Cellular Capacity Research Articles

Different phenotypes with different endings-Telomere biology disorders and cancer predisposition with long telomeres.

Rare germline pathogenic variants (GPVs) in genes essential in telomere length maintenance and function have been implicated in two broad classes of human disease. The telomere biology disorders (TBDs) are a spectrum of life-threatening conditions, including bone marrow failure, liver and lung disease, cancer and other complications caused by GPVs in telomere maintenance genes that result in short and/or dysfunctional telomeres and reduced cellular replicative capacity. In contrast, cancer predisposition with long telomeres (CPLT) is a disorder associated with elevated risk of a variety of cancers, primarily melanoma, thyroid cancer, sarcoma, glioma and lymphoproliferative neoplasms caused by GPVs in shelterin complex genes that lead to excessive telomere elongation and increased cellular replicative capacity. While telomeres are at the root of both disorders, the term TBD is used to convey the clinical phenotypes driven by critically short or otherwise dysfunctional telomeres and their biological consequences.

Immunological resilience of a temperate catshark to a simulated marine heat wave.

Marine heatwaves (MHW) have recently been proposed as more relevant in driving population changes than the continuous increase in average temperatures associated with climate change. The causal processes underpinning MHW effects in sharks are unclear but may be linked to changes in fitness caused by physiological trade-offs that influence the immune response. Considering the scarcity of data about the immune response of sharks under anomalous warming events, the present study analyzed several fitness indices and characterized the immune response (in the blood, epigonal organ, liver, spleen, and intestine) of temperate adult small-spotted catsharks (Scyliorhinus canicula) after a 30-day exposure to a Category II MHW. The results indicated that adult small-spotted catsharks have developed coping strategies for the MHW. Specifically, among the 35 parameters investigated, only the gonad-to-body ratio (GBR) and plasma glucose showed significant increases. In contrast, igm and tumor necrosis factor receptor (tnfr) gene expression in blood cells, tnfr in the epigonal organ, and the number of monocytes significantly decreased. Although a decline in immune function in small-spotted catsharks was revealed following the MHW exposure, energy mobilization restored homeostasis and indicated a shift in energy allocation towards reproduction. Group resilience may be due to the variable tolerance of individuals, the phenotypic plasticity of cellular immunity, thermal imprinting, and/or metabolic capacity of the individuals.

Isolated prolapse of the posterior mitral valve leaflet: phenotypic refinement, heritability and genetic etiology.

Isolated posterior leaflet mitral valve prolapse (PostMVP), a common form of MVP, often referred as fibroelastic deficiency, is considered a degenerative disease. PostMVP patients are usually asymptomatic and often undiagnosed until chordal rupture. The present study aims to characterize familial PostMVP phenotype and familial recurrence, its genetic background, and the pathophysiological processes involved. We prospectively enrolled 284 unrelated MVP probands, of whom 178 (63%) had bi-leaflet MVP and 106 had PostMVP (37%). Familial screening within PostMVP patients allowed the identification of 20 families with inherited forms of PostMVP for whom whole genome sequencing was carried out in probands. Functional in vivo and in vitro investigations were performed in zebrafishand in Hek293T cells. In the 20 families with inherited form of PostMVP, 38.8% of relatives had a MVP/prodromal form, mainly of the posterior leaflet, with transmission consistent with an autosomal dominant mode of inheritance. Compared with control relatives, PostMVP family patients have clear posterior leaflet dystrophy on echocardiography. Patients with PostMVP present a burden of rare genetic variants in ARHGAP24. ARHGAP24 encodes the filamin A binding RhoGTPase-activating protein FilGAP and its silencing in zebrafish leads to atrioventricular regurgitation. In vitro functional studies showed that variants of FilGAP, found in PostMVP families, are loss-of-function variants impairing cellular adhesion and mechano-transduction capacities. PostMVP should not only be considered an isolated degenerative pathology but as a specific heritable phenotypic trait with genetic and functional pathophysiological origins. The identification of loss-of-function variants in ARHGAP24 further reinforces the pivotal role of mechano-transduction pathways in the pathogenesis of MVP. Isolated posterior mitral valve prolapse (PostMVP), often called fibro-elastic deficiency MVP, is at least in some patients, a specific inherited phenotypic traitPostMVP has both genetic and functional pathophysiological origins Genetic variants in the ARHGAP24 gene, which encodes for the FilGAP protein, cause progressive Post MVP in familial cases, and impair cell adhesion and mechano-transduction capacities.

