Publications by authors named "Ralf Weiskirchen"

321 Publications

Flow Cytometry: A Blessing and a Curse.

Biomedicines 2021 Nov 4;9(11). Epub 2021 Nov 4.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, D-52074 Aachen, Germany.

Flow cytometry is a laser-based technology generating a scattered and a fluorescent light signal that enables rapid analysis of the size and granularity of a particle or single cell. In addition, it offers the opportunity to phenotypically characterize and collect the cell with the use of a variety of fluorescent reagents. These reagents include but are not limited to fluorochrome-conjugated antibodies, fluorescent expressing protein-, viability-, and DNA-binding dyes. Major developments in reagents, electronics, and software within the last 30 years have greatly expanded the ability to combine up to 50 antibodies in one single tube. However, these advances also harbor technical risks and interpretation issues in the identification of certain cell populations which will be summarized in this viewpoint article. It will further provide an overview of different potential applications of flow cytometry in research and its possibilities to be used in the clinic.
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http://dx.doi.org/10.3390/biomedicines9111613DOI Listing
November 2021

Cold traps as reliable devices for quantitative determination of SARS-CoV-2 load in aerosols.

Environ Monit Assess 2021 Nov 8;193(12):778. Epub 2021 Nov 8.

Medical Center Baden-Baden, Beethovenstr. 2, Baden-Baden, 76530, Germany.

Spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is a demanding challenge. This is of particular importance in schools and public areas of unavoidable access. New viral mutations may increase infectivity and require even better methods to identify areas of potential hazards. High-throughput SARS-CoV-2 testing and legal restrictions are not effective in order to get the current outbreak under control. The occurrence of new SARS-CoV-2 variants with a higher transmissibility requires efficient strategies for early detection and surveillance. Until today, testing focuses on nasal or pharyngeal mucosa swabs, neglecting the origin of aerosolic transmission, thus failing to detect the spread by carriers of the virus. Therefore, in this study, SARS-CoV-2 RNA levels were determined by quantitative real time PCR in aerosols collected by non-powered cold traps. SARS-CoV-2 spreading kinetics were recorded in indoor hotspots within a high-endemic area. These hotspots included a SARS-CoV-2 isolation unit, an outpatient endoscopy facility, a concert hall, and a shopping mall. For determination of viral presence aerosols were collected by cold traps positioned at different locations in the area of interest over a period of 4-6 h. Indoor SARS-CoV-2 hotspots were found in non-ventilated areas and in zones that are predisposed to a buoyancy (chimney) effect. SARS-CoV-2 RNA in those aerosols reached concentrations of 10 copies/mL, while extensive outdoor air ventilation reliably eliminated SARS-CoV-2 aerosol contamination. The method presented herein is effective for the identification of SARS-CoV-2 indoor hotspots and may help to characterize the spreading kinetics of SARS-CoV-2. Moreover, it can be used for the surveillance of emerging SARS-CoV-2 variants. Due to low costs and easy handling, the procedure might enable efficient algorithms for COVID-19 screening and prevention.
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http://dx.doi.org/10.1007/s10661-021-09580-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8573756PMC
November 2021

The potential pathophysiological role of altered lipid metabolism and electronegative low-density lipoprotein (LDL) in non-alcoholic fatty liver disease and cardiovascular diseases.

Clin Chim Acta 2021 Oct 19;523:374-379. Epub 2021 Oct 19.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, Aachen, Germany.

Non-alcoholic fatty liver disease (NAFLD) is an umbrella term for a range of conditions caused by a build-up of fat in the liver. It is usually seen in people who are overweight or obese. Increasingly common around the world, this disease is the most common chronic liver disease in the United States, affecting about a quarter of the population. Recently, the designation of NAFLD as 'metabolic dysfunction-associated fatty liver disease' (MAFLD) has been a subject of current debate. In this context, 'insulin resistance' is the underlying common and basic pathophysiological mechanism of metabolic dysfunction due to its association with obesity, type 2 diabetes mellitus (T2DM), metabolic syndrome, dyslipidemia and NAFLD. All these pathological conditions are among the metabolic risk factors for cardiovascular diseases, too. Also, due to the bidirectional causality between NAFLD and cardiovascular diseases, a liver-heart axis is suggested. Therefore, various factors such as insulin resistance as well as systemic inflammation, cytokines, oxidative stress, adipokines, hepatokines, genes and intestinal microbiota have been identified as possible pathogenic factors that play a role in the explanation of the complex NAFLD and cardiovascular risk relationship. Recent data and cumulative evidence show that electronegative low-density lipoprotein [LDL (-)/L5] cholesterol is a promising biomarker for complex organ interactions and diseases associated with liver-heart axis. In this mini review, we focus not only on recent data on NAFLD mechanisms, but also on the potential of the lipid mediator LDL (-)/L5 as a diagnostic and therapeutic target for liver-heart line diseases.
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http://dx.doi.org/10.1016/j.cca.2021.10.018DOI Listing
October 2021

