Label-free Quantitative Proteomic Analysis Research Articles

MiR-215 Modulates Ubiquitination to Impair Inflammasome Activation and Autophagy During Salmonella Typhimurium Infection in Porcine Intestinal Cells

The host response to S. Typhimurium infection can be post-transcriptionally regulated by miRNAs. In this study, we investigated the role of miR-215 using both in vivo porcine infection models and in vitro intestinal epithelial cell lines. Several miRNAs were found to be dysregulated in the porcine ileum during infection with wild-type and SPI2-defective mutant strains of S. Typhimurium, with some changes being SPI2-dependent. Notably, miR-215 was significantly downregulated during infection. To explore its functional role, gain-of-function experiments were performed by transfecting porcine intestinal epithelial cells (IPEC-J2) with a miR-215-5p mimic, followed by label-free quantitative (LFQ) proteomic analysis. This analysis identified 157 proteins, of which 35 were downregulated in response to miR-215 overexpression, suggesting they are potential targets of this miRNA. Among these, E2 small ubiquitin-like modifier (SUMO)-conjugating enzyme UBC9 and E3 ubiquitin-ligase HUWE1 were identified as key targets, both of which are upregulated during S. Typhimurium infection. The miR-215-mediated downregulation of these proteins resulted in a significant decrease in overall ubiquitination, a process crucial for regulating inflammasome activation and autophagy. Consistently, inflammasome markers caspase 1 (CASP1) and apoptosis-associated speck-like protein containing a CARD (ASC), as well as autophagy markers microtubule-associated protein 1A/1B-light chain 3 (LC3B) and Ras-related protein Rab-11 (RAB11A), showed decreased expression in miR-215 mimic-transfected and infected IPEC-J2 cells. To further validate these findings, human intestinal epithelial cells (HT29) were used as a complementary model, providing additional insights into conserved immune pathways and extending the observations made in the porcine system. Overall, our findings demonstrate that miR-215 plays a significant role in modulating host inflammasome activation and autophagy by targeting proteins involved in ubiquitination during S. Typhimurium infection.

Neutrophil Elastase Targets Select Proteins on Human Blood-Monocyte-Derived Macrophage Cell Surfaces.

Neutrophil elastase (NE) has been reported to be a pro-inflammatory stimulus for macrophages. The aim of the present study was to determine the impact of NE exposure on the human macrophage proteome and evaluate its impact on pro-inflammatory signals. Human blood monocytes from healthy volunteers were differentiated to macrophages and then exposed to either 500 nM of NE or control vehicle for 2 h in triplicate. Label-free quantitative proteomics analysis identified 41 differentially expressed proteins in the NE versus control vehicle datasets. A total of 26 proteins were downregulated and of those, 21 were cell surface proteins. Importantly, four of the cell surface proteins were proteoglycans: neuropilin 1 (NRP1), syndecan 2 (SDC2), glypican 4 (GPC4), and CD99 antigen-like protein 2 (CD99L2) along with neuropilin 2 (NRP2), CD99 antigen (CD99), and endoglin (ENG) which are known interactors. Additional NE-targeted proteins related to macrophage function were also measured including CD40, CD48, SPINT1, ST14, and MSR1. Collectively, this study provides a comprehensive unbiased view of selective NE-targeted cell surface proteins in chronically inflamed lungs.

Quantitative Proteomic Analysis of APP/PS1 Transgenic Mice.

