ABSTRACT
Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of these phospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.
Subject(s)
Membrane Glycoproteins , Neuronal Ceroid-Lipofuscinoses , Mice , Animals , Child , Humans , Membrane Glycoproteins/metabolism , Molecular Chaperones/metabolism , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/metabolism , Neuronal Ceroid-Lipofuscinoses/pathology , Lysosomes/metabolism , Phospholipases/metabolism , Glycerophospholipids/metabolism , Phospholipids/metabolismABSTRACT
Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer and is the major autoantigen in membranous nephropathy, a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts, and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1 and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by ß- or γ-secretase, suggesting that PLA2R1 may not be a substrate for regulated intramembrane proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C activator PMA, cytokines, and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer, and membranous nephropathy.
Subject(s)
ADAM10 Protein , ADAM17 Protein , Amyloid Precursor Protein Secretases , Membrane Proteins , Receptors, Phospholipase A2 , ADAM10 Protein/metabolism , ADAM10 Protein/genetics , Humans , ADAM17 Protein/metabolism , ADAM17 Protein/genetics , Amyloid Precursor Protein Secretases/metabolism , Amyloid Precursor Protein Secretases/genetics , Animals , Membrane Proteins/metabolism , Membrane Proteins/genetics , Mice , HEK293 Cells , Receptors, Phospholipase A2/metabolism , Receptors, Phospholipase A2/genetics , Podocytes/metabolism , Proteolysis , Protein Domains , Ionomycin/pharmacologyABSTRACT
In this study, we advance our understanding of the spatial relationship between the purinosome, a liquid condensate consisting of six enzymes involved in de novo purine biosynthesis, and mitochondria. Previous research has shown that purinosomes move along tubulin toward mitochondria, suggesting a direct uptake of glycine from mitochondria. Here, we propose that the purinosome is located proximally to the mitochondrial transporters SLC25A13 and SLC25A38, facilitating the uptake of glycine, aspartate, and glutamate, essential factors for purine synthesis. We utilized the proximity ligation assay (PLA) and APEX proximity labeling to investigate the association between purinosome proteins and mitochondrial transporters. Our results indicate that purinosome assembly occurs close to the mitochondrial membrane under purine-deficient conditions, with the transporters migrating to be adjacent to the purinosome. Furthermore, both targeted and non-targeted analyses suggest that the SLC25A13-APEX2-V5 probe accurately reflects endogenous cellular status. These findings provide insights into the spatial organization of purine biosynthesis and lay the groundwork for further investigations into additional proteins involved in this pathway.
ABSTRACT
ABCB5 is a member of the ABC transporter superfamily composed of 48 transporters, which have been extensively studied for their role in cancer multidrug resistance and, more recently, in tumorigenesis. ABCB5 has been identified as a marker of skin progenitor cells, melanoma, and limbal stem cells. It has also been associated with multidrug resistance in several cancers. The unique feature of ABCB5 is that it exists as both a full transporter (ABCB5FL) and a half transporter (ABCB5ß). Several studies have shown that the ABCB5ß homodimer does not confer multidrug resistance, in contrast to ABCB5FL. In this study, using three complementary techniques, (1) nanoluciferase-based bioluminescence resonance energy transfer, (2) coimmunoprecipitation, and (3) proximity ligation assay, we identified two novel heterodimers in melanoma: ABCB5ß/B6 and ABCB5ß/B9. Both heterodimers could be expressed in High-Five insect cells and ATPase assays revealed that both functional nucleotide-binding domains of homodimers and heterodimers are required for their basal ATPase activity. These results are an important step toward elucidating the functional role of ABCB5ß in melanocytes and melanoma.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B , Melanoma , Humans , Adenosine Triphosphatases/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/isolation & purification , ATP Binding Cassette Transporter, Subfamily B/metabolism , Melanoma/genetics , Melanoma/physiopathology , HEK293 CellsABSTRACT
The involvement of secreted phospholipase A2s in respiratory diseases, such as asthma and respiratory viral infections, is well-established. However, the specific role of secreted phospholipase A2 group IIE (PLA2G2E) during influenza virus infection remains unexplored. Here, we investigated the role of PLA2G2E during H1N1 influenza virus infection using a targeted mouse model lacking Pla2g2e gene (Pla2g2e-/-). Our findings demonstrated that Pla2g2e-/- mice had significantly lower survival rates and higher viral loads in lungs compared to wild-type mice following influenza virus infection. While Pla2g2e-/- mice displayed comparable innate and humoral immune responses to influenza virus challenge, the animals showed impaired influenza-specific cellular immunity and reduced T cell-mediated cytotoxicity. This indicates that PLA2G2E is involved in regulating specific T cell responses during influenza virus infection. Furthermore, transgenic mice expressing the human PLA2G2E gene exhibited resistance to influenza virus infection along with enhanced influenza-specific cellular immunity and T cell-mediated cytotoxicity. Pla2g2e deficiency resulted in perturbation of lipid mediators in the lung and T cells, potentially contributing to its impact on the anti-influenza immune response. Taken together, these findings suggest that targeting PLA2G2E could hold potential as a therapeutic strategy for managing influenza virus infections.IMPORTANCEThe influenza virus is a highly transmissible respiratory pathogen that continues to pose a significant public health concern. It effectively evades humoral immune protection conferred by vaccines and natural infection due to its continuous viral evolution through the genetic processes of antigenic drift and shift. Recognition of conserved non-mutable viral epitopes by T cells may provide broad immunity against influenza virus. In this study, we have demonstrated that phospholipase A2 group IIE (PLA2G2E) plays a crucial role in protecting against influenza virus infection through the regulation of T cell responses, while not affecting innate and humoral immune responses. Targeting PLA2G2E could therefore represent a potential therapeutic strategy for managing influenza virus infection.