Serum Perilipin 2 (PLIN2) Predicts Multiple Organ Dysfunction in Critically Ill Patients.

Biomedicines 2021 Sep 13;9(9). Epub 2021 Sep 13.

Department of Medicine III, University Hospital RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany.

Perilipin 2 (PLIN2) is a lipid droplet protein with various metabolic functions. However, studies investigating PLIN2 in the context of inflammation, especially in systemic and acute inflammation, are lacking. Hence, we assessed the relevance of serum PLIN2 in critically ill patients. We measured serum PLIN2 serum in 259 critically ill patients (166 with sepsis) upon admission to a medical intensive care unit (ICU) compared to 12 healthy controls. A subset of 36 patients underwent computed tomography to quantify body composition. Compared to controls, serum PLIN2 concentrations were elevated in critically ill patients at ICU admission. Interestingly, PLIN2 independently indicated multiple organ dysfunction (MOD), defined as a SOFA score > 9 points, at ICU admission, and was also able to independently predict MOD after 48 h. Moreover, serum PLIN2 levels were associated with severe respiratory failure potentially reflecting a moribund state. However, PLIN2 was neither a predictor of ICU mortality nor did it reflect metabolic dysregulation. Conclusively, the first study assessing serum PLIN2 in critical illness proved that it may assist in risk stratification because it is capable of independently indicating MOD at admission and predicting MOD 48 h after PLIN2 measurement. Further evaluation regarding the underlying mechanisms is warranted.
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http://dx.doi.org/10.3390/biomedicines9091210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8468514PMC
September 2021

Perilipin 5 Ameliorates Hepatic Stellate Cell Activation via SMAD2/3 and SNAIL Signaling Pathways and Suppresses STAT3 Activation.

Cells 2021 08 24;10(9). Epub 2021 Aug 24.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, D-52074 Aachen, Germany.

Comprehending the molecular mechanisms underlying hepatic fibrogenesis is essential to the development of treatment. The hallmark of hepatic fibrosis is the development and deposition of excess fibrous connective tissue forcing tissue remodeling. Hepatic stellate cells (HSC) play a major role in the pathogenesis of liver fibrosis. Their activation via the transforming growth factor-β1 (TGF-β1) as a key mediator is considered the crucial event in the pathophysiology of hepatic fibrogenesis. It has been shown that Perilipin 5 (PLIN5), known as a lipid droplet structural protein that is highly expressed in oxidative tissue, can inhibit such activation through various mechanisms associated with lipid metabolism. This study aimed to investigate the possible influence of PLIN5 on TGF-β1 signaling. Our findings confirm the importance of PLIN5 in maintaining HSC quiescence in vivo and in vitro. PLIN5 overexpression suppresses the TGF-β1-SMAD2/3 and SNAIL signaling pathways as well as the activation of the signal transducers and activators of transcription 3 (STAT3). These findings derived from experiments in hepatic cell lines LX-2 and Col-GFP, in which overexpression of PLIN5 was able to downregulate the signaling pathways SMAD2/3 and SNAIL activated previously by TGF-β1 treatment. Furthermore, TGF-β1-mediatedinduction of extracellular matrix proteins, such as collagen type I (COL1), Fibronectin, and α-smooth muscle actin (α-SMA), was suppressed by PLIN5. Moreover, STAT3, which is interrelated with TGF-β1 was already basally activated in the cell lines and inhibited by PLIN5 overexpression, leading to a further reduction in HSC activity shown by lowered α-SMA expression. This extension of the intervening mechanisms presents PLIN5 as a potent and pleiotropic target in HSC activation.
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http://dx.doi.org/10.3390/cells10092184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8467115PMC
August 2021

Chronic mineral oil administration increases hepatic inflammation in wild type mice compared to lipocalin 2 null mice.

