Clusterin (CLU) is one of the main genetic risk factors of late onset Alzheimer’s disease (AD), and different polymorphisms have been associated with the disease. A GWAS performed in a population isolate from eastern Canada has found the polymorphism rs11136000 of the CLU gene to be associated with increased risk for AD. For the present work, we used two different cohorts (the “at‐risk” pre‐symptomatic PREVENT‐AD and the symptomatic ROSMAP cohort) with the objective of exploring how the rs11136000 polymorphism is associated with clinical manifestations, biomarkers and neuropathology of AD at different disease stages.
A. Molfino, Francesca Ambrosani, Federica Tambaro
et al.
BACKGROUND & AIMS Anorexia is a disabling symptom in cancer and we aimed at investigating the role of changes in gene expression in lung cancer patients presenting with anorexia. METHODS Genome-wide transcriptomic profiling was assessed in PBMCs RNA from newly diagnosed lung cancer patients and in a control group. RT-qPCR was used for selected genes. RESULTS RNA-Seq analysis revealed among groups a large number of differentially expressed genes mainly implicated in immune system regulation, oxidative stress and cytokine-mediated inflammation signaling pathways. In particular, we identified a total of 983 DEGs (843 up-regulated; 140 down-regulated) in anorexic cancer compared to controls. A selected number of DEGs including ADAM8, SMAD4, CCR4 and CLU were differentially expressed within cancer group according to the presence/absence of anorexia. In terms of RT-qPCR, ADAM8 was less expressed in cancer patients than controls (p < 0.001), and in anorexic patients vs controls (p = 0.001). The expression of SMAD4 was lower in cancer vs controls (p = 0.005), and in anorexic patients vs controls (p = 0.009). We observed lower CCR4 expression in both anorexic and non-anorexic vs control (p = 0.004, p = 0.011, respectively) and a similar trend was present for CLU. CONCLUSIONS Our data shed new light on the role of specific genes and their associated molecular pathways as potential key mechanisms for the development of anorexia and may represent a novel landmark for understanding the complex pathophysiology of impaired appetite in cancer.
Anastasia Ricci, Federica Biancucci, Gianluca Morganti
et al.
Abstract Ataxia Telangiectasia is a rare neurodegenerative disease caused by biallelic mutations in the Ataxia Telangiectasia Mutated gene. No cure is currently available for these patients but positive effects on neurologic features in AT patients have been achieved by dexamethasone administration through autologous erythrocytes (EryDex) in phase II and phase III clinical trials, leading us to explore the molecular mechanisms behind the drug action. During these investigations new ATM variants, which originated from alternative splicing of ATM messenger, were discovered, and detected in vivo in the blood of AT patients treated with EryDex. Some of the new ATM variants, alongside an i n silico designed one, were characterized and examined in AT fibroblast cell lines. ATM variants were capable of rescuing ATM activity in AT cells, particularly in the nuclear role of DNA DSBs recognition and repair, and in the cytoplasmic role of modulating autophagy, antioxidant capacity and mitochondria functionality, all of the features that are compromised in AT but essential for neuron survival. These outcomes are triggered by the kinase and further functional domains of the tested ATM variants, that are useful for restoring cellular functionality. The in silico designed ATM variant eliciting most of the functionality recover may be exploited in gene therapy or gene delivery for the treatment of AT patients.
<p>In ATM network design, self-healing is the ability of the network to continue to provide service in the event of failures, and this compromises both planning and operational aspects. The planning aspect involves optimal/near-optimal network design problems while the operational aspect deals with the implementation of protection schemes using restoration mechanisms, for allocating spare capacity to the network to be used in case of a failure event. This project investigates the survivability (i.e. restoration ratio) - here defined by means of the aggregate restoration ratio - in existing ATM networks based on various spare capacity distribution schemes, with the goal to (1) compare the network survivability for link and path restorations, and (2) determine the effects of various traffic and design related patterns on the restoration ratio.</p>
Background There is a great need to discover factors that could protect pancreatic β-cells from apoptosis and thus prevent diabetes mellitus. Clusterin (CLU), a chaperone protein, plays an important role in cell protection in numerous cells and is involved in various cellular mechanisms, including autophagy. In the present study, we investigated the protective role of CLU through autophagy regulation in pancreatic β-cells. Methods To identify the protective role of CLU, mouse insulinoma 6 (MIN6) cells were incubated with CLU and/or free fatty acid (FFA) palmitate, and cellular apoptosis and autophagy were examined. Results Treatment with CLU remarkably upregulated microtubule-associated protein 1-light chain 3 (LC3)-II conversion in a dose- and time-dependent manner with a significant increase in the autophagy-related 3 (Atg3) gene expression level, which is a mediator of LC3-II conversion. Moreover, co-immunoprecipitation and fluorescence microscopy experiments showed that the molecular interaction of LC3 with Atg3 and p62 was markedly increased by CLU. Stimulation of LC3-II conversion by CLU persisted in lipotoxic conditions, and FFA-induced apoptosis and dysfunction were simultaneously improved by CLU treatment. Finally, inhibition of LC3-II conversion by Atg3 gene knockdown markedly attenuated the cytoprotective effect of CLU. Conclusion Taken together, these findings suggest that CLU protects pancreatic β-cells against lipotoxicity-induced apoptosis via autophagy stimulation mediated by facilitating LC3-II conversion. Thus, CLU has therapeutic effects on FFA-induced pancreatic β-cell dysfunction.