Subject(s)
Influenza A Virus, H1N1 Subtype , Lung , Orthomyxoviridae Infections , Animals , Mice , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/virology , Influenza A Virus, H1N1 Subtype/immunology , Lung/virology , Lung/immunology , Lung/pathology , Humans , Group II Phospholipases A2/genetics , Group II Phospholipases A2/immunology , T-Lymphocytes/immunology , Mice, Knockout , Immunity, Cellular , Mice, Inbred C57BL , Mice, Transgenic , Viral Load , Disease Models, Animal , Immunity, Humoral , Immunity, Innate , Influenza, Human/immunology , Influenza, Human/virology , FemaleABSTRACT
Secreted phospholipase A2s are involved in the development of obesity, type 2 diabetes mellitus (T2DM) and cardiovascular disease, which have become serious and growing health concerns worldwide. Integration of genome-wide association study and gene co-expression networks analysis showed that the secreted phospholipase A2 group XIIA (PLA2G12A) may participate in hepatic lipids metabolism. Nevertheless, the role of PLA2G12A in lipid metabolism and its potential mechanism remain elusive. Here, we used AAV9 vector carrying human PLA2G12A gene to exogenously express hPLA2G12A in the liver of mice. We demonstrated that the overexpression of hPLA2G12A resulted in a significant decrease in serum lipid levels in wild-type mice fed with chow diet or high-fat diet (HFD). Moreover, hPLA2G12A treatment protected against diet-induced obesity and insulin resistance in mice fed a HFD. Notably, we found that hPLA2G12A treatment confers protection against obesity and hyperlipidemia independent of its enzymatic activity, but rather by increasing physical activity and energy expenditure. Furthermore, we demonstrated that hPLA2G12A treatment induced upregulation of ApoC2 and Cd36 and downregulation of Angptl8, which contributed to the increase in clearance of circulating triglycerides and hepatic uptake of fatty acids without affecting hepatic de novo lipogenesis, very low-density lipoprotein secretion, or intestinal lipid absorption. Our study highlights the potential of PLA2G12A gene therapy as a promising approach for treating obesity, insulin resistance and T2DM.
Subject(s)
Diet, High-Fat , Energy Metabolism , Insulin Resistance , Mice, Inbred C57BL , Obesity , Triglycerides , Animals , Obesity/metabolism , Obesity/etiology , Mice , Triglycerides/metabolism , Triglycerides/blood , Male , Diet, High-Fat/adverse effects , Humans , Liver/metabolism , Lipid MetabolismABSTRACT
Melatonin (MLT), a conserved small indole compound, exhibits anti-inflammatory and antioxidant properties, contributing to its cardioprotective effects. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with atherosclerosis disease risk, and is known as an atherosclerosis risk biomarker. This study aimed to investigate the impact of MLT on Lp-PLA2 expression in the atherosclerotic process and explore the underlying mechanisms involved. In vivo, ApoE-/- mice were fed a high-fat diet, with or without MLT administration, after which the plaque area and collagen content were assessed. Macrophages were pretreated with MLT combined with ox-LDL, and the levels of ferroptosis-related proteins, NRF2 activation, mitochondrial function, and oxidative stress were measured. MLT administration significantly attenuated atherosclerotic plaque progression, as evidenced by decreased plaque area and increased collagen. Compared with those in the high-fat diet (HD) group, the levels of glutathione peroxidase 4 (GPX4) and SLC7A11 (xCT, a cystine/glutamate transporter) in atherosclerotic root macrophages were significantly increased in the MLT group. In vitro, MLT activated the nuclear factor-E2-related Factor 2 (NRF2)/SLC7A11/GPX4 signaling pathway, enhancing antioxidant capacity while reducing lipid peroxidation and suppressing Lp-PLA2 expression in macrophages. Moreover, MLT reversed ox-LDL-induced ferroptosis, through the use of ferrostatin-1 (a ferroptosis inhibitor) and/or erastin (a ferroptosis activator). Furthermore, the protective effects of MLT on Lp-PLA2 expression, antioxidant capacity, lipid peroxidation, and ferroptosis were decreased in ML385 (a specific NRF2 inhibitor)-treated macrophages and in AAV-sh-NRF2 treated ApoE-/- mice. MLT suppresses Lp-PLA2 expression and atherosclerosis processes by inhibiting macrophage ferroptosis and partially activating the NRF2 pathway.