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder affecting the central nervous system (CNS), with its etiology still shrouded in uncertainty. The interplay of extracellular amyloid-β (Aβ) deposition, intracellular neurofibrillary tangles (NFTs) composed of tau protein, cholinergic neuronal impairment, and other pathogenic factors is implicated in the progression of AD. The current study endeavors to delineate the proteomic landscape alterations in the hippocampus of an AD murine model, utilizing proteomic analysis to identify key physiological and pathological shifts induced by the disease. This endeavor aims to shed light on the underlying pathogenic mechanisms, which could facilitate early diagnosis and pave the way for novel therapeutic interventions for AD. To dissect the proteomic perturbations induced by Aβ and Presenilin-1 (PS1) in the AD pathogenesis, we undertook a label-free quantitative (LFQ) proteomic analysis focusing on the hippocampal proteome of the APP/PS1 transgenic mouse model. Employing a multi-faceted approach that included differential protein functional enrichment, cluster analysis, and protein-protein interaction (PPI) network analysis, we conducted a comprehensive comparative proteomic study between APP/PS1 transgenic mice and their wild-type C57BL/6 counterparts. Mass spectrometry identified a total of 4817 proteins in the samples, with 2762 proteins being quantifiable. Comparative analysis revealed 396 proteins with differential expression between the APP/PS1 and control groups. Notably, 35 proteins exhibited consistent temporal regulation trends in the hippocampus, with concomitant alterations in biological pathways and PPI networks. This study presents a comparative proteomic profile of transgenic (APP/PS1) and wild-type mice, highlighting the proteomic divergences. Furthermore, it charts the trajectory of proteomic changes in the AD mouse model across the developmental stages from 2 to 12 months, providing insights into the physiological and pathological implications of the disease-associated genetic mutations.

Label-free quantitative proteomic analysis reveals the characteristics of ovarian development from stage II to III in Chinese sturgeon (Acipenser sinensis)

Chinese sturgeon (Acipenser sinensis) is an endangered anadromous and rare fish. The ovary development from stage II to III is being challenged by captive conditions, which seriously hinders the artificial reproduction of Chinese sturgeon. A comparative proteomic analysis on the ovaries at stage II and stage III of Chinese sturgeon was conducted in this study using the label-free quantitative method to uncover the intricate physiological processes. The findings revealed 200 up-regulated differentially expressed proteins (DEPs) and 150 down-regulated DEPs in the ovary at stage III compared with stage II, and 11 DEPs (including UCHL1, VTG, CTSD, and ZP, etc.) involved in the vitellogenesis and oocyte growth. Moreover, the up-regulated DEPs exhibited significant enrichment in the pathways linked to protein synthesis (ribosome, protein processing in the endoplasmic), metabolism and energy production (valine, leucine and isoleucine degradation, TCA cycle, oxidative phosphorylation, and fatty acid degradation), suggesting more active protein processing and energy metabolism at stage III. Meanwhile, APOB was down-regulated while VTG was up-regulated in stage III ovary, indicating the uptake of yolk protein and lipid through VTG at this stage. The parallel reaction monitoring (PRM) quantification of 16 DEPs solidly validated the outcomes of label-free analysis. This study provided powerful information for understanding the physiological processes of ovarian development in Chinese sturgeon, and potential biomarkers in predicting the vitellogenic stage.

Proteomic Profiling of COVID-19 Patients Sera: Differential Expression with Varying Disease Stage and Potential Biomarkers.

Background/Objectives: SARS-CoV-2 is one of the viruses that caused worldwide health issues. This effect is mainly due to the wide range of disease prognoses it can cause. The aim of this study is to determine protein profiles that can be used as potential biomarkers for patients' stratification, as well as potential targets for drug development. Methods: Eighty peripheral blood samples were collected from heathy as well as SARS-CoV-2 patients admitted at a major tertiary care center in Riyadh, Saudi Arabia. A label-free quantitative mass spectrometry-based proteomic analysis was conducted on the extracted sera. Protein-protein interactions and functional annotations of identified proteins were performed using the STRING. Results: In total, two-hundred-eighty-eight proteins were dysregulated among all four categories. Dysregulated proteins were mainly involved in the network map of SARS-CoV-2, immune responses, complement activation, and lipid transport. Compared to healthy subjects, the most common upregulated protein in all three categories were CRP, LGALS3BP, SAA2, as well as others involved in SARS-CoV-2 pathways such as ZAP70 and IGLL1. Notably, we found fifteen proteins that significantly discriminate between healthy/recovered subjects and moderate/under medication patients, among which are the SERPINA7, HSPD1 and TTC41P proteins. These proteins were also significantly downregulated in under medication versus moderate patients. Conclusions: Our results emphasize the possible association of specific proteins with the SARS-CoV-2 pathogenesis and their potential use as disease biomarkers and drug targets. Our study also gave insights about specific proteins that are likely increased upon infection but are likely restored post recovery.

Label-Free Quantitative Proteomics Analysis of Nasal Lavage Fluid in Chronic Rhinosinusitis with Nasal Polyposis.