ELAVL1 regulates glycolysis in nasopharyngeal carcinoma cells through the HMGB3/β-catenin axis

BackgroundThe role of ELAVL1 in the progression of various tumors has been demonstrated. Our research aims to investigate how ELAVL1 controls the glycolytic process in nasopharyngeal carcinoma cells through the HMGB3/β-catenin pathway.MethodsThe expression of ELAVL1 was detected in clinical tumor samples and nasopharyngeal carcinoma cell lines. A subcutaneous tumor model was established in nude mice to investigate the role of ELAVL1 in tumor progression. The relationship between HMGB3 and ELAVL1 was validated by RNA pull down and RIP assays. TOPFlash/FOPFlash reporter assay was used to detect β-catenin activity. Assay kits were utilized to measure glucose consumption, lactate production, and G6PD activity in nasopharyngeal carcinoma cells. Western blot was conducted to detect the expression of glycolysis-related proteins. The glycolytic capacity was analyzed through extracellular acidification rate (ECAR).ResultsIn both clinical samples and nasopharyngeal carcinoma cell lines, the expression levels of ELAVL1 mRNA and protein were found to be upregulated. Knockdown of ELAVL1 significantly inhibited the in vivo proliferation of nasopharyngeal carcinoma and suppressed the glycolytic capacity of nasopharyngeal carcinoma cells. ELAVL1 interacts with HMGB3, leading to an increase in the stability of HMGB3 mRNA. Overexpression of HMGB3 elevated the reduced β-catenin activity caused by sh-ELAVL1 and reversed the inhibitory effect of sh-ELAVL1 on cellular glycolytic capacity. Treatment with β-catenin inhibitor (FH535) effectively suppressed the promotion of glycolytic capacity induced by HMGB3 overexpression.ConclusionsELAVL1 promotes glycolysis in nasopharyngeal carcinoma cells by interacting with HMGB3 to stabilize HMGB3 mRNA, thereby activating β-catenin pathway. Therefore, targeting the ELAVL1-HMGB3-β-catenin axis has the potential to be a novel approach for treating nasopharyngeal carcinoma.

Microfluidic post-insertion of polyethylene glycol lipids and KK or RGD high functionality and quality lipids in milk-derived extracellular vesicles

To achieve the desired delivery effect, extracellular vesicles (EVs) must bypass rapid clearance from circulation and exhibit affinity for target cells; however, it is difficult to simultaneously incorporate two materials into EVs. Post-insertion is a general modification method that can be performed by simply mixing different solutions. Previously, we have developed a microfluidic post-insertion method that supported fast and upscaled modification of EVs using KK-modified high-functionality and -quality (HFQ) lipids. Here, we used microfluidic post-insertion to achieve simultaneous incorporation of polyethylene glycol (PEG) lipids and KK or RGD-modified HFQ lipids into milk-derived EVs to avoid uptake from the reticuloendothelial system and increase the uptake into target cells. PEG lipid and HFQ lipids were formulated to produce micelles and subsequently mixed with EV solution using a microfluidic device. Compared to bulk mixing, microfluidic post-insertion showed higher cellular association. Altered cellular association capacities and endocytic pathways indicated simultaneous incorporation. The cellular association of modified EVs can be adjusted by altering the ratio of (EK)4-KK in micelles with slight changes in physicochemical properties. Furthermore, microfluidic post-insertion is also suitable for (SG)5-RGD, which is insoluble in phosphate-buffered saline (PBS). Our results may be valuable for the development and manufacture of functional EVs as drug delivery systems for clinical applications.

A combination of major histocompatibility complex (MHC) I overexpression and type I interferon induce mitochondrial dysfunction in human skeletal myoblasts.

