Protein Damage Research Articles

Thawing of Frozen Hairtail (Trichiurus lepturus) with Graphene Nanoparticles Combined with Radio Frequency: Variations in Protein Aggregation, Structural Characteristics, and Stability.

Efficient thawing can preserve the quality of frozen hairtail (Trichiurus lepturus) close to that of fresh hairtail. In contrast to air thawing (AT) and radio-frequency thawing (RT), this study looked at how graphene oxide (GO) and graphene magnetic (GM) nanoparticles paired with RT affect the microstructure and protein conformation of hairtails after thawing. The results suggested that GM-RT can reduce the myofibrillar protein (MP) damage and be more effective than other thawing treatments, like AT, RT, and GO-RT, in maintaining the microstructure of hairtail. The particle size and zeta potential showed that GM-RT could reduce the aggregation of MP during the thawing process compared to other thawing methods. Moreover, the texture of the hairtail after GM-RT exhibited higher hardness (1185.25 g), elasticity (2.25 mm), and chewiness (5.75 mJ) values compared to other thawing treatments. Especially compared with RT, the GM-RT treatment displayed significant improvements in hardness (27.24%), a considerable increase in springiness (92.23%), and an increase in chewiness (57.96%). GO-RT and GM-RT significantly reduced the centrifugal loss. The scanning electron microscopy results demonstrated that the effect of GM-RT was more akin to that of a fresh sample (FS) and characterized by a well-organized microstructure. In conclusion, GM-RT effectively diminished the MP aggregation and improved the texture of thawed fish. It can be regarded as a viable alternative thawing technique to enhance MP stability, which is vital for preserving meat quality.

Comparative Physiological and Transcriptomic Profiling Reveals the Characteristics of Tissue Tolerance Mechanisms in the japonica Rice Landrace Under Salt Stress

The aim of this study was to characterize the tissue tolerance mechanisms of rice under salt stress. Our preliminary experiment identified a japonica rice landrace Shuzenji-kokumai (SZK), which is considered to be tissue-tolerant because it can maintain better growth than salt-sensitive rice varieties while having a high-Na+ concentration in the shoots under salt stress. These mechanisms differ from those of most salt-tolerant rice varieties, which have low Na+ concentrations in the shoots. We compared the physiological and molecular characteristics of SZK with those of FL478, a salt-tolerant variety, and Kunishi, a salt-sensitive variety. Under salt stress conditions, SZK accumulated high levels of Na+ in the roots, leaf sheaths, and leaf blades, which were almost as high as those in the salt-sensitive Kunishi. Simultaneously, SZK maintained better growth and physiological status, as determined by its higher dry weight, lower electrolyte leakage ratio, and lower malondialdehyde concentration. Expressions of OsNHX1 and OsNHX2 were upregulated in the leaf sheaths of SZK, suggesting that Na+ might be compartmentalized in the vacuoles to avoid cytosolic Na+ toxicity. In contrast, FL478 showed upregulation of OsHKT1;5 and OsSOS1 in the roots, which may exclude Na+ from the shoots. RNA-seq analysis showed that 4623 and 1998 differentially expressed genes were detected in the leaf sheaths and leaf blades of SZK, respectively. Among them, the HSP (heat shock protein) gene expression was highly up-regulated only in SZK, indicating that SZK protects against the protein damage caused by Na+ toxicity. Our findings suggest that SZK has atypical survival mechanisms under salt stress conditions. These mechanisms offer potential traits for improving salt tolerance in rice in terms of tissue tolerance.

Physiological strategies of acute thermal conditions of Rhamdia voulezi collected in the Iguaçu river watershed, Paraná, Brazil: biochemical markers of metabolic and oxidative stress.

Thermal pollution creates substantial challenges that alter energy demand and produce reactive oxygen species that damage fish DNA, proteins, and lipids. Rhamdia voulezi is a species of fish native to the Iguaçu river, Paraná, Brazil, that does not have scientific records of minimum (CTmin) and maximum (CTmax) temperatures required for survival. As it is a top predator species in the food chain and lives at temperatures below 22°C, the loss of the species can cause functional problems in controlling the ecosystem and energy flow. The study evaluated the tissue metabolism of the brain, heart, and muscle of R. voulezi (n = 72) subjected to acute thermal stress of 31°C for 2, 6, 12, 24, and 96h after acclimatization to 21°C. The biochemical markers SOD, GPx, MDH, HK, and CK of the brain, PCO of the heart and CAT, glycogen, G6PDH, and ALT of muscle were significant. PCA, IBR, thermal sensitive, and condition factor suggested that R. voulezi has different physiological strategies for acclimatization to 31°C to mobilize and sustain the metabolic needs of oxygenation and energy allocation/utilization for tissue ATP production.

