in lambs fed with hazelnut skin by-products: Relationships with meat color

This study investigated the effect of agro-industrial hazelnut skin by-products supplementation on lamb meat color variation and the changes in the sarcoplasmic muscle proteome during post-mortem storage (0, 4 and 7 days). Gel-based proteomics and bioinformatics approaches were applied to better understand the potential role of feeding strategies in modulating the mechanisms underpinning meat discoloration and post-mortem changes during storage. Therefore, twenty-two Valle del Belice male lambs were randomly assigned to two dietary treatments: control (C), lambs fed with maize-barley diet, and hazelnut skin (H), lambs fed hazelnut skin by-product as maize partial replacer in the concentrate diet. Hazelnut dietary treatment led to better lamb meat color stability as evidenced by the lowest decrease in redness and saturation index values. Proteomics and bioinformatics results revealed changes in the abundance of 41 proteoforms, which were mainly involved in glycolytic processes, responses to oxidative stress, and immune and endocrine system. The proteins allowed revealing interconnected pathways to be behind meat color variation as a consequence of using hazelnut skin by-products to sustainable feed lamb. The proteins can be used as potential predictors of lamb meat color variation. Accordingly, the regression equations developed in this paper revealed triosephosphate isomerase (TPI1) as a reliable candidate biomarker of color stability in lamb meat. SignificanceThe use of agro-industrial by-products in animal feeding can be a potential sustainable strategy to reduce the environmental impacts of the food production chain and consequently improve animal welfare and product quality. The inclusion of hazelnut skin by-products in the animal's diet, due to the high concentration of polyphenols, represents an effective strategy to improve the oxidative stability of meat, with significant implications on color. The use of proteomics combined with bioinformatics on the sarcoplasmic proteome is a powerful approach to decipher the underlying mechanism. Accordingly, this approach allowed in this trial a deeper understanding of the molecular mechanisms involved in the post-mortem processes through the discovery of several biological pathways linked with lamb meat color variation. Glycolysis, followed by responses to oxidative stress, and other proteins involved in the immune and endocrine system were found as the major interconnected pathways that could act as potential predictors of lamb meat color stability. Candidate proteins biomarkers were further revealed in this study to be related with multiple meat color traits.

Better late than never: Optimising the proteomic analysis of field-collected octopus.

Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the sampling and preparation of animal tissues from the field. Although RNAlater is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require samples to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught samples. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNAlater can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNAlater immediately, 3 h, and 6 h after euthanasia. Processed tissue samples were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of sample treatment. However, nearly 10% additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that sampling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity.

OMICS and Other Advanced Technologies in Mycological Applications.

Fungi play many roles in different ecosystems. The precise identification of fungi is important in different aspects. Historically, they were identified based on morphological characteristics, but technological advancements such as polymerase chain reaction (PCR) and DNA sequencing now enable more accurate identification and taxonomy, and higher-level classifications. However, some species, referred to as "dark taxa", lack distinct physical features that makes their identification challenging. High-throughput sequencing and metagenomics of environmental samples provide a solution to identifying new lineages of fungi. This paper discusses different approaches to taxonomy, including PCR amplification and sequencing of rDNA, multi-loci phylogenetic analyses, and the importance of various omics (large-scale molecular) techniques for understanding fungal applications. The use of proteomics, transcriptomics, metatranscriptomics, metabolomics, and interactomics provides a comprehensive understanding of fungi. These advanced technologies are critical for expanding the knowledge of the Kingdom of Fungi, including its impact on food safety and security, edible mushrooms foodomics, fungal secondary metabolites, mycotoxin-producing fungi, and biomedical and therapeutic applications, including antifungal drugs and drug resistance, and fungal omics data for novel drug development. The paper also highlights the importance of exploring fungi from extreme environments and understudied areas to identify novel lineages in the fungal dark taxa.

Chronic fluoxetine treatment in socially-isolated rats modulates the prefrontal cortex synaptoproteome