Subject(s)
Atherosclerosis , Ferroptosis , Melatonin , NF-E2-Related Factor 2 , Animals , Ferroptosis/drug effects , NF-E2-Related Factor 2/metabolism , Melatonin/pharmacology , Mice , Atherosclerosis/metabolism , Atherosclerosis/drug therapy , Atherosclerosis/prevention & control , Atherosclerosis/pathology , Male , Amino Acid Transport System y+/metabolism , Amino Acid Transport System y+/genetics , Diet, High-Fat/adverse effects , Macrophages/metabolism , Macrophages/drug effects , Mice, Inbred C57BL , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism , Oxidative Stress/drug effects , Signal Transduction/drug effects , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Lipoproteins, LDL/metabolism , Antioxidants/pharmacologyABSTRACT
BACKGROUND: Breast cancer is a lethal malignancy affecting females worldwide. It has been reported that upregulated centromere protein A (CENPA) expression might indicate unfortunate prognosis and can function as a prognostic biomarker in breast cancer. This study aimed to investigate the accurate roles and downstream mechanisms of CENPA in breast cancer progression. METHODS: CENPA protein levels in breast cancer tissues and cell lines were analyzed by Western blot and immunohistochemistry assays. We used gain/loss-of-function experiments to determine the potential effects of CENPA and phospholipase A2 receptor (PLA2R1) on breast cancer cell proliferation, migration, and apoptosis. Co-IP assay was employed to validate the possible interaction between CENPA and DNA methyltransferase 1 (DNMT1), as well as PLA2R1 and hematopoietically expressed homeobox (HHEX). PLA2R1 promoter methylation was determined using methylation-specific PCR assay. The biological capabilities of CENPA/PLA2R1/HHEX axis in breast cancer cells was determined by rescue experiments. In addition, CENPA-silenced MCF-7 cells were injected into mice, followed by measurement of tumor growth. RESULTS: CENPA level was prominently elevated in breast cancer tissues and cell lines. Interestingly, CENPA knockdown and PLA2R1 overexpression both restrained breast cancer cell proliferation and migration, and enhanced apoptosis. On the contrary, CENPA overexpression displayed the opposite results. Moreover, CENPA reduced PLA2R1 expression through promoting DNMT1-mediated PLA2R1 promoter methylation. PLA2R1 overexpression could effectively abrogate CENPA overexpression-mediated augment of breast cancer cell progression. Furthermore, PLA2R1 interacted with HHEX and promoted HHEX expression. PLA2R1 knockdown increased the rate of breast cancer cell proliferation and migration but restrained apoptosis, which was abrogated by HHEX overexpression. In addition, CENPA silencing suppressed tumor growth in vivo. CONCLUSION: CENPA knockdown restrained breast cancer cell proliferation and migration and attenuated tumor growth in vivo through reducing PLA2R1 promoter methylation and increasing PLA2R1 and HHEX expression. We may provide a promising prognostic biomarker and novel therapeutic target for breast cancer.