The overexpression of major histocompatibility complex (MHC) I on the surface of muscle fibers is a characteristic hallmark of the idiopathic inflammatory myopathies (IIMs), collectively termed myositis. Alongside MHC-I overexpression, subtypes of myositis, display a distinct type I interferon (IFN) signature. This study examined the combinational effects of elevated MHC-I and type I IFNs (IFNα/β) on mitochondrial function, as mitochondrial dysfunction is often seen in IIMs. Human skeletal muscle myoblasts were transfected with an MHC-I isoform using the mammalian HLA-A2/Kb vector. Mitochondrial respiration, mitochondrial membrane potential, and reactive oxygen/nitrogen species generation were assessed with or without IFNα and IFNβ. We show that MHC-I overexpression in human skeletal muscle myoblasts led to decreased basal glycolysis and mitochondrial respiration, cellular spare respiratory capacity, adenosine triphosphate-linked respiration, and an increased proton leak, which were all exaggerated by type I IFNs. Mitochondrial membrane depolarization was induced by MHC-I overexpression both in absence and presence of type I IFNs. Human myoblasts overexpressing MHC-I showed elevated nitric oxide generation that was abolished when combined with IFN. MHC-I on its own did not result in an increased reactive oxygen species (ROS) production, but IFN on their own, or combined with MHC-I overexpression did induce elevated ROS generation. Surprisingly, we observed no gross changes in mitochondrial reticular structure or markers of mitochondrial dynamics. We present new evidence that MHC-I overexpression and type I IFNs aggravate the effects each has on mitochondrial function in human skeletal muscle cells, providing novel insights into their mechanisms of action and suggesting important implications in the further study of myositis pathogenesis.

DHMBA, a molecule from Pacific oyster (Crassostrea gigas) alleviates AD pathology by inhibiting ubiquination of Nrf2

Alzheimer's disease (AD) is an age-related, progressive neurodegenerative disorder with oxidative stress and infammation in brain. 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) is a newly identified molecule with antioxidant capacity in Pacific oyster (Crassostrea gigas), but its detailed mechanism of antioxidant activity remains unexplored. In this study, we identified that DHMBA inhibits Nrf2 degradation and alleviates AD Pathology in AD model mice. Treatment with DHMBA significantly reduced levels of β-amyloid (Aβ) and phosphorylated Tau protein in the hippocampus, while also improving anxiety-like behaviors, depressive-like behaviors, and working memory capabilities in AD model mice. Furthermore, DHMBA increased neuronal density within the brain. Additionally, both in the AD cell model and AD model mice, DHMBA was found to significantly upregulate nuclear factor erythroid 2-like 2 (Nrf2) protein levels along with its target genes. We revealed that DHMBA hinders the binding between Keap1 and Nrf2, thereby relieving Nrf2 ubiquitination and increasing Nrf2 protein levels to enhance cellular capacities for antioxidation and anti-inflammation. These findings demonstrate the mechanism by which DHMBA alleviates AD and provide a new insight into the treatment of AD.

Application of XBP1s Decoy Oligodeoxynucleotide Attenuates Cancerous Phenotype in Huh-7 Hepatocellular Carcinoma Cells.

The spliced form of X-box binding protein 1 (XBP1s) is a key transcription factor in the unfolded protein response (UPR), an adaptive mechanism for cell survival. Many studies demonstrated the induced expression of XBP1s in various cancers, including hepatocellular carcinoma (HCC). Such upregulated expression is linked to an enhancement of cell proliferation, migration, and improvement of the survival rate. In this study, we aimed to assess the therapeutic potential of targeting XBP1s, by specific decoy oligodeoxynucleotide (ODN) and evaluated the cancerous phenotypes in Huh-7 cells. In this experimental study, we transfected Huh-7 cells with XBP1s decoy oligonucleotide (ODN). Subsequently, we assess some cellular features, including viability, migration capacity, proliferation potential, and apoptosis. Therefore, various techniques included wound healing test, BrdU, and annexin/PI assays. Additionally, the colony formation capacity was evaluated. The mRNA expression levels of BAX, BCL-2, c-MYC, CCND1, MMP-9, CDH1, and CD133 were quantified by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Transfection of Huh-7 cells by XBP1s decoy ODN led to significant down-regulation of c-Myc, CCND1, MMP-9, BCL-2 and CD133 and up-regulation of CDH1 and BAX transcriptional expressions in comparison with the vehicle group. Our results also demonstrated that transfection of XBP1s-decoy reduced HCC cell viability, proliferation, migration capacity as well as colonization ability in comparison with the vehicle group. These findings proposed the potential application of XBP1s-decoy ODN to reduce cancerous phenotypes such as cell proliferation, cell migration and apoptosis induction in the Huh-7 cell line. More experiments on other cell lines and primary cells could validate our results.

