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Journal of the American Heart... Jul 2024Endothelial cell (EC) dysfunction involves reduced nitric oxide (NO) bioavailability due to NO synthase uncoupling linked to increased oxidation and reduced cofactor...
BACKGROUND
Endothelial cell (EC) dysfunction involves reduced nitric oxide (NO) bioavailability due to NO synthase uncoupling linked to increased oxidation and reduced cofactor availability. Loss of endothelial function and NO bioavailability are associated with inflammation, including leukocyte activation. Eicosapentaenoic acid (EPA) administered as icosapent ethyl reduced cardiovascular events in REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl-Intervention Trial) in relation to on-treatment EPA blood levels. The mechanisms of cardiovascular protection for EPA remain incompletely elucidated but likely involve direct effects on the endothelium.
METHODS AND RESULTS
In this study, human ECs were treated with EPA and challenged with the cytokine IL-6 (interleukin-6). Proinflammatory responses in the ECs were confirmed by ELISA capture of sICAM-1 (soluble intercellular adhesion molecule-1) and TNF-α (tumor necrosis factor-α). Global protein expression was determined using liquid chromatography-mass spectrometry tandem mass tag. Release kinetics of NO and peroxynitrite were monitored using porphyrinic nanosensors. IL-6 challenge induced proinflammatory responses from the ECs as evidenced by increased release of sICAM-1 and TNF-α, which correlated with a loss of NO bioavailability. ECs pretreated with EPA modulated expression of 327 proteins by >1-fold (<0.05), compared with IL-6 alone. EPA augmented expression of proteins involved in NO production, including heme oxygenase-1 and dimethylarginine dimethylaminohydrolase-1, and 34 proteins annotated as associated with neutrophil degranulation. EPA reversed the endothelial NO synthase uncoupling induced by IL-6 as evidenced by an increased [NO]/[peroxynitrite] release ratio (<0.05).
CONCLUSIONS
These direct actions of EPA on EC functions during inflammation may contribute to its distinct cardiovascular benefits.
PubMed: 38958135
DOI: 10.1161/JAHA.123.034076 -
Cytoskeleton (Hoboken, N.J.) Jul 2024Actin has been implicated in lens opacification; however, the specific actin-related pathways involved in cataracts remain unelucidated. In this study, actin-related...
INTRODUCTION
Actin has been implicated in lens opacification; however, the specific actin-related pathways involved in cataracts remain unelucidated. In this study, actin-related proteome changes and signaling pathways involved in the development of cataracts were evaluated.
METHODS
The anterior capsule and phacoemulsification (phaco) cassette contents were collected during cataract surgery from 11 patients with diabetic cataract (DC), 12 patients with age-related cataract (ARC), and seven patients with post-vitrectomy cataract (PVC). Untargeted, global identification and quantification of proteins was performed through liquid chromatography-mass spectrometry with the data-independent acquisition (DIA).
RESULTS
In phaco cassette samples, proteins with significantly lower expression in ARC than in DC and PVC were involved in various pathways, including actin binding, actin cytoskeleton reorganization, actin filament capping, cortical actin cytoskeleton organization, and small GTPase-mediated signal transduction pathways. In anterior capsules, proteins with significantly lower expression in ARC than in DC and PVC were involved in actin binding and actin cytoskeleton reorganization pathways.
CONCLUSION
Actin cytoskeleton and actin-binding proteins are involved in lens fiber elongation and differentiation. Rho GTPases contribute to actin cytoskeletal reorganization, and their inactivation is linked to abnormal lens fiber migration. These findings link actin binding to lens fiber integrity, lens opacification, and cataracts.
PubMed: 38958111
DOI: 10.1002/cm.21889 -
Advanced Materials (Deerfield Beach,... Jul 2024Label-free proteomics is widely used to identify disease mechanism and potential therapeutic targets. However, deep proteomics with ultratrace clinical specimen remains...