Revealing molecular dynamics of nanoparticles' antimicrobial activity, toxicities and coping up strategies

There is a growing interest in nanoformulations of medicinal drugs for antibacterial, antiviral, antifungal, antiprastic, and antioxidant drug delivery applications by researchers. Using nanoformulations, the conventional medicines have enhanced their functional properties and are more targeted to specific distribution sites. Some Nanoformulations gaining prominence in the pharmaceutical industry are hydrogels, nanocrystals, polymeric nanoparticles, liposomes, nanoemulsions, and micelles. The metal nanoparticles (silver, gold, iron oxide, and others) and nanoemulsions cause protein and DNA damage, producinge ROS (reactive oxygen species) as salient antimicrobial tools. Nanomaterials have been utilized in biomedical applications, spanning metals, nonmetallic/inorganic materials, and biodegradable materials. Human cells exhibit toxicity (e.g.Titanium dioxide) towards nanoparticles (NPs) due to the presence of synthetic chemicals as surface functional agents. Consequently, the nanoparticles alone can induce hepatotoxicity, skin sensitization, and damage. A focus on strategies such as coupling agents has been used in recent advancements aimed at reducing the toxicity of nanoformulations. These modified nanostructures provide safer and more efficient biomedical applications by reducing toxicity and enhancing antimicrobial activity.

A Revolution in Cellular Aging: A Narrative Review of the Promising Role of Nanorobots in Diagnosis, Treatment, and Regenerative Medicine

Nanorobots, also known as nanobots, are promising medical structures with significant potential for revolutionizing medical treatments and preventing their onset. Nanorobotics shows promise for diagnosing, treating, and regenerating age-related diseases. These tiny machines can repair cellular damage, deliver drugs, stimulate tissue regeneration, and monitor treatments. However, their application in controlling cellular aging is in its early stages, requiring more research to fully understand their potential and address safety concerns. Despite this, the benefits of nanorobots in age-related disease management justify further investigation. This narrative review article provides an overview of recent developments in nanorobotics, specifically focusing on its medical applications. PubMed and Google Scholar were utilized to conduct a comprehensive search using relevant keywords including "nanorobots", "cellular aging", "drug delivery systems", "cellular repair", "therapeutics", and "diagnosis". Only articles published between 2005-2023 were selected to support the arguments with up-to-date evidence. The extracted information includes nanorobot design, fabrication methods, applications, and key findings. A quality assessment was performed, and the data were categorized thematically to identify patterns and trends. The article concludes with an examination of the current state and future prospects of nanorobotics in different fields. Our research showed that nanorobots have significant potential in the diagnosis, treatment, and regenerative medicine of age-related diseases. They can detect and repair cellular damage caused by aging, targeting DNA damage and cellular protein misfolding. Nanorobots can also deliver drugs directly to affected areas, reducing side effects. Furthermore, they stimulate tissue regeneration and the growth of new blood vessels by delivering growth factors. Nanorobots can also monitor treatment effectiveness by measuring drug concentration in tumor cells. However, there are concerns about their safety in long-term use that require further research. Overall, nanorobots present an innovative approach to addressing cellular changes in aging, but more research is needed to understand their full potential and address safety concerns. The use of nanorobots in treating cellular aging has opened new possibilities in diagnosing and treating age-related diseases and it is a rapidly evolving field with the potential to revolutionize age-related disease treatment and improve the quality of life globally.

Prenatal per- and polyfluoroalkyl substances (PFAS) and maternal oxidative stress: Evidence from the LIFECODES study