(1) Background: Chronic rhinosinusitis (CRS) is a common chronic inflammation of the nasal mucosa and the paranasal sinuses. The pathogenesis of chronic rhinosinusitis (CRS) is multifactorial and, as of yet, not well understood. (2) Methods: Nasal lavage fluid samples were collected from patients diagnosed with chronic sinusitis with nasal polyposis (CRSwNP) (n = 10) and individuals without sinusitis (control group) (n = 10) who had no nasal complaints. In the present study, we used an untargeted label-free LC-MS/MS mass spectrometric approach combined with bioinformatics and network pathway analysis to compare the changes in the proteomic profiles of the CRSwNP group and the control group. Data from LC-MS/MS underwent univariate and multivariate analyses. (3) Results: The proteomic analyses revealed distinct differences in the abundances of nasal lavage fluid proteins between the CRSwNP and control groups: a total of 234 proteins, 151 up- and 83 down-regulated in CRSwNP. Functional Gene Ontology (GO) analysis showed that dysregulated proteins were involved in airway inflammatory reaction, immune response, and oxidative stress. The biomarkers were evaluated using the Receiver Operating Characteristic (ROC) curve; an Area Under the Curve (AUC) of 0.999 (95% CI) identified potential biomarkers between the CRSwNP and control group. EMILIN-3 and RAB11-binding protein RELCH were down-regulated, and Macrophage migration inhibitory factor and deoxyribonuclease-1 were up-regulated, in CRSwNP compared to the control group. (4) Conclusions: These differentially expressed proteins identified in CRSwNP are involved in airway inflammatory reaction, immune response, and oxidative stress. In particular, the identification of increased interleukin-36 gamma (IL-36γ), which contributes to inflammatory response, and a decrease in SOD, in this group are notable findings. In the future, several of these proteins may prove useful for exploring the pathogenesis of nasal polyps and chronic sinusitis or as objective biomarkers for quantitatively monitoring disease progression or response to therapy.

Characterisation of proteomic alterations in worker bees in response to amitraz treatment during summer to winter transition in Apis mellifera colonies

The application of treatments to reduce/control the number of Varroa destructor mites within Apis mellifera colonies is a common apicultural practice. The effectiveness of the treatments in reducing Varroa mites from colonies has been extensively studied, however, the effects of these treatments on the bees within the colonies are poorly characterised. This work utilised label-free quantitative proteomic analysis to investigate how the presence of the anti-Varroa treatment amitraz, affected worker bees. Samples of A. mellifera were isolated from colonies one week before amitraz treatment (T0) and 1, 3, 6 and 8 weeks post-treatment application. The trial was conducted during the transition period from short-lived summer workers to long-lived winter workers. The results highlight two large proteomic shifts; a decrease in the abundance of cuticular proteins and an increase in ribosomal protein abundance in Weeks 3, 6, and 8. The changes in the protein abundance in Week 8 may not be a response to amitraz exposure alone but may also be attributed to a potential change in the population of short-lived to long-lived winter bees. The work provides insight into the effect of amitraz on the honey bee proteome during the transition period from summer to winter colony and how it may contribute to inducing a stress response in individual bees.

Chicoric acid exerts therapeutic effects in DSS-induced ulcerative colitis by targeting the USP9X/IGF2BP2 axis.

Chicoric acid, a hydroxycinnamic acid, exhibits anti-inflammation activities. However, the specific mechanisms underlying the effects of chicoric acid on dextran sulfate sodium (DSS)-induced colitis remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the protective effects of chicoric acid in DSS-induced colitis. Mice with DSS-induced colitis (UC mice) were treated for a week with chicoric acid. Symptoms of colitis, colonic pathology, inflammation-related indicators, and intestinal mucosal barrier function were evaluated. RNA sequencing was performed on colon tissues to obtain differentially expressed genes. The deubiquitinating enzyme USP9X was selected, and the inhibitory and targeting effects of chicoric acid on USP9X were subsequently determined. In vivo and in vitro, DSS-induced colitis was treated with USP9X inhibitors WP1130 and EOAI3402143. Ubiquitination label-free quantitative proteomic analysis was performed to identify protein peptides that may undergo de-ubiquitination by USP9X. Co-immunoprecipitation (Co-IP), immunohistochemistry and western blotting were used to validate in vivo and in vitro results. Chicoric acid significantly alleviated clinical activity and histological changes, inhibited pro-inflammatory cytokine production and improved integrity of the intestinal barrier in UC mice. Moreover, chicoric acid suppressed USP9X expression in colonic tissues from UC mice. Furthermore, USP9X contributed to promoting the onset of UC and that insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) was deubiquitinated by USP9X. Chicoric acid ameliorated DSS-induced colitis by targeting the USP9X/IGF2BP2 axis, indicating that targeting the USP9X/IGF2BP2 axis presents a promising and innovative therapeutic approach for the treatment of UC.