Label-free proteomics is widely used to identify disease mechanism and potential therapeutic targets. However, deep proteomics with ultratrace clinical specimen remains a major technical challenge due to extensive contact loss during complex sample pretreatment. Here, a hybrid of four boronic acid-rich lanthanide metal-organic frameworks (MOFs) with high protein affinity is introduced to capture proteins in ultratrace samples jointly by nitrogen-boronate complexation, cation-π and ionic interactions. A MOFs Aided Sample Preparation (MASP) workflow that shrinks sample volume and integrates lysis, protein capture, protein digestion and peptide collection steps into a single PCR tube to minimize sample loss caused by non-specific absorption, is proposed further. MASP is validated to quantify ≈1800 proteins in 10 HEK-293T cells. MASP is applied to profile cerebrospinal fluid (CSF) proteome from cerebral stroke and brain damaged patients, and identified ≈3700 proteins in 1 µL CSF. MASP is further demonstrated to detect ≈9600 proteins in as few as 50 µg mouse brain tissues. MASP thus enables deep, scalable, and reproducible proteome on precious clinical samples with low abundant proteins.
PubMed: 38958107
DOI: 10.1002/adma.202401559 -
Autophagy Jul 2024Autophagosome biogenesis is a complex process orchestrated by dynamic interactions between Atg (autophagy-related) proteins and characterized by the turnover of specific...
Autophagosome biogenesis is a complex process orchestrated by dynamic interactions between Atg (autophagy-related) proteins and characterized by the turnover of specific cargoes, which can differ over time and depending on how autophagy is stimulated. Proteomic analyses are central to uncover protein-protein interaction networks and when combined with proximity-dependent biotinylation or proximity labeling (PL) approaches, they also permit to detect transient and weak interactions. However, current PL procedures for yeast , one of the leading models for the study of autophagy, do not allow to keep temporal specificity and thus identify interactions and cargoes at a precise time point upon autophagy induction. Here, we present a new ascorbate peroxidase 2 (APEX2)-based PL protocol adapted to yeast that preserves temporal specificity and allows uncovering neighbor proteins by either western blot or proteomics. As a proof of concept, we applied this new method to identify Atg8 and Atg9 interactors and detected known binding partners as well as potential uncharacterized ones in rich and nitrogen starvation conditions. Also, as a proof of concept, we confirmed the spatial proximity interaction between Atg8 and Faa1. We believe that this protocol will be a new important experimental tool for all those researchers studying the mechanism and roles of autophagy in yeast, but also other cellular pathways in this model organism.: APEX2, ascorbate peroxidase 2, Atg, autophagy-related; BP, biotin phenol; Cvt, cytoplasm-to-vacuole targeting; ER, endoplasmic reticulum; LN2, liquid nitrogen; MS, mass spectrometry; PAS, phagophore assembly site; PL, proximity labeling; PE, phosphatidylethanolamine; PPINs, protein-protein interaction networks; PPIs, protein-protein interactions; RT, room temperature; SARs, selective autophagy receptors; WT, wild-type.
PubMed: 38958087
DOI: 10.1080/15548627.2024.2366749 -
Aging Cell Jul 2024Age-related chronic inflammatory lung diseases impose a threat on public health, including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease...
Age-related chronic inflammatory lung diseases impose a threat on public health, including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). However, their etiology and potential targets have not been clarified. We performed genome-wide meta-analysis for IPF with the largest sample size (2883 cases and 741,929 controls) and leveraged the summary statistics of COPD (17,547 cases and 617,598 controls). Transcriptome-wide and proteome-wide Mendelian randomization (MR) designs, together with genetic colocalization, were implemented to find robust targets. The mediation effect was assessed using leukocyte telomere length (LTL). The single-cell transcriptome analysis was performed to link targets with cell types. Individual-level data from UK Biobank (UKB) were used to validate our findings. Sixteen genetically predicted plasma proteins were causally associated with the risk of IPF and 6 proteins were causally associated with COPD. Therein, genetically-elevated plasma level of SCARF2 protein should reduce the risk of both IPF (odds ratio, OR = 0.9974 [0.9970, 0.9978]) and COPD (OR = 0.7431 [0.6253, 0.8831]) and such effects were not mediated by LTL. Genetic colocalization further corroborated these MR results of SCARF2. The transcriptome-wide MR confirmed that higher expression level of SCARF2 was associated with a reduced risk of both. However, the single-cell RNA analysis indicated that SCARF2 expression level was only relatively lower in epithelial cells of COPD lung tissue compared to normal lung tissue. UKB data implicated an inverse association of serum SCARF2 protein with COPD (hazard ratio, HR = 1.215 [1.106, 1.335]). The SCARF2 gene should be a novel target for COP.