Subject(s)
Neoplasms , Receptors, Phospholipase A2 , Female , Animals , Mice , Centromere Protein A/metabolism , Receptors, Phospholipase A2/genetics , Receptors, Phospholipase A2/metabolism , Genes, Homeobox , Cell Line, Tumor , DNA Methylation/genetics , Biomarkers/metabolism , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Neoplasms/geneticsABSTRACT
Phospholipase A2 receptor 1 (PLA2R1) plays a crucial role in various diseases, including membranous nephropathy. However, the precise implications of PLA2R1 deficiency remain poorly understood. In this study, we created PLA2R1 knockout rats to explore potential consequences resulting from the loss of the PLA2R1 gene. Unexpectedly, our PLA2R1 knockout rats exhibited symptoms resembling those of chronic kidney disease after an 8-week observation period. Notably, several rats developed persistent proteinuria, a hallmark of renal dysfunction. Immunohistochemical and immunofluorescence analyses revealed insignificant glomerular fibrosis, reduced podocyte count, and augmented glomerular expression of complement C3 (C3) compared to immunoglobin A (IgA) and immunoglobin G(IgG) in the rat model. These findings suggest that the loss of PLA2R1 may contribute to the pathogenesis of membranous nephropathy and related conditions. Our knockout rat model provides a valuable tool for investigating the underlying pathology of PLA2R1-associated diseases, and may facilitate the development of targeted therapies for membranous nephropathy and other related disorders.
Subject(s)
Glomerulonephritis, Membranous , Receptors, Phospholipase A2 , Animals , Rats , Autoantibodies , Glomerulonephritis, Membranous/genetics , Glomerulonephritis, Membranous/diagnosis , Glomerulonephritis, Membranous/metabolism , Receptors, Phospholipase A2/genetics , Receptors, Phospholipase A2/metabolismABSTRACT
Although many important discoveries have been made regarding the jasmonate signaling pathway, how jasmonate biosynthesis is initiated is still a major unanswered question in the field. Previous evidences suggest that jasmonate biosynthesis is limited by the availability of fatty acid precursor, such as âº-linolenic acid (âº-LA). This indicates that the lipase responsible for releasing α-LA in the chloroplast, where early steps of jasmonate biosynthesis take place, is the key initial step in the jasmonate biosynthetic pathway. Nicotiana benthamiana glycerol lipase A1 (NbGLA1) is homologous to N. attenuata GLA1 (NaGLA1) which has been reported to be a major lipase in leaves for jasmonate biosynthesis. NbGLA1 was studied for its potential usefulness in a species that is more common in laboratories. Virus-induced gene silencing of both NbGLA1 and NbGLA2, another homolog, resulted in more than 80% reduction in jasmonic acid (JA) biosynthesis in wounded leaves. Overexpression of NbGLA1 utilizing an inducible vector system failed to increase JA, indicating that transcriptional induction of NbGLA1 is insufficient to trigger JA biosynthesis. However, co-treatment with wounding in addition to NbGLA1 induction increased JA accumulation several fold higher than the gene expression or wounding alone, indicating an enhancement of the enzyme activity by wounding. Domain-deletion of a 126-bp C-terminal region hypothesized to have regulatory roles increased NbGLA1-induced JA level. Together, the data show NbGLA1 to be a major lipase for wound-induced JA biosynthesis in N. benthamiana leaves and demonstrate the use of inducible promoter-driven construct of NbGLA1 in conjunction with its transient expression in N. benthamiana as a useful system to study its protein function.
Subject(s)
Lipase , Nicotiana , Oxylipins , Nicotiana/genetics , Lipase/genetics , Chloroplasts , Cyclopentanes , GlycerolABSTRACT
Non-small cell lung cancer (NSCLC) stands prominent among the prevailing and formidable oncological entities. The immune and metabolic-related molecule Phospholipase A2, group IID (PLA2G2D) exerts promotional effects on tumor progression. However, its involvement in cancer angiogenesis remains elusive. Therefore, this investigation delved into the functional significance of PLA2G2D concerning angiogenesis in NSCLC. This study analyzed the expression and enriched pathways of PLA2G2D in NSCLC tissues through bioinformatics analysis, and measured the expression of PLA2G2D in NSCLC cells using qRT-PCR and western blot (WB). Subsequently, the viability and angiogenic potential of NSCLC cells were assessed employing CCK-8 and angiogenesis assays, respectively. The expression profile of angiogenic factors was analyzed through WB. Finally, the expression of glycolysis pathway-related genes, extracellular acidification rate and oxygen consumption rate, and the levels of pyruvate, lactate, citrate, and malate were analyzed in NSCLC cells using qRT-PCR, Seahorse XF 96, and related kits. Bioinformatics analysis revealed the upregulation of PLA2G2D in NSCLC tissues and its association with VEGF and glycolysis signaling pathways. Molecular and cellular experiments demonstrated that upregulated PLA2G2D promoted the viability, angiogenic ability, and glycolysis pathway of NSCLC cells. Rescue assays revealed that the effects of high expression of PLA2G2D on the viability, angiogenic ability, and glycolysis of NSCLC cells were weakened after the addition of the glycolysis inhibitor 2-DG. In summary, PLA2G2D plays a key role in NSCLC angiogenesis through aerobic glycolysis, displaying great potential as a target for anti-angiogenesis therapy.