Study on the mechanism of echinacoside in preventing and treating hypoxic pulmonary hypertension based on proteomic analyses.

Hypoxic pulmonary hypertension (HPH), a chronic condition affecting the cardiopulmonary system, has high mortality. Echinacoside (ECH) is a phenylethanoid glycoside, which is used to ameliorate pulmonary vascular remodeling and pulmonary vasoconstriction in rats. Accordingly, we aimed to explore the mechanism of ECH in preventing and treating HPH. Sprague Dawley rats were housed in a hypobaric hypoxia chamber for 28 days to obtain the HPH model. The experimental rats were randomly allocated into the following several groups: normoxia group, chronic hypoxia group, and ECH group. The therapeutic results of ECH (10, 20, and 40 mg/kg) showed that ECH reduced mPAP, Hb, Hct, and RVHI in HPH rats. Then this work employed label-free quantitative proteomic analysis, western blotting, and RT-PCR to investigate the mechanism by which ECH prevents HPH. The results found that in the chronic hypoxia group, the levels of ACSL1, COL6A1, COL4A2, COL1A1, and PC increased compared to the normoxia group. However, the opposite effect was observed in the chronic hypoxia group treated with ECH. The study indicates that the administration of ECH may slow the pathological progression of HPH by suppressing the inflammatory response, inhibiting smooth muscle cell proliferation, and minimizing the deposition of extracellular matrix.

Characterizing Leishmania infantum-induced resistance to trivalent stibogluconate (SbIII) through deep proteomics

Leishmania infantum belongs to the L. donovani complex, which includes species associated with visceral leishmaniasis. Traditionally, antimonial compounds have served as the primary antiparasitic treatment for all clinical forms of leishmaniasis. However, the global spread of resistance to these compounds has posed a significant challenge in the treatment in some regions. In this study, we aimed to investigate resistance to trivalent sodium stibogluconate in vitro using promastigotes from a wild strain of L. infantum. We compared the growth rates and proteomic profiles of wild-type and resistant line conducting label-free quantitative mass spectrometry-based proteomic analyses. Statistical and bioinformatics analyses were employed to evaluate the significance of protein concentration changes, protein identity annotation, GO term analysis, biosynthetic pathways, and protein-protein interactions. Our findings revealed that the resistant line displayed a notable reduction in growth rate. Proteomic data unveiled similar protein concentrations per cell in both groups but with differing molecule copy numbers. We identified 165 proteins with increased concentration, these were associated with transcription and translation activities, lipid metabolism, energy metabolism, and peroxisome biogenesis. In the decreased protein groups were 56 proteins linked to metal acquisition and metabolism, particularly iron. These results suggest a novel perspective on antimonial resistance, highlighting the importance of post-transcriptional and post-translational regulation, alongside energy expenditure compensation and alterations in organelle membrane lipid composition in antimonial-resistant parasites. Overall, our study provides insights into the proteomic profile of stibogluconate-resistant strain, contributing to our general understanding of the complex landscape of antiparasitic resistance in L. infantum. SignificanceSpecies within the Leishmania donovani complex are implicated in cases of visceral leishmaniasis in the world. Leishmania infantum is a species that predominates in regions spanning the Mediterranean Basin, the Middle East, Central Asia, South and Central America. Antimonials were the first treatment for leishmaniasis, however in the last decades, the resistance has emerged in subregions like India, where it is not a therapeutic option. In contrast, sodium stibogluconate (SbIII) remains the first-line treatment in the Americas. Unfortunately, the emergence of resistance has outpaced the development of new therapeutic options, thereby becoming a critical point in the struggle against the disease. In this study we performed an in-depth proteomic analysis with liquid chromatography mass-mass spectrometry (LC-MS/MS) on L. infantum with Sb-induced resistance in vitro. Results showed a complex proteomic adaptation in the resistant line, involving transcriptional and translational proteins, energy compensation, and homeostasis maintenance. These insights contribute to understanding the molecular adaptation in the parasite and provide information to new investigations related to therapeutics development.