Exposure to chronic social isolation (CSIS) and synapse dysfunction have been implicated in the etiology of major depressive disorder (MDD). Fluoxetine (Flx) has been widely used to treat MDD, but its mechanisms of action remain elusive. We employed comparative synaptoproteomics to investigate the changes in the levels of proteins and molecular signaling pathways in prefrontal cortical samples of adult male Wistar rats exposed to CSIS, a rat model of depression, and CSIS rats treated with chronic Flx and controls, using liquid chromatography coupled to tandem mass spectrometry. Flx-treated control rats showed a decreased level of proteins involved in vesicle-mediated transport, and a predominantly increased level of exocytosis-associated proteins. CSIS significantly reduced the level of proteins involved in the ATP metabolic process, clathrin-dependent endocytosis, and proteolysis. Flx treatment in CSIS rats stimulated synaptic vesicle trafficking by increasing the regulation of exo/endocytosis-associated proteins, proteins involved in synaptic plasticity including neurogenesis, Cox5a, mitochondria-associated proteins involved in oxidative phosphorylation, and ion transport proteins (Slc8a2, Atp1b2). Flx treatment resulted in an increased synaptic vesicle dynamic, plasticity and mitochondrial functionality, and a suppression of CSIS-induced impairment of these processes. Biological significanceIdentifying biomarkers of MDD and treatment response is the goal of many studies. Contemporary studies have shown that many molecular alterations associated with the pathophysiology of MDD reside within the synapse. As part of this research, a growing importance is the use of proteomics, as monitoring the changes in protein levels enables the identification of (possible) biochemical pathways and processes of importance for the development of depressive-like behavior and the efficacy of antidepressant treatments. We profiled proteomic changes representative of the development of CSIS-induced depressive-like behavior and the antidepressant effects of Flx. Our study has identified synaptosomal proteins and altered molecular pathways that may be potential markers of prefrontal cortical synaptic dysfunction associated with depressive-like behavior, and further clarified the mechanisms of depressive-like behavior and mode of action of Flx. Our findings indicate potential PFC synaptic targets for antidepressant treatment.

Control of TurboID-dependent biotinylation intensity in proximity ligation screens

Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system's inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilized avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions. SignificanceProximity biotinylation screens using the last generation biotin ligase TurboID represent a powerful approach for the characterisation of transient protein-protein interaction and signaling networks. However, a constant and high basal biotinylation rate and the associated cytotoxicity often preclude the use of this method in proteomic studies. We report a protocol based on modulation of free biotin levels that prevents the deleterious effects of TurboID while allowing inducible biotinylation, even in subcellular compartments such as the endoplasmic reticulum. This optimised protocol greatly expands the applications of TurboID in proteomic screens.

Magnetic responsive hydroxyapatite scaffold modulated macrophage polarization through PPAR/JAK-STAT signaling and enhanced fatty acid metabolism

Despite the general observations of bone repair with magnetic cues, the mechanisms of magnetic cues in macrophage response during bone healing have not been systematically investigated. Herein, by introducing magnetic nanoparticles into hydroxyapatite scaffolds, an appropriate and timely transition from proinflammatory (M1) to anti-inflammatory (M2) macrophages during bone healing is achieved. The combined use of proteomics and genomics analysis reveals the underlying mechanism of magnetic cue-mediated macrophage polarization form the perspective of protein corona and intracellular signal transduction. Our results suggest that intrinsically-present magnetic cues in scaffold contribute to the upregulated peroxisome proliferator-activated receptor (PPAR) signals, and the activation of PPAR signal transduction in macrophages results in the downregulation of the Janus Kinase-Signal transducer and activator of transcription (JAK-STAT) signals and the enhancement of fatty acid metabolism, thus facilitating M2 polarization of macrophages. Magnetic cue-dependent changes in macrophage benefit from the upregulation of adsorbed proteins associated with “hormone” and “response to hormone”, as well as the downregulation of adsorbed proteins related to “enzyme-linked receptor signaling” in the protein corona. In addition, magnetic scaffolds may also act cooperatively with the exterior magnetic field, showing further inhibition of M1-type polarization. This study demonstrates that magnetic cues play critical roles on M2 polarization, coupling protein corona, intracellular PPAR signals and metabolism.

The Need for Biomarkers in the ALS-FTD Spectrum: A Clinical Point of View on the Role of Proteomics.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are severely debilitating and progressive neurodegenerative disorders. A distinctive pathological feature of several neurodegenerative diseases, including ALS and FTD, is the deposition of aberrant protein inclusions in neuronal cells, which leads to cellular dysfunction and neuronal damage and loss. Despite this, to date, the biological process behind developing these protein inclusions must be better clarified, making the development of disease-modifying treatment impossible until this is done. Proteomics is a powerful tool to characterize the expression, structure, functions, interactions, and modifications of proteins of tissue and biological fluid, including plasma, serum, and cerebrospinal fluid. This protein-profiling characterization aims to identify disease-specific protein alteration or specific pathology-based mechanisms which may be used as markers of these conditions. Our narrative review aims to highlight the need for biomarkers and the potential use of proteomics in clinical practice for ALS-FTD spectrum disorders, considering the emerging rationale in proteomics for new drug development. Certainly, new data will emerge in the near future in this regard and support clinicians in the development of personalized medicine.