Previous evidence revealed significant elevated liver cancer mortality in the areas where water was contaminated with hexavalent chromium [Cr(vi)], which highlighted that we should pay more attention to Cr(vi)-induced cytotoxicity in hepatocytes. We found that Clusterin (CLU) was up-regulated in Cr(vi)-exposed L-02 hepatocytes, but the role CLU played in Cr(vi)-induced cytotoxicity has never been explored. In the present study, we demonstrate Cr(vi) targeted mitochondrial respiratory chain complex I (MRCC I) activity and induced reactive oxygen species (ROS) accumulation, which caused mitochondrial damage that was characterized by the increase of permeability transition pore (PTP) open rate, the collapse of mitochondrial membrane potential (MMP), and the release of apoptosis-inducing factor (AIF) and Cytochrome C (Cyt C) from mitochondria to cytoplasm, which then induced cell viability loss and increased aspartate transaminase (AST)/alanine transaminase (ALT) leakage. We reveal that Cr(vi) may regulate CLU expression through the ROS-ataxia telangiectasia mutant (ATM)-insulin-like growth factor 1 (IGF-1) axis, and CLU expression was positively correlated to MRCC I activity. We further confirmed that CLU may regulate MRCC I activity via modulating its subunit nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3) expression. By the establishment of CLU over-expression cells, we found that over-expression of CLU alleviated Cr(vi)-induced MRCC I inhibition and further rescued cell viability loss and reduced AST and ALT leakage. Thus, we reached the conclusion that the CLU-induced increase of MRCC I activity protected against Cr(vi)-induced cytotoxicity. The present research will provide new experimental evidence for thoroughly clarifying the cytotoxicity and the carcinogenic mechanism of Cr(vi).
Background: Left ventricular remodeling (LVR) after myocardial infarction is associated with an increased risk of heart failure and death. In spite of a modern therapeutic approach, LVR remains relatively frequent and difficult to predict in clinical practice. Our aim was to identify new biomarkers of LVR and understand their involvement in its development. Methods and Results: Proteomic analysis of plasma from the REVE-2 study (Remodelage Ventriculaire)—a study dedicated to the analysis of LVR which included 246 patients after a first anterior myocardial infarction—identified increased plasma levels of CLU (clusterin) in patients with high LVR. We used a rat model of myocardial infarction to analyze CLU expression in the LV and found a significant increase that was correlated with LVR parameters. We found increased CLU expression and secretion in primary cultures of rat neonate cardiomyocytes hypertrophied by isoproterenol. Silencing of CLU in hypertrophied neonate cardiomyocytes induced a significant decrease in cell size, ANP (atrial natriuretic peptide), and BNP (B-type natriuretic peptide) expression, associated with a decreased ERK (extracellular signal-regulated kinase) 1/2 activity, suggesting a prohypertrophic role of CLU. We then confirmed a significant increase of both intracellular p-CLU (precursor form of CLU) and m-CLU (mature form of CLU) in failing human hearts. Finally, the circulating levels of CLU (secreted form) were increased in patients with chronic heart failure who died from cardiovascular cause during a 3-year follow-up (n=99) compared with survivors (n=99). Conclusions: Our results show for the first time that plasma CLU levels are associated with LVR post–myocardial infarction, have in part a cardiac origin, and are a predictor of early death in heart failure patients.