Antioxidant Peptides Derived from Cheese Products via Single and Mixed Lactobacillus Strain Fermentation.

Probiotics are used in cheese fermentation to endow the product with unique functional properties, such as enhanced flavor and aroma development through proteolysis and lipolysis. In this study, two probiotic Lactobacillus strains, Lactobacillus plantarum A3 and Lactobacillus reuteri WQY-1, were selected to develop new probiotic cheeses in the form of single- and mixed-strain starters. The results demonstrated that the L. plantarum A3 single-strain group and the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group exhibited superior product performance, particularly the release of functional hydrolysates during cheese ripening. Furthermore, Label-free quantitative proteomic analysis revealed 26 unique antioxidant peptides in the L. plantarum A3 single-strain group and 53 in the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group. Among these, CMENSAEPEQSLACQCL (β-lactoglobulin), CMENSAEPEQSLVCQCL (β-lactoglobulin), and IQYVLSR (κ-casein) have been found to possess potential antioxidant properties both in vitro and in vivo. This confirmed that milk-derived protein peptides in cheese products exhibit potential antioxidant functions through the hydrolysis of probiotic strains.

Label-Free Quantitative Proteomics Analysis of COVID-19 Vaccines by Nano LC-HRMS.

A nanoliter liquid chromatography-high resolution mass spectrometry-based method was developed for quantitative proteomics analysis of COVID-19 vaccines. It can be used for simultaneous qualitative and quantitative analysis of target proteins and host cell proteins (HCPs) in vaccine samples. This approach can directly provide protein information at the molecular level. Based on this, the proteomes of 15 batches of COVID-19 inactivated vaccine samples from two companies and 12 batches of COVID-19 recombinant protein vaccine samples from one company were successfully analyzed, which provided a significant amount of valuable information. Samples produced in different batches or by different companies can be systematically contrasted in this way, offering powerful supplements for existing quality standards. This strategy paves the way for profiling proteomics in complex samples and provides a novel perspective on the quality evaluation of bio-macromolecular drugs.

205 An autosomal recessive mutation in PYGM causes myophosphorylase deficiency in composite cattle

Abstract Between 2020 and 2022, eight calves (6 heifers, 2 bulls) in a Nebraska herd (composite Simmental, Red Angus, Gelbvieh) experienced exercise intolerance during extended physical activity. These calves exhibited an inability to keep pace with the rest of the herd, with some eventually succumbing to physical collapse from which they did not recover. Increased creatine kinase, an indication of muscle degeneration, was confirmed in biopsy and necropsy samples. Available sire pedigrees revealed a common paternal ancestor within 2 to 4 generations in all the affected calves. Dam pedigrees were unavailable; however, the herd retained replacement females that occasionally shared common ancestors with breeding bulls. Thus, a de novo autosomal recessive mutation was hypothesized as the cause of exercise intolerance in these calves. Genomic data (100K SNP) from 6 affected calves and 715 herd mates were initially used for a whole genome-wide association study, pinpointing a significant region on chromosome 29. Whole-genome sequencing of 2 affected calves combined with 145 other sequenced cattle led to the identification of a nonsense mutation in the PYGM gene, also located on chromosome 29. The mutation, confirmed to be present in the skeletal muscle transcriptome, was predicted to produce a premature stop codon. The protein product of PYGM, myophosphorylase, has a crucial role in metabolizing glycogen stored within skeletal muscle to generate energy for muscle cells. As a result, the recessive genotype led to greater (P < 0.05) glycogen concentrations in affected calves (n = 2) compared with heterozygous animals (n = 3) and wild-type controls (n = 2). Immunohistochemistry and label-free quantitative proteomics analysis confirmed the absence of the PYGM protein product in skeletal muscle. Myophosphorylase is needed for efficient glycogen utilization, which is crucial for producing high-quality beef. In the 2 affected calves harvested, increased skeletal muscle glycogen persisted postmortem, resulting in increased pH and dark-cutting beef, which is associated with a negative consumer perception and economic losses to the industry. In addition to identifying the cause of exercise intolerance and welfare concerns in these cattle, this study is among the first to identify a specific genetic mutation associated with dark-cutting beef. Carriers of the mutation did not display any discernible differences in meat quality or measures of animal well-being. While cattle heterozygous for the mutation may not immediately impact the beef industry, identifying carriers is paramount for implementing proactive selection and breeding strategies to prevent the production of affected calves.