Microalgal astaxanthin ameliorates cypermethrin-induced necroptosis and inflammation via targeting mitochondrial Ca2+ homeostasis and the ROS-NF-κB-RIPK3/MLKL axis in carp hepatocytes (Cyprinus carpio)

Cypermethrin is a toxic pesticide that has infiltrated water bodies due to its widespread use. This contamination has led to detrimental effects on the immune organs of aquatic species, including fish. The natural fat-soluble orange-red carotenoid, astaxanthin (MAT), derived from microalgae, possesses anti-inflammatory, antioxidant, and immunomodulatory properties. To elucidate the mechanism of CY induced damage to carp liver cells and assess the potential protective effects of MAT, we established a carp hepatocyte model exposed to CY and/or MAT. Hepatocytes from carp (Cyprinus carpio) were treated with either 8 μM CY or 60 μM MAT for 24 h. Upon exposure CY, a significant increase in reactive oxygen species (ROS) was observed alongside a diminution in the activities of key antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), suggesting an impairment of cellular antioxidant capacity. Subsequently, acridine orange/ethidium bromide (AO/EB) staining and flow cytometry analysis revealed that hepatocytes exposed to CY exhibited a higher incidence of necroptosis, associated with an elevated mitochondrial Ca2+ concentration, which contributed to cellular dysfunction. Furthermore, exposure to CY also activated the ROS-NF-κB-RIPK3/MLKL signaling pathway, increasing the levels of necroptosis-related regulatory factors (RIP1, RIP3, and MLKL) in hepatocytes and the expression of inflammatory genes (IL-6, IFN-γ, IL-4, IL-1β, and TNF-α), which led to immune dysfunction in hepatocytes. The immunotoxic effects induced by CY were mitigated by MAT treatment, suggesting its potential in alleviating the aforementioned changes caused by CY. Overall, the data suggested that MAT therapy could enhance hepatocyte defenses against CY-induced necroptosis and inflammatory responses by regulating mitochondrial Ca2+ homeostasis and inhibiting the ROS-NF-κB-RIPK3/MLKL signaling cascade. This study elucidated the potential benefits of employing MAT to protect farmed fish from agrobiological hazards during CY exposure, underscoring the practical applications of MAT in aquaculture.

Maternal stress and the early embryonic microenvironment: investigating long-term cortisol effects on bovine oviductal epithelial cells using air–liquid interface culture

The oviduct epithelium is the initial maternal contact site for embryos after fertilization, offering the microenvironment before implantation. This early gestation period is particularly sensitive to stress, which can cause reduced fertility and reproductive disorders in mammals. Nevertheless, the local impact of elevated stress hormones on the oviduct epithelium has received limited attention to date, except for a few reports on polyovulatory species like mice and pigs. In this study, we focused on the effects of chronic maternal stress on cattle, given its association with infertility issues in this monoovulatory species. Bovine oviduct epithelial cells (BOEC) differentiated at the air–liquid interface (ALI) were stimulated with 250 nmol/L cortisol for 1 or 3 weeks. Subsequently, they were assessed for morphology, bioelectrical properties, and gene expression related to oviduct function, glucocorticoid pathway, cortisol metabolism, inflammation, and apoptosis. Results revealed adverse effects of cortisol on epithelium structure, featured by deciliation, vacuole formation, and multilayering. Additionally, cortisol exposure led to an increase in transepithelial potential difference, downregulated mRNA expression of the major glucocorticoid receptor (NR3C1), upregulated the expression of cortisol-responsive genes (FKBP5, TSC22D3), and significant downregulation of oviductal glycoprotein 1 (OVGP1) and steroid receptors PGR and ESR1. The systematic comparison to a similar experiment previously performed by us in porcine oviduct epithelial cells, indicated that bovine cultures were more susceptible to elevated cortisol levels than porcine. The distinct responses between both species are likely linked to their divergence in the cortisol-induced expression changes of HSD11B2, an enzyme controlling the cellular capacity to metabolise cortisol. These findings provide insights into the species-specific reactions and reproductive consequences triggered by maternal stress.