Clusterin (CLU) is considered one of the most important roles for pathogenesis of Alzheimer’s Disease (AD). The early genome-wide association studies (GWAS) identified the CLU rs11136000 polymorphism is significantly associated with AD in Caucasian. However, the subsequent studies are unable to replicate these findings in different populations. Although two independent meta-analyses show evidence to support significant association in Asian and Caucasian populations by integrating the data from 18 and 25 related GWAS studies, respectively, many of the following 18 studies also reported the inconsistent results. Moreover, there are six missed and a misclassified GWAS studies in the two meta-analyses. Therefore, we suspected that the small-scale and incompletion or heterogeneity of the samples maybe lead to different results of these studies. In this study, large-scale samples from 50 related GWAS studies (28,464 AD cases and 45,784 controls) were selected afresh from seven authoritative sources to reevaluate the effect of rs11136000 polymorphism to AD risk. Similarly, we identified that the minor allele variant of rs11136000 significantly decrease AD risk in Caucasian ethnicity using the allele, dominant and recessive model. Different from the results of the previous studies, however, the results showed a negligible or no association in Asian and Chinese populations. Collectively, our analysis suggests that, for Asian and Chinese populations, the variant of rs11136000 may be irrelevant to AD risk. We believe that these findings can help to improve the understanding of the AD’s pathogenesis.
Newer urinary protein kidney safety biomarkers can outperform the conventional kidney functional biomarkers blood urea nitrogen (BUN) and serum creatinine (SCr) in rats. However, there is far less experience with the relative performance of these biomarkers in dogs and nonhuman primates. Here, we report urine protein biomarker performance in tenofovir-treated cynomolgus monkeys and beagle dogs. Tenofovir intravenous daily dosing in monkeys for 2 or 4 weeks at 30 mg/kg/day resulted in minimal to moderate tubular degeneration and regeneration, and tenofovir disoproxil fumarate oral dosing in dogs for 10 days at 45 mg/kg/day resulted in mild to marked tubular degeneration, necrosis, and regeneration. Among biomarkers tested, kidney injury molecule 1 (Kim-1) and clusterin (CLU) clearly outperformed BUN and SCr and were the most reliable in detecting the onset and progression of tenofovir-induced tubular injury. Cystatin C, retinol binding protein 4, β2-microglobulin, neutrophil gelatinase–associated lipocalin, albumin, and total protein also performed better than BUN and SCr and added value when considered together with Kim-1 and CLU. These findings demonstrate the promising utility of these urinary safety biomarkers in monkeys and dogs and support their further evaluation in human to improve early detection of renal tubular injury.
Mohammad Javad Ehsani Ardakani, Akram Safaei, Afsaneh Arefi Oskouie
et al.
Aim: In the current study, we analysised only the articles that investigate serum proteome profile of cirrhosis patients or HCC patients versus healthy controls. Background: Increased understanding of cancer biology has enabled identification of molecular events that lead to the discovery of numerous potential biomarkers in diseases. Protein-protein interaction networks is one of aspect that could elevate the understanding level of molecular events and protein connections that lead to the identification of genes and proteins associated with diseases. Methods: Gene expression data, including 63 gene or protein names for hepatocellular carcinoma and 29 gene or protein names for cirrhosis, were extracted from a number of previous investigations. The networks of related differentially expressed genes were explored using Cytoscape and the PPI analysis methods such as MCODE and ClueGO. Centrality and cluster screening identified hub genes, including APOE, TTR, CLU, and APOA1 in cirrhosis. Results: CLU and APOE belong to the regulation of positive regulation of neurofibrillary tangle assembly. HP and APOE involved in cellular oxidant detoxification. C4B and C4BP belong to the complement activation, classical pathway and acute inflammation response pathway. Also, it was reported TTR, TFRC, VWF, CLU, A2M, APOA1, CKAP5, ZNF648, CASP8, and HSP27 as hubs in HCC. In HCC, these include A2M that are corresponding to platelet degranulation, humoral immune response, and negative regulation of immune effector process. CLU belong to the reverse cholesterol transport, platelet degranulation and human immune response. APOA1 corresponds to the reverse cholesterol transport, platelet degranulation and humoral immune response, as well as negative regulation of immune effector process pathway. Conclusion: In conclusion, this study suggests that there is a common molecular relationship between cirrhosis and hepatocellular cancer that may help with identification of target molecules for early treatment that is essential in cancer therapy.
Stefano Amente, G. Milazzo, M. C. Sorrentino
et al.
The chromatin-modifying enzyme lysine-specific demethylase 1, KDM1A/LSD1 is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells and its overexpression correlated with aggressive disease, poor differentiation and infaust outcome. Here, we show that LSD1 physically binds MYCN both in vitro and in vivo and that such an interaction requires the MYCN BoxIII. We found that LSD1 co-localizes with MYCN on promoter regions of CDKN1A/p21 and Clusterin (CLU) suppressor genes and cooperates with MYCN to repress the expression of these genes. KDM1A needs to engage with MYCN in order to associate with the CDKN1A and CLU promoters. The expression of CLU and CDKN1A can be restored in MYCN-amplified cells by pharmacological inhibition of LSD1 activity or knockdown of its expression. Combined pharmacological inhibition of MYCN and LSD1 through the use of small molecule inhibitors synergistically reduces MYCN-amplified Neuroblastoma cell viability in vitro. These findings demonstrate that LSD1 is a critical co-factor of the MYCN repressive function, and suggest that combination of LSD1 and MYCN inhibitors may have strong therapeutic relevance to counteract MYCN-driven oncogenesis.