BackgroundPer- and polyfluoroalkyl substances (PFAS) are synthetic chemicals linked to adverse pregnancy outcomes. Although their underlying biological mechanisms are not fully understood, evidence suggests PFAS may disrupt endocrine functions and contribute to oxidative stress (OS) and inflammation. ObjectiveWe examined associations between early pregnancy PFAS exposure and OS biomarkers, exploring potential effect modifications by fetal sex and maternal race. MethodsWe used data from 469 LIFECODES participants with measured plasma PFAS (median 10 weeks gestation) and repeated measures (median 10, 18, 26, and 35 weeks gestation) of urinary OS biomarkers [8-iso-prostaglandin-F2α (8-isoprostane) and 8-hydroxydeoxyguanosine (8-OHdG)]. Protein damage biomarkers (chlorotyrosine, dityrosine, and nitrotyrosine) were additionally measured in plasma from a subset (N = 167) during the third visit. Associations between each PFAS and OS biomarkers were examined using linear mixed-effects models and multivariable linear regressions, adjusting for potential confounders, including maternal age, race, education level, pre-pregnancy BMI, insurance status, and parity. Effect modifications were evaluated by including an interaction term between each PFAS and fetal sex or maternal race in the models. ResultsWe observed significant positive associations between PFOS and 8-isoprostane, with a 9.68% increase in 8-isoprostane levels (95% CI: 0.10%, 20.18%) per interquartile range increase in PFOS. In contrast, PFUA was negatively associated [9.32% (95% CI: −17.68%, −0.11%)], while there were suggestive positive associations for MPAH and PFOA with 8-isoprostane. The associations of several PFAS with 8-OHdG varied by fetal sex, showing generally positive trends in women who delivered females, but negative or null in those who delivered males. No significant effect modification by maternal race was observed. ConclusionsThis study provides evidence linking PFAS exposure to OS during pregnancy, with potential sex-specific effects of certain PFAS on 8-OHdG. Further research should explore additional OS/inflammatory biomarkers and assess the modifying effects of dietary and behavioral patterns across diverse populations.

Failure of Autophagy in Pompe Disease.

Autophagy is an evolutionarily conserved lysosome-dependent degradation of cytoplasmic constituents. The system operates as a critical cellular pro-survival mechanism in response to nutrient deprivation and a variety of stress conditions. On top of that, autophagy is involved in maintaining cellular homeostasis through selective elimination of worn-out or damaged proteins and organelles. The autophagic pathway is largely responsible for the delivery of cytosolic glycogen to the lysosome where it is degraded to glucose via acid α-glucosidase. Although the physiological role of lysosomal glycogenolysis is not fully understood, its significance is highlighted by the manifestations of Pompe disease, which is caused by a deficiency of this lysosomal enzyme. Pompe disease is a severe lysosomal glycogen storage disorder that affects skeletal and cardiac muscles most. In this review, we discuss the basics of autophagy and describe its involvement in the pathogenesis of muscle damage in Pompe disease. Finally, we outline how autophagic pathology in the diseased muscles can be used as a tool to fast track the efficacy of therapeutic interventions.

Oxidative DNA damage promotes vascular aging associated with changes in extracellular matrix-regulating proteins.

Vascular aging is characterized by vessel stiffening, with increased deposition of extracellular matrix (ECM) proteins including collagens. Oxidative DNA damage occurs in vascular aging, but how it regulates ECM proteins and vascular stiffening is unknown. We sought to determine the relationship between oxidative DNA damage and ECM regulatory proteins in vascular aging. We examined oxidative DNA damage, the major base excision repair (BER) enzyme 8-Oxoguanine DNA Glycosylase (Ogg1) and its regulators, multiple physiological markers of aging, and ECM proteomics in mice from 22-72w. Vascular aging was associated with increased oxidative DNA damage, and decreased expression of Ogg1, its active acetylated form, its acetylation regulatory proteins P300 and CBP, and the transcription factor Foxo3a. Vascular stiffness was examined in vivo in control, Ogg1-/-, or mice with vascular smooth muscle cell-specific expression of Ogg1+ (Ogg1) or an inactive mutation (Ogg1KR). Ogg1-/- and Ogg1KR mice showed reduced arterial compliance and distensibility, and increased stiffness and pulse pressure, whereas Ogg1 expression normalised all parameters to 72w. ECM proteomics identified major changes in collagens with aging, and downregulation of the ECM regulatory proteins Protein 6-lysyl oxidase (LOX) and WNT1-inducible-signaling pathway protein 2 (WISP2). Ogg1 overexpression upregulated LOX and WISP2 both in vitro and in vivo, and downregulated Transforming growth factor β1 (TGFb1) and Collagen 4α1 in vivo compared with Ogg1KR. Foxo3a activation induced Lox, while Wnt3 induction of Wisp2 also upregulated LOX and Foxo3a, and downregulated TGFβ1 and fibronectin 1. In humans, 8-oxo-G increased with vascular stiffness, while active OGG1 reduced with both age and stiffness. Vascular aging is associated with oxidative DNA damage, downregulation of major BER proteins, and changes in multiple ECM structural and regulatory proteins. Ogg1 protects against vascular aging, associated with changes in ECM regulatory proteins including LOX and WISP2.