YiMu-QingGong san alleviates lipopolysaccharide-induced endometritis in mice via inhibiting inflammation and oxidative stress through regulating macrophage polarization

Ethnopharmacological relevanceEndometritis is a common reproductive disease in dairy cattle after calving, characterized by persistent inflammation of the endometrium. YiMu-QingGong San (YMQGS), a patented Chinese medicine formulation, has demonstrated efficacy in the treatment of clinical endometritis in dairy cattle. However, the potential mechanisms by which YMQGS alleviates endometritis remain unclear. Aim of the studyThe objective of this study is to elucidate the underlying therapeutic mechanisms of YMQGS in the context of endometritis. Materials and methodsThe chemical components of YMQGS were analyzed using UHPLC/MS. The anti-inflammatory capabilities of YMQGS were assessed by inducing an inflammatory response in RAW264.7 cells with lipopolysaccharide (LPS). Moreover, a clinical model of endometritis was simulated via vaginal injection of LPS into the uterine horns to investigate the therapeutic mechanisms of YMQGS. ResultsYMQGS could downregulate the expression of inflammatory mediators (TNF-α, IL-6, IL-1β) by suppressing the activation of the TLRs/MyD88/TRAF6/NF-κB signaling pathway in RAW264.7 cells. Additionally, YMQGS facilitated the polarization of RAW264.7 cells towards M2-like macrophages (Mφs) while inhibiting M1-like Mφs polarization, thereby exerting anti-inflammatory effects. Further investigation revealed that YMQGS could diminish ROS production, augment cellular antioxidant capacity, preserve mitochondrial membrane potential, and reduce intracellular [Ca2+]i accumulation, thereby minimizing cellular damage. In the mouse model of endometritis, YMQGS similarly inhibited the activation of the NF-κB signaling pathway in uterine tissues and modulated Mφ polarization in the endometrium, leading to a decrease in the expression of inflammatory factors and subsidence of the inflammatory response in the endometrium. Histological staining results showed that YMQGS reduced endometrial fibrosis, increased the expression of endometrial ZO-1, Occludin and glycoprotein, protect the endometrial barrier function, and prevent the further development of endometritis. ConclusionCollectively, the present study confirms that YMQGS possesses notable anti-inflammatory and antioxidant properties. The suppression of the NF-κB signaling pathway and the modulation of Mφ polarization emerge as the pivotal mechanisms by which YMQGS alleviates endometritis.

CDK14 is regulated by IGF2BP2 and involved in osteogenic differentiation via Wnt/β-catenin signaling pathway in vitro

AimsCyclin-dependent kinase (CDK) family proteins involve in various cellular processes via regulating the cell cycle; however, their expression during osteogenic differentiation and postmenopausal osteoporosis remains poorly understood. Main methodsUsing bioinformatics, we screened for CDK14 bound to Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and explored its expression in vitro with time-gradient model and in a mouse model of postmenopausal osteoporosis, building on prior research. Subsequently, we investigated its effect on osteoblast proliferation, cell cycle dynamics, and osteogenic differentiation by administering CDK14 siRNA and the covalent inhibitor FMF-04-159-2. Furthermore, we examined the interaction between IGF2BP2 and CDK14. Finally, we validated the regulatory role of CDK14 on the Wnt/β-catenin pathway. Key findingsOur findings demonstrate a time-dependent CDK14 expression patterns during osteogenic differentiation of MC3T3-E1 cell line, with an initial increase followed by gradual decline over time. Notably, CDK14 expression exhibited significant reduction in bone tissue of postmenopausal osteoporosis mouse model. CDK14 inhibition altered osteoblast cell cycle dynamics, significantly reduced cellular proliferation capacity, and impaired osteogenic differentiation ability. IGF2BP2 interacted with CDK14 mRNA, and stabilizing mRNA's structure and inhibiting its degradation. Additionally, CDK14 facilitated Low-density lipoprotein receptor-related protein 6 (LRP6) and Glycogen synthase kinase 3β (GSK3β) phosphorylation, thus regulating β-catenin levels. SignificanceThese findings provide further insight into the molecular mechanisms governing osteoblast proliferation, differentiation and osteoporosis.

Comparative investigation of exosome extraction from rat bone marrow mesenchymal stem cells using three different methodologies