Proteomic Profile of Circulating Extracellular Vesicles in the Brain after Δ9-Tetrahydrocannabinol Inhalation.

Given the increasing use of cannabis in the US, there is an urgent need to better understand the drug's effects on central signaling mechanisms. Extracellular vesicles (EVs) have been identified as intercellular signaling mediators that contain a variety of cargo, including proteins. Here, we examined whether the main psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), alters EV protein signaling dynamics in the brain. We first conducted in vitro studies, which found that THC activates signaling in choroid plexus epithelial cells, resulting in transcriptional upregulation of the cannabinoid 1 receptor and immediate early gene c-fos, in addition to the release of EVs containing RNA cargo. Next, male and female rats were examined for the effects of either acute or chronic exposure to aerosolized ('vaped') THC on circulating brain EVs. Cerebrospinal fluid was extracted from the brain, and EVs were isolated and processed with label-free quantitative proteomic analyses via high-resolution tandem mass spectrometry. Interestingly, circulating EV-localized proteins were differentially expressed based on acute or chronic THC exposure in a sex-specific manner. Taken together, these findings reveal that THC acts in the brain to modulate circulating EV signaling, thereby providing a novel understanding of how exogenous factors can regulate intercellular communication in the brain.

VEGF-B prevents chronic hyperglycemia-induced retinal vascular leakage by regulating the CDC42-ZO1/VE-cadherin pathway.

Non-proliferative diabetic retinopathy (NPDR) is the early stage of diabetic retinopathy (DR) and is a chronic oxidative stress-related ocular disease. Few treatments are approved for early DR. This study aimed to investigate the pathogenic mechanisms underlying the retinal micro-vasculopathy induced by diabetes and to explore an early potential for treating early DR in a mouse model. The mouse model of type 1 diabetes was established by intraperitoneal injection of streptozotocin (STZ, 180 mg/kg), which was used as the early DR model. The body weight and blood glucose mice were measured regularly; The retinal vascular leakage in the early DR mice was determined by whole-mount staining; Label-free quantitative proteomic analysis and bioinformatics were used to explore the target proteins and signaling pathways associated with the retinal tissues of early DR mice; To detect the effects of target protein on endothelial cell proliferation, migration, and tube formation, knockdown and overexpression of VEGF-B were performed in human retinal vascular endothelial cells (HRECs); Western blotting was used to detect the expression of target proteins invitro and invivo; Meanwhile, the therapeutic effect of VEGF-B on vascular leakage has also been evaluated invitro and invivo. The protein expressions of vascular endothelial growth factor (VEGF)-B and the Rho GTPases family member CDC42 were reduced in the retinal tissues of early DR. VEGF-B upregulated the expression of CDC42/ZO1/VE-cadherin and prevented hyperglycemia-induced vascular leakage in HRECs. Standard intravitreal VEGF-B injections improved the retinal vascular leakage and neurovascular response in early DR mice. Our findings demonstrated, for the first time, that in diabetes, the retinal vessels are damaged due to decreased VEGF-B expression through downregulation of CDC42/ZO1/VE-cadherin expression. Therefore, VEGF-B could be used as a novel therapy for early DR.