Shedding light on the shadows: oxidative stress and its pivotal role in prostate cancer progression.

Data on oxidative protein damage, total antioxidant capacity (TAC) and lipid peroxidation in progression of prostate cancer remain elusive. So far, the influence of the presence of perineural invasion on the level of oxidative stress has not been described. Additionally, there is limited data on the level of oxidative stress in patients' urine. We compared the levels of oxidative stress markers in serum and urine in 50 patients with prostate cancer depending on the tumor stage and histological grade, the Gleason score, and the presence of perineural invasion. We found a significantly de-creased level of serum thiol groups and TAC in participants with prostate cancer. Similarly, serum Amadori products and malondialdehyde (MDA) were higher in patients than in healthy men. There was a significantly decrease in TAC and a significantly increased MDA in the urine of prostate cancer patients. As the stage of cancer increased, a decrease in the thiol group concentration and TAC as well as an increase in the concentration of lipid peroxidation products in the serum was observed. The serum level of advanced oxidation protein products (AOPP) increased in the group with Gleason scores greater than 7. Furthermore, serum thiol groups and TAC were reduced in the group with Gleason >7 as compared to Gleason <7. The presence of perineural invasion significantly reduced serum and urinary TAC and increased urinary AOPP concentration. These results indicate a significant role for oxidative damage in prostate carcinogenesis and its progression. Characterizing oxidative and nitrosative damage to proteins may be useful in designing targeted therapies for prostate cancer patients.

Protein degrons and degradation: Exploring substrate recognition and pathway selection in plants.

Proteome composition is dynamic and influenced by many internal and external cues, including developmental signals, light availability, or environmental stresses. Protein degradation, in synergy with protein biosynthesis, allows cells to respond to various stimuli and adapt by reshaping the proteome. Protein degradation mediates the final and irreversible disassembly of proteins, which is important for protein quality control and to eliminate misfolded or damaged proteins, as well as entire organelles. Consequently, it contributes to cell resilience by buffering against protein or organellar damage caused by stresses. Moreover, protein degradation plays important roles in cell signaling, as well as transcriptional and translational events. The intricate task of recognizing specific proteins for degradation is achieved by specialized systems that are tailored to the substrate's physicochemical properties and subcellular localization. These systems recognize diverse substrate cues collectively referred to as "degrons," which can assume a range of configurations. They are molecular surfaces recognized by E3 ligases of the ubiquitin-proteasome system but can also be considered as general features recognized by other degradation systems, including autophagy or even organellar proteases. Here we provide an overview of the newest developments in the field, delving into the intricate processes of protein recognition and elucidating the pathways through which they are recruited for degradation.

Albumin Oxidation and Albumin Glycation Discordance During Type 2 Diabetes Therapy: Biological and Clinical Implications.

Aims: Cys34 albumin redox modifications (reversible "cysteinylation" and irreversible "di/trioxidation"), besides being just oxidative stress biomarkers, may have primary pathogenetic roles to initiate and/or aggravate cell, tissue, and vascular damage in diabetes. In an exploratory "proof-of-concept" pilot study, we examined longitudinal changes in albumin oxidation during diabetes therapy. Methods: Mass spectrometric analysis was utilized to monitor changes in human serum albumin (HSA) post-translational modifications {glycation [glycated albumin (GA)], cysteinylation [cysteinylated albumin (CA) or human non-mercaptalbumin-1; reversible], di/trioxidation (di/trioxidized albumin or human non-mercaptalbumin-2; irreversible), and truncation (truncated albumin)} during ongoing therapy. Four informative groups of subjects were evaluated [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity, and healthy controls] at baseline, and subjects with diabetes were followed for a period up to 280 days. Results: At baseline, T2DM was associated with relatively enhanced albumin cysteinylation (CA% total) compared with T1DM (P = 0.004), despite comparable mean hyperglycemia (P values: hemoglobin A1c = 0.09; GA = 0.09). T2DM, compared with T1DM, exhibited selectively and significantly higher elevations of all the "individual" glycated cum cysteinylated ("multimodified") albumin isoforms (P values: CysHSA+1G = 0.003; CysHSA+2G = 0.007; and CysHSA+3G = 0.001). Improvements in glycemic control and decreases in albumin glycation during diabetes therapy in T2DM were not always associated with concurrent reductions of albumin cysteinylation, and in some therapeutic situations, albumin cysteinylation worsened (glycation-cysteinylation discordance). Important differences were observed between the effects of sulfonylureas and metformin on albumin molecular modifications. Conclusions: T2DM was associated with higher oxidative (cysteinylation) and combined (cysteinylation plus glycation) albumin molecular modifications, which are not ameliorated by improved glucose control alone. Further studies are required to establish the clinical significance and optimal therapeutic strategies to address oxidative protein damage and resulting consequences in diabetes.