AbstractExosomes have been identified as crucial mediators in numerous physiological and pathological processes, emerging as a focal point of scientific inquiry. This study aims to compare three methods for isolating exosomes from rat bone marrow mesenchymal stem cells: ultracentrifugation (UC), ultrafast separation system (EXODUS), and commercial precipitation kit (EXO‐kit). First, the investigation compared exosomal morphology, particle size distribution, and expression of marker proteins. Subsequently, the RNA content, protein concentration, and purity of exosomes were evaluated. Finally, the impact of these exosomes on cellular metabolic viability and migration capacity was assessed. Results indicated that exosomes exhibited spherical or elliptical membrane structures, and most of the exosomes extracted by the three methods were in the range of 30to 200 nm. UC‐extracted exosomes demonstrated the least impurities and clearest background, followed by EXODUS‐extracted exosomes, and lastly EXO‐kit‐extracted exosomes. The EXO‐kit‐extracted exosomes yielded the highest RNA and protein content, whereas those isolated through UC exhibited superior purity. Furthermore, exosomes extracted from EXODUS and EXO‐kit methods effectively enhanced the metabolic viability and migratory ability of osteoblast precursor cells compared to UC‐extracted exosomes. In conclusion, each of the three methodologies presents advantages and limitations. Therefore, the selection of an appropriate exosome extraction technique should be based on specific experimental objectives and requirements.

Activity and gene expression analysis of the NADP-dependent isocitrate dehydrogenase (NADP-ICDH) through pepper fruit ripening and its modulation by nitric oxide (NO). Molecular characterization of the peroxisomal isozyme

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) is one of the main sources of cellular reductant capacity in the form of NADPH. Although there is significant knowledge about the relevance of this enzyme during some physiological and stress processes, the available information about its involvement in fruit ripening is scarce. Using sweet green pepper (Capsicum annuum L.) fruits, a 50–75 % ammonium-sulfate-enriched protein fraction containing the NADP-ICDH activity allowed its biochemical characterization. The enzyme displayed a typical Michaelis-Menten kinetics and exhibited Vmax and Km values of 97 μUnits and 78 µM for isocitrate, and 92 μUnits and 46 µM for NADP+. Three NADP-ICDH isozymes were identified by non-denaturing PAGE designated as NADP-ICDH I to III, each representing 33 %, 24 %, and 43 %, respectively, of the total activity. Based on our previous transcriptome (RNA-Seq), three CaICDH genes (CaNADP-ICDH1, CaNADP-ICDH2, and CaNADP-ICDH3) were identified in sweet pepper fruits encoding isozymes potentially distributed in the cytosol, cytosol/mitochondrion, and peroxisome, according to their percentage of identity with the Arabidopsis isozymes. The time-course expression analysis of these genes during different fruit ripening stages including green immature (G), breaking point (BP), and red ripe (R), and in fruits subjected to nitric oxide (NO) treatments, showed dissimilar expression patterns. During ripening from green to red fruits, CaNADP-ICDH1 and CaNADP-ICDH2 were upregulated but were negatively affected by NO; however, CaNADP-ICDH3 was downregulated during ripening but unaffected by NO treatment. Furthermore, during ripening, the NADP-ICDH activity increased in red ripe fruits whereas the NO gas treatment produced a significant inhibition. These findings provide, to our knowledge, the first characterization of the NADP-ICDH family in this non-climacteric fruit and suggest that NADP-ICDH must play an important role in maintaining the supply of NADPH during pepper fruit ripening and that NO partially modulates this NADPH-generating system.

Screening and preparation of highly active antioxidant peptides of apricot and their inhibitory effect on ultraviolet radiation

In today's social environment, the objective reality of people's increasing life pressure, environmental deterioration, and enhanced ultraviolet rays caused by the destruction of the ozone layer has led to the aggravation of people's oxidative stress. Therefore, exogenous antioxidant peptides have become a hot topic in research. In the context of insufficient protein supply and resource recycling, almond meal was used as raw material in this study. As a by-product of oil processing, it has a protein content of 68 % and antioxidant-related amino acids accounted for 84.62 %, which can be used as a high-quality natural source of antioxidant peptides. Taking antioxidant activity as the only indicator, papain was screened as a hydrolase, and 7 antioxidant peptides such as YLSF, LPSYVN and SPHWNVN were separated and purified. The affinity energy of docking with Keap1-Nrf2-ARE protein molecules was −7.5—-8.9 kal/mol, and hydrophobic stacking, hydrogen bonding and intermolecular forces were maintained. Seven antioxidant peptides were synthesized in solid phase, and the IC50 values of in vitro ABTS+ scavenging rates were 3.59 μg/mL-6.73 μg/mL, and the antioxidant capacity was stronger than that of glutathione and ascorbic acid. In the in vitro cellular ROS scavenging capacity, all seven peptides had the effect of scavenging intracellular ROS, among which YLSF and ESWNPRDPQF had stronger scavenging capacity than glutathione. Finally, the mouse skin staining method determined that apricot antioxidant peptides had a significant inhibitory effect on UV damage to mouse skin, and targeted proteomics was used to clarify that apricot antioxidant peptides inhibited UV damage by mainly affecting three pathways, including the base excision repair pathway. This study not only improved the economic value of processing by-products, but also obtained 7 highly active almond antioxidant peptides, tapping the potential ability of apricot antioxidant peptides to be incorporated into functional food or cosmetic formulations.