Background/Aims: Clusterin (CLU), also known as Apolipoprotein J (ApoJ) is a highly glycosylated extracellular chaperone. In humans it is expressed from a broad spectrum of tissues and related to a plethora of physiological and pathophysiological processes, such as Alzheimer's disease, atherosclerosis and cancer. In its dominant form it is expressed as a secretory protein (secreted CLU, sCLU). During its maturation, the sCLU-precursor is N-glycosylated and cleaved into an α- and a β-chain, which are connected by five symmetrical disulfide bonds. Recently, it has been demonstrated that besides the predominant sCLU, rare intracellular CLU forms are expressed in stressed cells. Since these forms do not enter or complete the secretory pathway, they are not proteolytically modified and show either no or only core glycosylation. Due to their sparsity, these intracellular forms are functionally poorly characterized. To evaluate the function(s) of these stress-related intracellular forms, we investigate for the first time the impact of proteolytic cleavage, differential glycosylation and the influence of the redox environment on the chaperone activity of CLU. Methods: Non-cleavable sCLU was generated by expression from a mutant construct of sCLU, in which the furin-like proprotein convertase (PC) recognition site was modified. After purification of recombinant uncleaved sCLU from the medium of over-expressing cells, we performed chaperone activity assays to compare the activities of wild-type (cleaved) and uncleaved mutant sCLU. Additionally, this approach enabled us to investigate the role of carbohydrates, the proteolytic maturation and reducing conditions on CLU chaperone activity. Further, we characterized the differentially treated CLU forms by using MALDI-TOF, CD-spectroscopy and Western blotting in addition to the functional assay. Results: We show that the PC-cleavage is dispensable for sCLU chaperone activity. Moreover, our data demonstrate that while fully deglycosylated sCLU lacks chaperone activity, partially deglycosylated sCLU is still capable of solubilizing target proteins. Most importantly, we here demonstrate for the first time that uncleaved sCLU is highly sensitive towards reducing conditions. Conclusions: Our study provides evidence that unglycosylated intracellular CLU forms cannot exhibit a chaperone activity compared to sCLU. Additionally, we support recent postulates that glycosylated intracellular CLU forms may act as a redox sensor under oxidative stress conditions. Furthermore, we conclude that the proteolytic cleavage of sCLU is important to maintain full chaperone activity, i.e. in the presence of necrosis.
Abstract. Open biomass burning is a significant source of primary air pollutants such as particulate matter and non-methane organic gases. However, the physical and chemical atmospheric processing of these emissions during transport is poorly understood. Atmospheric transformations of biomass burning emissions have been investigated in environmental chambers, but there have been limited opportunities to investigate these transformations in the atmosphere. In this study, we deployed a suite of real-time instrumentation on a Twin Otter aircraft to sample smoke from prescribed fires in South Carolina, conducting measurements at both the source and downwind to characterize smoke evolution with atmospheric aging. Organic aerosol (OA) within the smoke plumes was quantified using an Aerosol Mass Spectrometer (AMS), along with refractory black carbon (rBC) using a Single Particle Soot Photometer and carbon monoxide (CO) and carbon dioxide (CO2) using a Cavity Ring-Down Spectrometer. During the two fires for which we were able to obtain aerosol aging data, normalized excess mixing ratios and "export factors" of conserved species (rBC, CO, CO2) were unchanged with increasing sample age. Investigation of AMS mass fragments indicated that the in-plume fractional contribution (fm/z) to OA of the primary fragment (m/z 60) decreased downwind, while the fractional contribution of the secondary fragment (m/z 44) increased. Increases in f44 are typically interpreted as indicating chemical production of secondary OA (SOA). Likewise, we observed an increase in the O : C elemental ratio downwind, which is usually associated with aerosol aging. However, the rapid mixing of these plumes into the background air suggests that these chemical transformations may be attributable to the different volatilities of the compounds that fragment to these m/z in the AMS. The gas-particle partitioning behavior of the bulk OA observed during the study was consistent with the predictions from a parameterization developed for open biomass burning emissions in the laboratory. Furthermore, we observed no statistically-significant increase in total organic mass with atmospheric transport. Hence, our results suggest that dilution-driven evaporation likely dominated over chemical production of SOA within our smoke plumes, likely due to the fast dilution and limited aging times (