Systemic administration of a novel Beclin 1-derived peptide significantly upregulates autophagy in the spinal motor neurons of autophagy reporter mice

Autophagy, an intracellular degradation system, plays a vital role in protecting cells by clearing damaged organelles, pathogens, and protein aggregates. Autophagy upregulation through pharmacological interventions has gained significant attention as a potential therapeutic avenue for proteinopathies. Here, we report the development of an autophagy-inducing peptide (BCN4) derived from the Beclin 1 protein, the master regulator of autophagy. To deliver the BCN4 into cells and the central nervous system (CNS), it was conjugated to our previously developed cell and blood–brain barrier-penetrating peptide (CPP). CPP-BCN4 significantly upregulated autophagy and reduced protein aggregates in motor neuron (MN)-like cells. Moreover, its systemic administration in a reporter mouse model of autophagy resulted in a significant increase in autophagy activity in the spinal MNs. Therefore, this novel autophagy-inducing peptide with a demonstrated ability to upregulate autophagy in the CNS has significant potential for the treatment of various neurodegenerative diseases with protein aggregates as a characteristic feature.

Effect of N-Acetyl-Carnosine on Body Weight and Composition

With a globally aging population, the need for treatments that address age related comorbidities are more relevant than ever. Previous research done in our lab has shown that treatment of mice with the compound N-Acetyl-Carnosine (N-Ac-Carn) could partially prevent protein damage caused by lipid peroxidation, and subsequent muscle atrophy and weakness in a mouse-model of inactivity. N-Ac-Carn is endogenously produced, safe, and affordable making it a potentially promising strategy for age-related functional decline. Our current pre-clinical trial aims to comprehensively determine the effcacy of N-Ac-Carn treatment in preventing age-related functional decline. To this end, 18-month-old mice were treated with either normal water (CON) or water supplemented with 80mM N-Ac-Carn (N-Ac-Carn) for 6 months. Targeted metabolomics verified that this treatment enriched all tissues with N-Ac-Carn. Mice treated with N-Ac-Carn maintained higher body weights longer than CON (Interaction: p=0.022). This effect was driven specifically by higher fat mass over time (p=0.016) in female mice. These changes in the N Acetyl Carnosine mice were not explained by an increase in overall food consumption, which remained similar across both groups. Metabolic cage analysis revealed lower VO2 in N-Ac-Carn mice (Group: p=0.002) although this effect was observed in both male and female mice (Interaction: p=0.878). Despite higher fat mass, IPGTT revealed equivocal glucose tolerance in CON and N-Ac-Carn mice. Therefore, this preserved fat mass with aging is not detrimental to metabolic regulation and may be protective against developing other symptoms of frailty. Future analysis will pursue the mechanisms of preserved adipose tissue mass with N-Ac-Carn treatment and if this phenotype is associated with improved health span. R01AG074535-03. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Stress-Induced Proteasome Sub-Cellular Translocation in Cardiomyocytes Causes Altered Intracellular Calcium Handling and Arrhythmias.