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Neovascularization, Pathologic , Humans , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/blood supply , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/blood supply , Lung Neoplasms/genetics , Neovascularization, Pathologic/metabolism , Cell Line, Tumor , Glycolysis , Group II Phospholipases A2/metabolism , Group II Phospholipases A2/genetics , Gene Expression Regulation, Neoplastic , Cell Proliferation , Signal Transduction , AngiogenesisABSTRACT
Microplastics, such as polylactic acid (PLA), are ubiquitous environmental pollutants with unclear implications for health impact. This study aims to elucidate the mechanisms of PLA-induced inflammatory liver injury, focusing on disturbance of bile acid metabolism. The in vitro PLA exposure experiment was conducted using HepG2 cells to assess cell viability, cytokine secretion, and effects on bile acid metabolism. In vivo, male C57BL/6 J mice were exposed to PLA for ten days continuously, liver function and histopathological assessment were evaluated after the mice sacrificed. Molecular analyses including quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting, were applied to evaluate the expression of bile acid metabolizing enzymes and transporters. PLA exposure resulted in decreased cell viability in HepG2 cells, increased inflammation and altered bile acid metabolism. In mice, PLA exposure resulted in decreased body weight and food intake, impaired liver function, increased hepatic inflammation, altered bile acid profiles, and dysregulated expression of bile acid metabolic pathways. PLA exposure disrupts bile acid metabolism through inhibition of the CYP7A1 enzyme and activation of the FGF-JNK/ERK signaling pathway, contributing to liver injury. These findings highlight the potential hepatotoxic effects of environmentally friendly plastics PLA and underscore the need for further research on their biological impact.
ABSTRACT
Berberine, isolated from Coptis chinensis and Phellodendron amurense, can attenuate colonic injury and modulate gut microbiota disorders in ulcerative colitis (UC). However, the mechanism and causal relationship between gut microbiota and the efficacy of Berberine on UC are still unclear, which were investigated by pseudo-germ-free (PGF) mice, 16S rRNA gene analysis and transcriptome analysis in this study. The results demonstrated that Berberine improved gut microbiota disorders, colon damage, tight-junction proteins, inflammatory and anti-inflammatory cytokines in DSS-induced colitis mice with intact gut microbiota but not in PGF mice. Besides, immune-related and inflammation-related pathways were closely related to the efficacy that Berberine alleviated colitis by regulating gut microbiota. Furthermore, Berberine reduced PGE2, PLA2, COX-2, Ptges, EP2 and p-Stat3 only in colitis mice with intact gut microbiota. In summary, our study confirms that Berberine inhibits PLA2-COX-2-PGE2-EP2 pathway in UC through gut microbiota, leading to the alleviation of inflammation in colon, which further elucidates the underlying mechanism and promotes the application of Berberine in UC.
Subject(s)
Berberine , Colitis, Ulcerative , Colitis , Gastrointestinal Microbiome , Animals , Mice , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/drug therapy , Berberine/pharmacology , Berberine/therapeutic use , Cyclooxygenase 2 , Dinoprostone , RNA, Ribosomal, 16S , Inflammation/drug therapy , Phospholipases A2 , Dextran Sulfate , Disease Models, Animal , Colon , Mice, Inbred C57BLABSTRACT
RATIONALE & OBJECTIVE: Proteinuria and anti-phospholipase A2 receptor 1 (anti-PLA2R1) antibody titers are associated with primary membranous nephropathy (MN) outcomes. We evaluated the association of antibodies against the cysteine-rich (CysR) and C-type lectin 1, 7, and 8 (CTLD1, CTLD7, and CTLD8) domains of PLA2R1 with MN outcomes. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: One-hundred-thirteen consecutive, consenting patients referred to the Nephology Unit of the Azienda-Socio-Sanitaria-Territoriale (ASST) Papa Giovanni XXIII (Bergamo, Italy) with PLA2R1-related, biopsy-proven MN whose persistent nephrotic syndrome (NS) was managed conservatively for>6 months and were monitored with serial evaluations of proteinuria, autoantibodies (by enzyme-linked immunosorbent assay), and clinical outcomes. EXPOSURE: Rituximab. OUTCOME: Complete (proteinuria<0.3g/24h) or partial (proteinuria≥0.3g/24h and<3.0g/24h with>50% reduction vs basal) NS remission. ANALYTICAL APPROACH: Univariable and multivariable Cox regression analyses. RESULTS: All patients had anti-CysR antibodies; 62 (54.9%) were multidomain recognizers. Anti-PLA2R1 and anti-CysR antibody titers were strongly correlated at baseline (P<0.001, r=0.934), 6 months (P<0.001, r=0.964), and 12 months (P<0.001, r=0.944). During a median follow-up of 37.1 (IQR, 20.3-56.9) months, 71 patients (62.8%) achieved either complete or partial remission of their NS. Lower baseline anti-PLA2R1 (HR, 0.997 [95% CI, 0.996-0.999], P=0.002) and anti-CysR [HR, 0.996 [95% CI, 0.993-0.998], P=0.001) titers were associated with a higher probability of remission, along with female sex, lower proteinuria, and lower serum creatinine levels (P<0.05 for all comparisons). Anti-CTLD antibodies were not associated with outcomes. At 6 and 12 months, compared to baseline, anti-PLA2R1 and anti-CysR antibody titers decreased more in patients progressing to partial or complete remission than in those without remission (P<0.05 for all comparisons). LIMITATIONS: Observational design. CONCLUSIONS: In PLA2R1-related MN, anti-PLA2R1 and anti-CysR antibodies similarly predict rituximab efficacy independent of PLA2R1 domain recognition. The choice between these tests should be dictated by feasibility and costs. Evaluating anti-CTLD antibodies appears unnecessary. PLAIN-LANGUAGE SUMMARY: Primary membranous nephropathy (MN), a leading cause of nephrotic syndrome (NS) in adults, is an autoimmune disease caused by autoantibodies binding to the podocyte antigen phospholipase A2 receptor 1 (PLA2R1). We assessed whether the effects of anti-CD20 cytolytic therapy with the monoclonal antibody rituximab are associated with detection rates and levels of anti-PLA2R1 antibodies and antibodies against PLA2R1 domains such as cysteine-rich (CysR), and C-type lectin 1, 7, and 8 (CTLD1, 7, and 8), in patients with PLA2R1-related MN and persistent NS. The probability of rituximab-induced complete or partial NS remission was associated with baseline anti-PLA2R1 and anti-CysR antibody titers, but not with anti-CTLD1, 7 and 8 antibodies or multidomain recognition. Integrated evaluation of anti-PLA2R1 or anti-CysR antibodies with proteinuria and kidney function may play a role in monitoring the effects of rituximab in patients with PLA2R1-related NS and MN.
Subject(s)
Autoantibodies , Glomerulonephritis, Membranous , Receptors, Phospholipase A2 , Rituximab , Adult , Aged , Female , Humans , Male , Middle Aged , Autoantibodies/blood , Autoantibodies/immunology , Cohort Studies , Cysteine , Glomerulonephritis, Membranous/drug therapy , Glomerulonephritis, Membranous/immunology , Immunologic Factors/therapeutic use , Prospective Studies , Proteinuria/drug therapy , Receptors, Phospholipase A2/immunology , Rituximab/therapeutic use , Treatment OutcomeABSTRACT
It is urgent to explore factors affecting immunotherapy efficacy to benefit non-small cell lung cancer (NSCLC) patient survival. Bioinformatics predicted genes associated with programmed cell death ligand 1 (PD-L1) expression and analysed phospholipase A2 group IID (PLA2G2D) expression in NSCLC. BODIPY 493/503 dye staining and kits detected lipids, triglycerides, and phospholipids in H1299 cells, respectively. Extracellular vesicles (EVs) were extracted for morphology and size assessment using electron microscopy. Western blot assayed CD9, CD63, HSP90, EVs-PD-L1, PD-L1, and PLA2G2D expression. CCK-8, LDH, and ELISA tested proliferation and toxicity of CD8+ T cells, interleukin-2, and interferon-gamma secretion, respectively. PLA2G2D, PD-L1, and Ki67 expression was detected by immunohistochemistry. Immunofluorescence assayed PLA2G2D localisation and CD8+ T cell content. Flow cytometry assessed PD-L1 and CD8 expression. In NSCLC, upregulated EVs-PD-L1 and clinical characteristics showed a strong correlation. H1299 cells with overexpression PD-L1 significantly reduced proliferation, toxicity of CD8+ T cells, and interleukin-2 and interferon-gamma levels. Bioinformatics revealed positive correlations between PLA2G2D and overexpressed PD-L1. PLA2G2D was expressed in macrophages and dendritic cells in NSCLC tissue. Overexpression PLA2G2D (oe-PLA2G2D) increased lipids, triglycerides, and phospholipids contents in H1299 cells. oe-PLA2G2D significantly reduced proliferation, toxicity of CD8+ T cells, and interleukin-2 and interferon-gamma levels. si-PD-L1 restored inhibition of oe-PLA2G2D on CD8+ T cells. oe-PLA2G2D significantly increased mice tumour volume and weight, upregulated expression of blood EVs-PD-L1 and tissue PD-L1, PLA2G2D, Ki67, and decreased CD8+ T cell content. PLA2G2D facilitated immune escape in NSCLC by regulating CD8+ T cell immune function by upregulating EVs-PD-L1.