Emergence of dysfunctional neutrophils with a defect in arginase-1 release in severe COVID-19.

Neutrophilia occurs in patients infected with SARS-CoV-2 (COVID-19) and is predictive of poor outcomes. Here, we link heterogenous neutrophil populations to disease severity in COVID-19. We identified neutrophils with features of cellular aging and immunosuppressive capacity in mild COVID-19 and features of neutrophil immaturity and activation in severe disease. The low-density neutrophil (LDN) number in circulating blood correlated with COVID-19 severity. Many of the divergent neutrophil phenotypes in COVID-19 were overrepresented in the LDN fraction and were less detectable in normal-density neutrophils. Functionally, neutrophils from patients with severe COVID-19 displayed defects in neutrophil extracellular trap formation and reactive oxygen species production. Soluble factors secreted by neutrophils from these patients inhibited T cell proliferation. Neutrophils from patients with severe COVID-19 had increased expression of arginase-1 protein, a feature that was retained in convalescent patients. Despite this increase in intracellular expression, there was a reduction in arginase-1 release by neutrophils into serum and culture supernatants. Furthermore, neutrophil-mediated T cell suppression was independent of arginase-1. Our results indicate the presence of dysfunctional, activated, and immature neutrophils in severe COVID-19.

Nanoparticle-delivered quercetin exhibits enhanced efficacy in eliminating iron-overloaded senescent chondrocytes.

Aim: The therapeutic potential of senolytic drugs in osteoarthritis (OA) is poorly known. Quercetin, a senolytic agent exhibits promising potential to treat OA, having limited bioavailability. We investigated the effects of Quercetin-loaded nanoparticles (Q-NP) with enhanced bioavailability in human chondrocytes mimicking OA phenotype.Materials & methods: The C-20/A4 chondrocytes were exposed to ferric ammonium citrate to induce OA phenotype, followed by treatment with free Quercetin/Q-NP for 24 and 48-h. Q-NP were synthesized by nanoprecipitation method. Following treatment chondrocytes were assessed for drug cellular bioavailability, viability, cell cycle, apoptosis, oxidative stress and expression of key senescence markers.Results: Q-NP exhibited 120.1±1.2nm particle size, 81±2.4% encapsulation efficiency, increased cellular bioavailability and selective apoptosis of senescent chondrocytes compared with free Quercetin. Q-NP treatment also induced oxidative stress and reduced the expressions of senescence markers, including TRB3, p16, p62 and p21 suggesting their ability to eliminate senescent cells. Last, Q-NP arrested the cell cycle in the sub-G0 phase, potentially creating a beneficial environment for tissue repair.Conclusion: Q-NP propose a promising delivery system for treating OA by eliminating senescent chondrocytes through apoptosis. Furthermore, their enhanced cellular bioavailability and capacity to modify cell cycle and senescent pathways warrant further investigations.

Pretreatment with aqueous Moringa oleifera Lam. leaf extract prevents cadmium-induced hepatotoxicity by improving cellular antioxidant machinery and reducing cadmium accumulation

Cadmium (Cd) is a highly harmful pollutant that poses a serious threat to human health. The liver is the primary organ for Cd accumulation, and Cd-induced hepatotoxicity has been shown to be strongly correlated with an oxidative imbalance in hepatocytes. Our previous studies in the eukaryotic model organism Saccharomyces cerevisiae revealed that not only co-treatment but also pretreatment with aqueous Moringa oleifera Lam. leaf extract (AMOLE) effectively mitigated Cd toxicity by reducing intracellular Cd accumulation and Cd-mediated oxidative stress. In this study, we therefore investigated the preventive effect of AMOLE against Cd toxicity in human HepG2 hepatocytes. The results showed that, similar to the case of the yeast model, pretreatment with AMOLE prior to Cd exposure also significantly inhibited Cd-induced oxidative stress in HepG2 cells. Untargeted LC-MS/MS-based metabolomic analysis of AMOLE revealed that its major phytochemical constituents were organic acids, particularly phenolic acids and carboxylic acids. Additionally, DPPH-HPTLC fingerprints suggested that quercetin and other flavonoids possibly contribute to the antioxidant activities of AMOLE. Based on our findings, it appears that pretreatment with AMOLE prevented Cd-induced hepatotoxicity via three possible mechanisms: i) direct elimination of free radicals by AMOLE antioxidant compounds; ii) upregulation of antioxidant defensive machinery (GPx1, and HO-1) via Nrf2 signaling cascade to improve cellular antioxidant capacity; and iii) reduction of intracellular Cd accumulation, probably by suppressing Cd uptake. These data strongly suggest the high potential of AMOLE for clinical utility in the prevention of Cd toxicity.