The ubiquitin-proteasome system (UPS) is an essential mechanism responsible for the selective degradation of substrate proteins via their conjugation with ubiquitin. Since cardiomyocytes have very limited self-renewal capacity, as they are prone to protein damage due to constant mechanical and metabolic stress, the UPS has a key role in cardiac physiology and pathophysiology. While altered proteasomal activity contributes to a variety of cardiac pathologies, such as heart failure and ischemia/reperfusion injury (IRI), the environmental cues affecting its activity are still unknown, and they are the focus of this work. Following a recent study by Ciechanover's group showing that amino acid (AA) starvation in cultured cancer cell lines modulates proteasome intracellular localization and activity, we tested two hypotheses in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs, CMs): (i) AA starvation causes proteasome translocation in CMs, similarly to the observation in cultured cancer cell lines; (ii) manipulation of subcellular proteasomal compartmentalization is associated with electrophysiological abnormalities in the form of arrhythmias, mediated via altered intracellular Ca2+ handling. The major findings are: (i) starving CMs to AAs results in proteasome translocation from the nucleus to the cytoplasm, while supplementation with the aromatic amino acids tyrosine (Y), tryptophan (W) and phenylalanine (F) (YWF) inhibits the proteasome recruitment; (ii) AA-deficient treatments cause arrhythmias; (iii) the arrhythmias observed upon nuclear proteasome sequestration(-AA+YWF) are blocked by KB-R7943, an inhibitor of the reverse mode of the sodium-calcium exchanger NCX; (iv) the retrograde perfusion of isolated rat hearts with AA starvation media is associated with arrhythmias. Collectively, our novel findings describe a newly identified mechanism linking the UPS to arrhythmia generation in CMs and whole hearts.

Ultrasound‐activated in situ click chemistry to trigger autophagosome tracking for enhanced autophagy blockade and synergistic cancer therapy

AbstractThe blockade of cytoprotective autophagy has been demonstrated to effectively enhance the efficacy of sonodynamic therapy (SDT). However, the limited recognition of antiautophagy agents for autophagosomes impedes the clinical application of autophagy inhibition. To efficiently deliver hydroxychloroquine (HCQ), an autophagy inhibitor, to autophagosomes, we utilized a strategy based on in situ click chemistry between sulfhydryl (‐SH) and maleimide (Mal) groups to trigger autophagosomes tracking and suppress tumor growth synergistically. A cascade nanoreactor was synthesized by encapsulating Mal‐modified HCQ (MHCQ) into a manganese porphyrin‐based metal‐organic framework with sonosensitizer properties, followed by poly(ethylene glycol)ylated liposomal membrane coating. After ultrasound irradiation, SDT‐induced apoptotic cells released damaged proteins with free ‐SH groups, which MHCQ rapidly captured in situ via a Mal‐thiol click reaction. When autophagosomes actively wrapped damaged proteins for detoxification, they simultaneously internalized HCQ anchored on proteins. In this scenario, antiautophagy drugs could actively track intracellular autophagosomes instead of undergoing passive diffusion in the cytosol. The interaction between HCQ and autophagic vesicles was greatly enhanced, which strengthened the blocking efficiency of autophagy and resulted in complete cell death. Overall, this study with smart design provides a promising strategy for improving intracellular targeted delivery to autophagosomes, thereby enhancing antitumor therapy.

Oxidative stress, defective proteostasis and immunometabolic complications in critically ill patients.

Oxidative stress (OS) develops in critically ill patients as a metabolic consequence of the immunoinflammatory and degenerative processes of the tissues. These induce increased and/or dysregulated fluxes of reactive species enhancing their pro-oxidant activity and toxicity. At the same time, OS sustains its own inflammatory and immunometabolic pathogenesis, leading to a pervasive and vitious cycle of events that contribute to defective immunity, organ dysfunction and poor prognosis. Protein damage is a key player of these OS effects; it generates increased levels of protein oxidation products and misfolded proteins in both the cellular and extracellular environment, and contributes to forms DAMPs and other proteinaceous material to be removed by endocytosis and proteostasis processes of different cell types, as endothelial cells, tissue resident monocytes-macrophages and peripheral immune cells. An excess of OS and protein damage in critical illness can overwhelm such cellular processes ultimately interfering with systemic proteostasis, and consequently with innate immunity and cell death pathways of the tissues thus sustaining organ dysfunction mechanisms. Extracorporeal therapies based on biocompatible/bioactive membranes and new adsorption techniques may hold some potential in reducing the impact of OS on the defective proteostasis of patients with critical illness. These can help neutralizing reactive and toxic species, also removing solutes in a wide spectrum of molecular weights thus improving proteostasis and its immunometabolic corelates. Pharmacological therapy is also moving steps forward which could help to enhance the efficacy of extracorporeal treatments. This narrative review article explores the aspects behind the origin and pathogenic role of OS in intensive care and critically ill patients, with a focus on protein damage as a cause of impaired systemic proteostasis and immune dysfunction in critical illness.