PubMed: 38958042
DOI: 10.1111/acel.14266 -
Biopreservation and Biobanking Jul 2024Anthropogenic actions, especially inadequate waste disposal, cause permanent effects on aquatic fauna, resulting in a significant loss in their population. In this...
Anthropogenic actions, especially inadequate waste disposal, cause permanent effects on aquatic fauna, resulting in a significant loss in their population. In this scenario, and conservation strategies have been developed for these species. Among these strategies is the formation of somatic cell and tissue banks derived from skin collection that act complementarily to other biotechnologies. These banks contain all the information for genomic, genetic, and proteomic analyses. They are useful in the assessment of the toxicity of pollutants on the physiology of the species and regenerative and reproductive biotechnologies. The formation of these cryobanks involves different steps, including cryopreservation, with the optimization of all steps occurring in a species-specific manner. There is a diversity of studies on aquatic mammals; however, a low quantity compared to the number of studies on land mammals, with more than 80% of species still unexplored. This is mainly due to the difficulty of execution and asepsis in collecting skin from aquatic mammals and the culture, which seems to require more particularities for it to be successful. Therefore, this review aims to address the current scenario and the steps involved in the conservation of somatic cells and tissues derived from aquatic mammal skin, as well as results that have been achieved in recent years and the prospects.
PubMed: 38957968
DOI: 10.1089/bio.2023.0168 -
Frontiers in Genetics 2024has been at the forefront of genetic studies and biochemical modeling for over a century. Yet, the functions of many genes are still unknown, mainly because no...
has been at the forefront of genetic studies and biochemical modeling for over a century. Yet, the functions of many genes are still unknown, mainly because no phenotypic data are available. Herein, we present the first evidence data regarding the particular molecular and other quantifiable phenotypes, such as viability and anatomical anomalies, induced by a novel insertional mutant allele of the gene. So far, the functions have only been theorized based on electronic annotation and presumptive associations inferred upon high-throughput proteomics or RNA sequencing experiments. The descendants of individuals harboring the allele were scored in order to assess mutant embryonic, larval, and pupal viability Canton Special (CantonS). Our results revealed that the homozygous / genotype determines significant lethality both at the inception of the larval stage and during pupal development. The very few imago escapers that either breach or fully exit the puparium exhibit specific eye depigmentation, wing abnormal unfolding, strong locomotor impairment with apparent spasmodic leg movements, and their maximum lifespan is shorter than 2 days. Using the quantitative real-time PCR (qRT-PCR) method, we found that is upregulated in male flies, but an unexpected gene upregulation was also detected in heterozygous mutants compared to wild-type flies, probably because of regulatory perturbations induced by the transposon. Our work provides the first phenotypic evidence for the essential role of , a scenario in accordance with the putative role of this gene in carbohydrate-binding processes.
PubMed: 38957808
DOI: 10.3389/fgene.2024.1355368 -
Frontiers in Immunology 2024There is a reciprocal relationship between extracellular matrix (ECM) remodelling and inflammation that could be operating in the progression of severe COVID-19. To...