Lysis of human erythrocytes due to Piezo1-dependent cytosolic calcium overload as a mechanism of circulatory removal

Hematopoietic stem cells surrender organelles during differentiation, leaving mature red blood cells (RBC) devoid of transcriptional machinery and mitochondria. The resultant absence of cellular repair capacity limits RBC circulatory longevity, and old cells are removed from circulation. The specific age-dependent alterations required for this apparently targeted removal of RBC, however, remain elusive. Here, we assessed the function of Piezo1, a stretch-activated transmembrane cation channel, within subpopulations of RBC isolated based on physical properties associated with aging. We subsequently investigated the potential role of Piezo1 in RBC removal, using pharmacological and mechanobiological approaches. Dense (old) RBC were separated from whole blood using differential density centrifugation. Tolerance of RBC to mechanical forces within the physiological range was assessed on single-cell and cell population levels. Expression and function of Piezo1 were investigated in separated RBC populations by monitoring accumulation of cytosolic Ca2+ and changes in cell morphology in response to pharmacological Piezo1 stimulation and in response to physical forces. Despite decreased Piezo1 activity with increasing cell age, tolerance to prolonged Piezo1 stimulation declined sharply in older RBC, precipitating lysis. Cell lysis was immediately preceded by an acute reversal of density. We propose a Piezo1-dependent mechanism by which RBC may be removed from circulation: Upon adherence of these RBC to other tissues, they are uniquely exposed to prolonged mechanical forces. The resultant sustained activation of Piezo1 leads to a net influx of Ca2+, overpowering the Ca2+-removal capacity of specifically old RBC, which leads to reversal of ion gradients, dysregulated cell hydration, and ultimately osmotic lysis.

Stimuli-Responsive Phosphate Hydrogel: A Study on Swelling Behavior, Mechanical Properties, and Application in Expansion Microscopy.

Phosphorus-based stimuli-responsive hydrogels have potential in a wide range of applications due to their ionizable phosphorus groups, biocompatibility, and tunable swelling capacity utilizing hydrogel design parameters and external stimuli. In this study, poly(2-methacryloyloxyethyl phosphate) (PMOEP) hydrogels were synthesized via aqueous activators regenerated by electron transfer atomic transfer radical polymerization using ascorbic acid as the reducing agent. Swelling and deswelling behaviors of PMOEP hydrogels were examined in different salt solutions, pH conditions, and temperatures. The degree of swelling in salt solutions followed CaCl2 < MgCl2 < KCl < NaCl with a decrease in swelling rate at higher concentrations until reaching a saturation point. In water, the degree of swelling increased significantly around neutral pH and remained constant at basic pH values. The effects of polymerization conditions, including pH, temperature (30, 40, 50 °C), and MOEP concentration (40, 50, 60% v/v MOEP/H2O), on the hydrogel swelling behavior in various salt solutions were also investigated. PMOEP hydrogels showed a decrease in the degree of swelling as the pH was increased above the native pH of the monomer solution. Scanning electron microscopy and energy-dispersive spectroscopy were utilized to examine the microstructure and chemical composition of the dried hydrogel after salt solution swelling. Cytotoxicity testing using rat bone marrow stem cells confirmed the biocompatibility of the PMOEP hydrogels. A unique feature of this effort was evaluation of these phosphate hydrogels for use in expansion microscopy where a significant twofold enhancement in cellular expansion capacity was showcased utilizing 4T1 mouse breast cancer cells. This comprehensive study provides valuable insights into the stimuli-responsive behavior and expansion characteristics of phosphate hydrogels, highlighting their potential in diverse biomedical applications.