Serum and cerebrospinal fluid neurofilament light chain and glial fibrillary acid protein levels in early and advanced stages of cerebral amyloid Angiopathy

BackgroundNeurofilament light chain (NFL) is a biomarker for neuroaxonal damage and glial fibrillary acidic protein (GFAP) for reactive astrocytosis. Both processes occur in cerebral amyloid angiopathy (CAA), but studies investigating the potential of NFL and GFAP as markers for CAA are lacking.We aimed to investigate NFL and GFAP as biomarkers for neuroaxonal damage and astrocytosis in CAA.MethodsFor this cross-sectional study serum and cerebrospinal fluid (CSF) samples were collected between 2010 and 2020 from controls, (pre)symptomatic Dutch-type hereditary (D-CAA) mutation-carriers and participants with sporadic CAA (sCAA) from two prospective CAA studies at two University hospitals in the Netherlands. NFL and GFAP levels were measured with Simoa-assays. The association between NFL and GFAP levels and age, cognitive performance (MoCA), CAA-related MRI markers (CAA-CSVD-burden) and Aβ40 and Aβ42 levels in CSF were assessed with linear regression adjusted for confounders. The control group was divided in age < 55 and ≥55 years to match the specific groups.ResultsWe included 187 participants: 28 presymptomatic D-CAA mutation-carriers (mean age 40 years), 29 symptomatic D-CAA participants (mean age 58 years), 59 sCAA participants (mean age 72 years), 33 controls < 55 years (mean age 42 years) and 38 controls ≥ 55 years (mean age 65 years).In presymptomatic D-CAA, only GFAP in CSF (7.7*103pg/mL vs. 4.4*103pg/mL in controls; P<.001) was increased compared to controls. In symptomatic D-CAA, both serum (NFL:26.2pg/mL vs. 12.5pg/mL; P=0.008, GFAP:130.8pg/mL vs. 123.4pg/mL; P=0.027) and CSF (NFL:16.8*102pg/mL vs. 7.8*102pg/mL; P=0.01 and GFAP:11.4*103pg/mL vs. 7.5*103pg/mL; P<.001) levels were higher than in controls and serum levels (NFL:26.2pg/mL vs. 6.7pg/mL; P=0.05 and GFAP:130.8pg/mL vs. 66.0pg/mL; P=0.004) were higher than in pre-symptomatic D-CAA. In sCAA, only NFL levels were increased compared to controls in both serum (25.6pg/mL vs. 12.5pg/mL; P=0.005) and CSF (20.0*102pg/mL vs 7.8*102pg/mL; P=0.008). All levels correlated with age. Serum NFL correlated with MoCA (P=0.008) and CAA-CSVD score (P<.001). NFL and GFAP in CSF correlated with Aβ42 levels (P=0.01/0.02).ConclusionsGFAP level in CSF is an early biomarker for CAA and is increased years before symptom onset. NFL and GFAP levels in serum and CSF are biomarkers for advanced CAA.

Elevated levels of iodide promote peroxidase-mediated protein iodination and inhibit protein chlorination

At inflammatory sites, immune cells generate oxidants including H₂O₂. Myeloperoxidase (MPO), released by activated leukocytes employs H₂O₂ and halide/pseudohalides to form hypohalous acids that mediate pathogen killing. Hypochlorous acid (HOCl) is a major species formed. Excessive or misplaced HOCl formation damages host tissues with this linked to multiple inflammatory diseases. Previously (Redox Biology, 2020, 28, 101331) we reported that iodide (I⁻) modulates MPO-mediated protein damage by decreasing HOCl generation with concomitant hypoiodous acid (HOI) formation. HOI may however impact on protein structure, so in this study we examined whether and how HOI, from peroxidase/H₂O₂/I⁻ systems ± Cl⁻, modifies proteins. Experiments employed MPO and lactoperoxidase (LPO) and multiple proteins (serum albumins, anastellin), with both chemical (intact protein and peptide mass mapping, LC-MS) and structural (SDS-PAGE) changes assessed. LC-MS analyses revealed dose-dependent iodination of anastellin and albumins by LPO/H2O2 with increasing I⁻. Incubation of BSA with MPO/H2O2/Cl⁻ revealed modest chlorination (Tyr286, Tyr475, ∼4 %) and Met modification. Lower levels of these species, and extensive iodination at specific Tyr and His residues (>20 % modification with ≥10 μM I⁻) were detected with increasing I⁻. Anastellin dimerization was inhibited by increasing I⁻, but less marked changes were observed with albumins. These data confirm that I⁻ competes with Cl⁻ for MPO and is an efficient HOCl scavenger. These processes decrease protein chlorination and oxidation, but result in extensive iodination. This is consistent with published data on the presence of iodinated Tyr on neutrophil proteins. The biological implications of protein iodination relative to chlorination require further clarification.