Unmasking of molecular players: proteomic profiling of vitreous humor in pathologic myopia

BackgroundThis study aimed to identify the differentially expressed proteins in the vitreous humor (VH) of eyes with and without pathologic myopia (PM), providing insights into the molecular pathogenesis.MethodsA cross-sectional, observational study was conducted. VH samples were collected from patients undergoing vitrectomy for idiopathic epiretinal membrane (ERM), macular hole (MH), or myopic retinoschisis (MRS). Label-free quantitative proteomic analysis identified differential protein expression, with validation using ELISA.ResultsThe proteomic profiling revealed significantly higher expressions of tubulin alpha 1a (TUBA1A) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) in PM groups (MH-PM, MRS-PM) compared to controls (MH, ERM). Conversely, xylosyltransferase 1 (XYLT1), versican core protein (VCAN), and testican-2 (SPOCK2) expressions were lower in PM. ELISA validation confirmed these findings.ConclusionsOur study provides novel insights into the molecular mechanisms of PM. The differentially expressed proteins EEF1A1, TUBA1A, XYLT1, VCAN, and SPOCK2 may play crucial roles in chorioretinal cell apoptosis, scleral extracellular matrix (ECM) synthesis, and scleral remodeling in PM. These proteins represent potential new targets for therapeutic intervention in PM, highlighting the importance of further investigations to elucidate their functions and underlying mechanisms in disease pathogenesis.

Comparative Proteomic Analysis Provides New Insights into Improved Grain-filling in Ratoon Season Rice

Grain-filling of rice spikelets (particularly for the later flowering inferior spikelets) is an important characteristic that affects both quality and yield. Rice ratooning technology is used to cultivate a second crop from dormant buds that sprout from stubble left after the first harvest. This study used two rice varieties, the conventional indica rice ‘Jinhui 809’ and the hybrid indica-japonica rice ‘Yongyou 1540’, to assess the impact of rice ratooning on grain-filling. The results indicated that the grain-filling process in inferior spikelets of ratoon season rice (ISR) showed significant improvement compared to inferior spikelets of main crop (late season) rice (ISL). This improvement was evident in the earlier onset of rapid grain-filling, higher seed-setting percentage, and improved grain quality. A label-free quantitative proteomic analysis using mass spectrometry identified 1724 proteins with significant abundance changes, shedding light on the molecular mechanisms behind the improved grain-filling in ISR. The functional analysis of these proteins indicated that ratooning stimulated the metabolic processes of sucrose-starch, trehalose, and hormones in rice inferior spikelets, leading to enhanced enzyme activities related to starch synthesis, elevated concentrations of trehalose-6-phosphate (T6P), indole-3-acetic acid (IAA) and zeatin riboside (ZR) during the active grain-filling phase. This research highlighted the importance of the GF14f protein as a key regulator in the grain-filling process of ISR. It revealed that GF14f transcriptional and protein levels declined more rapidly in ISR compared to ISL during grain-filling. Additionally, the GF14f-RNAi plants specific to the endosperm exhibited improved quality in inferior spikelets. These findings suggest that the enhancement of starch synthesis, increased levels of IAA, ZR, and T6P, along with the rapid decrease in GF14f protein, play a role in enhancing grain-filling in ratoon season rice.

Multiomics-Based Biocargo Components Analysis in Enterococcus faecium Membrane Vesicles.

Enterococcus spp. have been shown to have gastrointestinal tract protective functions; our recent results suggest that membrane vesicles (MVs) play an important role in the gastric protection of Enterococcus faecium (E. faecium). The specific function is determined by molecular compositions of MVs. To resolve biocargo components in E. faecium MVs (EfmMVs), MVs were isolated from E. faecium culture. Transcriptomics, label-free quantitative proteomics, and untargeted metabolomics were performed to obtain information about the complexity of ribonucleic acids (RNAs), proteins, and metabolites biocargo they carry, respectively. RNA-sequencing identified a total of 2122 transcripts. The top 20 transcripts accounted for 27.63% of total counts, which, including enzymes, participate in glycolysis, ribosomal proteins, DNA-directed RNA polymerases, protein-synthesizing relative enzymes, molecules associated with protein post-translational processing and transport, and peptidoglycan lyases. Label-free quantitative proteomics analysis identified a total of 711 proteins. The top 20 proteins accounted for 48.02% of all identified proteins, which including ribosomal proteins, enzymes participate in glycolysis, DNA-directed RNA polymerases, protein-synthesizing relative enzymes, peptidoglycan lyases, and autolysin. Untargeted metabolomics analysis identified a total of 519 metabolites. The top 20 metabolites accounted for 79.55% of all identified metabolites, which included amino acids, substrates, or products in the metabolism of amino acids, natural organic acids, products in the metabolism of organic acids, ketone compounds, and two other compounds. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that the identified biocargo components enriched in metabolism, genetic, and environmental information processing. Overall, we hope that the current exploration of multiple "-omics" analyses of this EfmMVs will provide useful information and further groundwork for future studies on E. faecium application.