Subject(s)
B7-H1 Antigen , CD8-Positive T-Lymphocytes , Carcinoma, Non-Small-Cell Lung , Extracellular Vesicles , Lung Neoplasms , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/metabolism , Humans , B7-H1 Antigen/metabolism , Lung Neoplasms/immunology , Extracellular Vesicles/immunology , Extracellular Vesicles/metabolism , Animals , Mice , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Tumor Escape/immunology , Female , Male , Cell Proliferation , Middle AgedABSTRACT
This study synthesized poly(3-hydroxypropionate) [P(3HP)]-containing polyhydroxyalkanoate (PHA) block copolymers, P(3HP)-b-P[2-hydroxybutyrate (2HB)] and P(3HP)-b-P(D-lactate) (PDLA), using Escherichia coli. The cells expressing an evolved sequence-regulating PHA synthase, PhaCARNDFH, and propionyl-CoA transferase were cultured with the supplementation of the corresponding monomer precursors in the medium. The block structure of P(3HP)-b-PDLA was confirmed by proton nuclear magnetic resonance analysis and solvent fractionation. The molecular weights of the polymers were in the range of 0.8-2.8 × 105. The solvent-cast polymer films were subjected to isothermal treatment to promote phase separation and crystallization and were subsequently melt-quenched to produce an amorphous phase. The melt-quenched P(3HP)-b-P(2HB) film exhibited a high elongation at break (1153%), resulting in a toughness of 181 MJ/m3. The solvent-cast film of P(3HP)-b-65 mol% PDLA exhibited partial elastic deformation, in which the P(3HP) phase functioned as a soft segment. The melt-quenching of the polymer resulted in embrittlement presumably due to the high lactate fraction. Overall, the P(3HP)-based block copolymers exhibited several mechanical properties depending on the higher-order structure of the polymer and the properties of the P(2-hydroxyalkanoate) segments. This study findings show that P(3HP)-b-P(2HB) and P(3HP)-b-PDLA can function excellently if their microstructures are properly controlled.
ABSTRACT
In this study, we investigated the mechanism of curcumin (CUR) release from poly(lactic-co-glycolic acid) (PLGA) and poly(lactic acid) (PLA) nanoparticles (NPs) by evaluating the temperature-dependent CUR release. NPs were prepared by the nanoprecipitation method using various PLGA/PLA polymers with different lactic:glycolic ratios (L:G ratios) and molecular weights. Increasing the polymer molecular weight resulted in a decrease in the particle size of NPs. The wet glass transition temperature (Tg) of PLGA/PLA NPs was lower than the intrinsic polymer Tg, which can be derived from the water absorption and nanosizing of the polymer. The reduction in Tg was more significant for the PLGA/PLA NPs with lower polymer L:G ratios and lower polymer molecular weight. The greater decrease of Tg in the lower polymer L:G ratios was possibly caused by the higher water absorption due to the more hydrophilic nature of the glycolic acid segment than that of the lactic acid segment. The efficient water absorption in PLGA/PLA NPs with lower molecular weight could cause a significant reduction of Tg as it has lower hydrophobicity. CUR release tests from the PLGA/PLA NPs exhibited enhanced CUR release with increasing temperatures, irrespective of polymer species. By fitting the CUR release profiles into mathematical models, the CUR release process was well described by an initial burst release followed by a diffusion-controlled release. The wet Tg and particle size of the PLGA/PLA NPs affected the amount and temperature dependence of the initial burst release of CUR. Above the wet Tg of NPs, the initial burst release of CUR increased sharply. Smaller particle sizes of PLGA/PLA NPs led to a higher fraction of initial CUR burst release, which was more pronounced above the wet Tg of NPs. The wet Tg and particle sizes of the PLGA/PLA NPs also influenced the diffusion-controlled CUR release. The diffusion rate of CUR in the NPs increased as the wet Tg values of the NPs decreased. The diffusion path length of CUR was affected by the particle size, with larger particle size resulting in a prolonged diffusion-controlled release of CUR. This study highlighted that for the formulation development of PLGA/PLA NPs, suitable PLGA/PLA polymers should be selected considering the physicochemical properties of PLGA/PLA NPs and their correlation with the release behavior of encapsulated drugs at the application temperature.