Inhibition of Usp14 ameliorates renal ischemia-reperfusion injury by reducing Tfap2a stabilization and facilitating mitophagy

Mitochondrial dysfunction is recognized as a pivotal contributor to the pathogenesis of renal ischemia-reperfusion (IR) injury. Mitophagy, the process responsible for removing damaged protein aggregates, stands as a critical mechanism safeguarding cells against IR injury. Currently, the role of deubiquitination in regulating mitophagy still needs to be completely elucidated. This study aimed to evaluate the impact of ubiquitin-specific peptidase 14 (Usp14), a deubiquitinase, in IR injury by influencing mitophagy. Utilizing a murine model of renal IR injury, Usp14 silencing was found to ameliorate kidney injury, leading to decreased levels of serum creatinine and blood urea nitrogen, alongside diminished oxidative stress and inflammation. In renal epithelial cells subjected to hypoxia/reoxygenation (H/R), Usp14 knockdown increased cell viability and reduced apoptosis. Further mechanistic studies revealed that Usp14 interacted with and deubiquitinated transcription factor AP-2 alpha (Tfap2a), thereby suppressing its downstream target gene, TANK binding kinase 1 (Tbk1), to influence mitophagy. Tfap2a overexpression or Tbk1 inhibition reversed the protective effects of Usp14 silencing on renal tubular cell injury and its facilitation of mitophagy. In summary, our study demonstrated the renoprotective role of Usp14 knockdown in mitigating renal IR injury by promoting Tfap2a-mediated Tbk1 upregulation and mitophagy. These findings advocate for exploring Usp14 inhibition as a promising therapeutic avenue for mitigating IR injury, primarily by enhancing the clearance of damaged mitochondria through augmented mitophagy.

Evidence for persistent UV-induced DNA damage and altered DNA damage response in xeroderma pigmentosa patient corneas

Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in the cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts that are repaired by the NER (Nucleotide Excision Repair) pathway. Mutations in the genes coding for NER proteins, as reported in XP patients, would lead to sub-optimal damage repair resulting in clinical signs varying from photo-keratitis to cancerous lesions on the ocular surface. Here, we aimed to provide evidence for the accumulation of DNA damage and activation of DNA repair pathway proteins in the corneal cells of patients with XP. Corneal buttons of patients who underwent penetrating keratoplasty were stained to quantify DNA damage and the presence of activated DNA damage response proteins (DDR) using specific antibodies. Positive staining for pH2A.X and thymidine dimers confirmed the presence of DNA damage in the corneal cells. Positive cells were found in both control corneas and XP samples however, unlike normal tissues, positive cells were found in all cell layers of XP samples indicating that these cells were sensitive to very low levels of UV. pH2A.X-positive cells were significantly more in XP corneas (p < 0.05) indicating the presence of double strand breaks in these tissues. A positive expression of phosphorylated-forms of DDR proteins was noted in XP corneas (unlike controls) such as ataxia telangiectasia mutated/Rad-3 related proteins (ATM/ATR), breast cancer-1 and checkpoint kinases-1 and -2. Nuclear localization of XPA was noted in XP samples which co-localized (calculated using Pearson's correlation) with pATM (0.9 ± 0.007) and pATR (0.6 ± 0.053). The increased presence of these in the nucleus confirms that unresolved DNA damage was accumulating in these cells thereby leading to prolonged activation of the damage response proteins. An increase in pp53 and TUNEL positive cells in the XP corneas indicated cell death likely driven by the p53 pathway. For comparison, cultured normal corneal epithelial cells were exposed to UV-radiation and stained for DDR proteins at 3, 6 and 24 h after irradiation to quantify the time taken by cells with intact DDR pathway to repair damage. These cells, when exposed to UV showed nuclear translocation of DDR proteins at 3 and 6 h which reduced significantly by 24 h confirming that the damaged DNA was being actively repaired leading to cell survival. The persistent presence of the DDR proteins in XP corneas indicates that damage is being actively recognized and DNA replication is stalled, thereby causing accumulation of damaged DNA leading to cell death, which would explain the cancer incidence and cell loss reported in these patients.