Sodium-iodate injection can replicate retinal and choroid degeneration in pigmented mice: Using multimodal imaging and label-free quantitative proteomics analysis

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the elderly population. Sodium iodate (NaIO3), a stable oxidizing agent, has been injected to establish a reproducible model of oxidative stress-induced RPE and photoreceptor death. The aim of our study was to evaluate the morphological and molecular changes of retina and retinal pigment epithelium (RPE)-choroid in NaIO3-treated mouse using multimodal fundus imaging and label-free quantitative proteomics analysis. Here, we found that following NaIO3 injection, retinal degeneration was evident. Fundus photographs showed numerous scattered yellow-white speckled deposits. Optical coherence tomography (OCT) images indicated disruption of the retinal layers, damage of the RPE layer and accumulation of hyper-reflective matter in multiple layers of the outer retina. Widespread foci of a high fundus autofluorescence (FAF) signal were noticed. Fundus fluorescein angiography (FFA) revealed diffuse intense transmitted fluorescence mixed with scattered spot-like blocked fluorescence. Indocyanine green angiography (ICGA) presented punctate hyperfluorescence. Due to the atrophy of the RPE and Bruch's membrane and choroidal capillary complex, the larger choroidal vessels become more prominent in ICGA and optical coherence tomography angiography (OCTA). Transmission electron microscope (TEM) illustrated abnormal material accumulation and damaged mitochondria. Bioinformatics analysis of proteomics revealed that the differentially expressed proteins participated in diverse biological processes, encompassing phototransduction, NOD-like receptor signaling pathway, phagosome, necroptosis, and cell adhesion molecules. In conclusion, by multimodal imaging, we described the phenotype of NaIO3-treated mouse model mimicking oxidative stress-induced RPE and photoreceptor death in detail. In addition, proteomics analysis identified differentially expressed proteins and significant enrichment pathways, providing insights for future research, although the exact mechanism of oxidative stress-induced RPE and photoreceptor death remains incompletely understood.

Proteomic changes orchestrate metabolic acclimation of a unicellular diazotrophic cyanobacterium during light-dark cycle and nitrogen fixation states.

Cyanobacteria have developed an impressive array of proteins and pathways, each tailored for specific metabolic attributes, to execute photosynthesis and biological nitrogen (N2)-fixation. An understanding of these biologically incompatible processes provides important insights into how they can be optimized for renewable energy. To expand upon our current knowledge, we performed label-free quantitative proteomic analysis of the unicellular diazotrophic cyanobacterium Crocosphaera subtropica ATCC 51142 grown with and without nitrate under 12-hour light-dark cycles. Results showed significant shift in metabolic activities including photosynthesis, respiration, biological nitrogen fixation (BNF), and proteostasis to different growth conditions. We identified 14 nitrogenase enzymes which were among the most highly expressed proteins in the dark under nitrogen-fixing conditions, emphasizing their importance in BNF. Nitrogenase enzymes were not expressed under non nitrogen fixing conditions, suggesting a regulatory mechanism based on nitrogen availability. The synthesis of key respiratory enzymes and uptake hydrogenase (HupSL) synchronized with the synthesis of nitrogenase indicating a coordinated regulation of processes involved in energy production and BNF. Data suggests alternative pathways that cells utilize, such as oxidative pentose phosphate (OPP) and 2-oxoglutarate (2-OG) pathways, to produce ATP and support bioenergetic BNF. Data also indicates the important role of uptake hydrogenase for the removal of O2 to support BNF. Overall, this study expands upon our knowledge regarding molecular responses of Crocosphaera 51142 to nitrogen and light-dark phases, shedding light on potential applications and optimization for renewable energy.