There is a reciprocal relationship between extracellular matrix (ECM) remodelling and inflammation that could be operating in the progression of severe COVID-19. To explore the immune-driven ECM remodelling in COVID-19, we in this explorative study analysed these interactions in hospitalised COVID-19 patients. RNA sequencing and flow analysis were performed on peripheral blood mononuclear cells. Inflammatory mediators in plasma were measured by ELISA and MSD, and clinical information from hospitalised COVID-19 patients (N=15) at admission was included in the analysis. Further, we reanalysed two publicly available datasets: (1) lung tissue RNA-sequencing dataset (N=5) and (2) proteomics dataset from PBCM. ECM remodelling pathways were enriched in PBMC from COVID-19 patients compared to healthy controls. Patients treated at the intensive care unit (ICU) expressed distinct ECM remodelling gene profiles compared to patients in the hospital ward. Several markers were strongly correlated to immune cell subsets, and the dysregulation in the ICU patients was positively associated with plasma levels of inflammatory cytokines and negatively associated with B-cell activating factors. Finally, our analysis of publicly accessible datasets revealed (i) an augmented ECM remodelling signature in inflamed lung tissue compared to non-inflamed tissue and (ii) proteomics analysis of PBMC from severe COVID-19 patients demonstrated an up-regulation in an ECM remodelling pathway. Our results may suggest the presence of an interaction between ECM remodelling, inflammation, and immune cells, potentially initiating or perpetuating pulmonary pathology in severe COVID-19.
Topics: Humans; COVID-19; Leukocytes, Mononuclear; Extracellular Matrix; Male; Female; Middle Aged; SARS-CoV-2; Aged; Cytokines; Proteomics; Lung; Adult
PubMed: 38957465
DOI: 10.3389/fimmu.2024.1379570 -
PNAS Nexus Jul 2024The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated...
The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A but not mutation renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair (1, 2), though the decision making between those remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair, we intercrossed mice. A combined mutation ( ) was found embryonically lethal. RNA-seq of primary double-mutant (DM) mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints. To exclude stress-induced confounders, we utilized a knock-down to obtain a model to study ICL repair in depth. Regarding ICL-induced cell toxicity, cell cycle arrest, and replication fork progression, single-mutant and DM MEFs were found equally sensitive, establishing PCNA-Ub to be critical for FA-ICL repair. Immunoprecipitation and spectrometry-based analysis revealed an unknown role of PCNA-Ub in excluding mismatch recognition complex MSH2/MSH6 from being recruited to ICLs. In conclusion, our results uncovered a dual function of PCNA-Ub in ICL repair, i.e. exclude MSH2/MSH6 recruitment to channel the ICL toward canonical FA repair, in addition to its established role in coordinating TLS opposite the unhooked ICL.
PubMed: 38957451
DOI: 10.1093/pnasnexus/pgae242 -
Evolutionary Applications Jul 2024The Neolithic transition introduced major diet and lifestyle changes to human populations across continents. Beyond well-documented bioarcheological and genetic effects,...
The Neolithic transition introduced major diet and lifestyle changes to human populations across continents. Beyond well-documented bioarcheological and genetic effects, whether these changes also had molecular-level epigenetic repercussions in past human populations has been an open question. In fact, methylation signatures can be inferred from UDG-treated ancient DNA through postmortem damage patterns, but with low signal-to-noise ratios; it is thus unclear whether published paleogenomes would provide the necessary resolution to discover systematic effects of lifestyle and diet shifts. To address this we compiled UDG-treated shotgun genomes of 13 pre-Neolithic hunter-gatherers (HGs) and 21 Neolithic farmers (NFs) individuals from West and North Eurasia, published by six different laboratories and with coverage c.1×-58× (median = 9×). We used epiPALEOMIX and a Monte Carlo normalization scheme to estimate methylation levels per genome. Our paleomethylome dataset showed expected genome-wide methylation patterns such as CpG island hypomethylation. However, analyzing the data using various approaches did not yield any systematic signals for subsistence type, genetic sex, or tissue effects. Comparing the HG-NF methylation differences in our dataset with methylation differences between hunter-gatherers versus farmers in modern-day Central Africa also did not yield consistent results. Meanwhile, paleomethylome profiles did cluster strongly by their laboratories of origin. Using larger data volumes, minimizing technical noise and/or using alternative protocols may be necessary for capturing subtle environment-related biological signals from paleomethylomes.
PubMed: 38957308
DOI: 10.1111/eva.13743