Subject(s)
Curcumin , Nanoparticles , Polylactic Acid-Polyglycolic Acid Copolymer , Curcumin/chemistry , Polyglycolic Acid/chemistry , Temperature , Delayed-Action Preparations , Glycols , Polyesters , Particle Size , Nanoparticles/chemistry , WaterABSTRACT
Calcium-independent phospholipase A2ß (iPLA2ß), a member of the phospholipase A2 (PLA2s) superfamily, is encoded by the PLA2G6 gene. Mutations in the PLA2G6 gene have been identified as the primary cause of infantile neuroaxonal dystrophy (INAD) and, less commonly, as a contributor to Parkinson's disease (PD). Recent studies have revealed that iPLA2ß deficiency leads to neuroinflammation, iron accumulation, mitochondrial dysfunction, lipid dysregulation, and other pathological changes, forming a complex pathogenic network. These discoveries shed light on potential mechanisms underlying PLA2G6-associated neurodegeneration (PLAN) and offer valuable insights for therapeutic development. This review provides a comprehensive analysis of the fundamental characteristics of iPLA2ß, its association with neurodegeneration, the pathogenic mechanisms involved in PLAN, and potential targets for therapeutic intervention. It offers an overview of the latest advancements in this field, aiming to contribute to ongoing research endeavors and facilitate the development of effective therapies for PLAN.
Subject(s)
MutationABSTRACT
PURPOSE: There is evidence that lower activity of the RAF/MEK/ERK network is associated with positive outcomes in mild and moderate courses of COVID-19. The effect of this cascade in COVID-19 sepsis is still undetermined. Therefore, we tested the hypothesis that activity of the RAF/MEK/ERK network in COVID-19-induced sepsis is associated with an impact on 30-day survival. METHODS: We used biomaterial from 81 prospectively recruited patients from the multicentric CovidDataNet.NRW-study cohort (German clinical trial registry: DRKS00026184) with their collected medical history, vital signs, laboratory parameters, microbiological findings and patient outcome. ERK activity was measured by evaluating ERK phosphorylation using a Proximity Ligation Assay. RESULTS: An increased ERK activity at 4 days after diagnosis of COVID-19-induced sepsis was associated with a more than threefold increased chance of survival in an adjusted Cox regression model. ERK activity was independent of other confounders such as Charlson Comorbidity Index or SOFA score (HR 0.28, 95% CI 0.10-0.84, p = 0.02). CONCLUSION: High activity of the RAF/MEK/ERK network during the course of COVID-19 sepsis is a protective factor and may indicate recovery of the immune system. Further studies are needed to confirm these results.
ABSTRACT
In this paper, we present a novel polylactic-acid/flax-composite substrate and the implementation of a demonstrator: a microcontroller board based on commercial design. The substrate is developed for printed circuit board (PCB) applications. The pre-preg is biodegradable, reinforced, and flame-retarded. The novel material was developed to counter the increasing amount of e-waste and to improve the sustainability of the microelectronics sector. The motivation was to present a working circuit in commercial complexity that can be implemented on a rigid substrate made of natural, bio-based materials with a structure very similar to the widely used Flame Retardant Class 4 (FR4) substrate at an early technological readiness level (2-3). The circuit design is based on the Arduino Nano open-source microcontroller board design so that the demonstration could be programmable and easy to fit into education, IoT applications, and embedded designs. During the work, the design was optimized at the level of layout. The copper-clad pre-preg was then prepared and processed with subtractive printed wiring technology and through hole plating. The traditional surface mounting methodology was applied for assembly. The resulting yield of PCB production was around 50%. Signal analysis was successful with analogue data acquisition (voltage) and low-frequency (4 kHz) tests, indistinguishable from sample FR4 boards. Eventually, the samples were subjected to highly accelerated stress test (HAST). HAST tests revealed limitations compared to traditional FR4 printed circuit materials. After six cycles, the weight loss was around 30% in the case of PLA/Flax, and as three-point bending tests showed, the possible ultimate strength (25 MPa at a flexural state) was reduced by 80%. Finally, the sustainability aspect was assessed, where we found that â¼95 vol% and â¼90 wt% of the traditional substrate can be substituted, significantly easing the load of waste on the environment.