Lab Invest 2021 Dec 13;101(12):1528-1539. Epub 2021 Sep 13.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH Aachen University Hospital, Aachen, Germany.

Lipocalin 2 (LCN2), an acute-phase protein produced during acute liver injury, plays an important role in the innate immune response against bacterial infection via iron scavenging. LCN2 further influences neutrophil development and physiology leading to increased inflammatory responses. We investigated the roles of LCN2 in chronic inflammation and fibrosis, using repeated carbon tetrachloride (CCl) in mineral-oil injection. Surprisingly, mice treated with the mineral oil vehicle alone showed liver inflammation, evidenced by neutrophil and monocyte-macrophage infiltration. Fluorescence-activated cell sorting (FACS) of isolated liver leukocytes showed significantly high CD45 leukocyte concentrations in CCl mice, but no difference of Ly6G neutrophils between mineral oil and CCl application. Liver CD11b F4/80 cells counted higher in CCl mice, but the proportions of Gr1, an indicator of inflammation, were significantly higher in mineral oil groups. Liver myeloperoxidase (MPO), expressed in neutrophils and monocytes, showed higher levels in wild type mice compared to Lcn2 in both mineral-oil and CCl treated groups. Hepatic and serum LCN2 levels were remarkably higher in the mineral oil-injected wild type group compared to the CCl. Wild type animals receiving mineral oil showed significantly higher inflammatory cytokine- and chemokine mRNA levels compared to Lcn2 mice, with no differences in the CCl treated groups. RNA sequencing (RNA-Seq) confirmed significant downregulation of gene sets involved in myeloid cell activation and immune responses in Lcn2 null mice receiving chronic mineral oil versus wild-type. We observed significant upregulation of gene sets and proteins involved in cell cycle DNA replication, with downregulation of collagen-containing extracellular matrix genes in Lcn2 mice receiving CCl compared to the wild type. Consequently, the wild type mice developed slightly more liver fibrosis compared to Lcn2 mice, evidenced by higher levels of collagen type I in the CCl groups and no liver fibrosis in mineral oil-treated mice. Our findings indicate that serum and hepatic LCN2 levels correlate with hepatic inflammation rather than fibrosis.
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http://dx.doi.org/10.1038/s41374-021-00672-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8590977PMC
December 2021

Single-cell omics: Overview, analysis, and application in biomedical science.

J Cell Biochem 2021 Nov 30;122(11):1571-1578. Epub 2021 Aug 30.

Department of Hematology, Oncology, and Tumor Immunology, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Single-cell sequencing methods provide the highest resolution insight into cellular heterogeneity. Owing to their rapid growth and decreasing cost, they are now widely accessible to scientists worldwide. Single-cell technologies enable analysis of a large number of cells, making them powerful tools to characterise rare cell types and refine our understanding of diverse cell states. Moreover, single-cell application in biomedical sciences helps to unravel mechanisms related to disease pathogenesis and outcome. In this Viewpoint, we briefly describe existing single-cell methods (genomics, transcriptomics, epigenomics, proteomics, and mulitomics), comment on available analysis tools, and give examples of method applications in the biomedical field.
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http://dx.doi.org/10.1002/jcb.30134DOI Listing
November 2021

The Unfolded Protein Response Is a Major Driver of LCN2 Expression in BCR-ABL- and JAK2V617F-Positive MPN.

Cancers (Basel) 2021 Aug 21;13(16). Epub 2021 Aug 21.

Department of Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Faculty of Medicine, RWTH Aachen University, 520674 Aachen, Germany.

Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression >100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun -terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1-JNK-NF-κB-C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.
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http://dx.doi.org/10.3390/cancers13164210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8391615PMC
August 2021

Physalin B attenuates liver fibrosis via suppressing LAP2α-HDAC1 mediated deacetylation of glioma-associated oncogene 1 and hepatic stellate cell activation.

Authors:
Ralf Weiskirchen

Br J Pharmacol 2021 10 18;178(20):4045-4047. Epub 2021 Aug 18.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, Aachen, Germany.

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http://dx.doi.org/10.1111/bph.15588DOI Listing
October 2021

The biotin interference within interference suppressed immunoassays.

J Clin Lab Anal 2021 Sep 4;35(9):e23940. Epub 2021 Aug 4.

Institute for Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), University Hospital RWTH, Aachen, Germany.

Background: Reports of false laboratory findings due to a biotin supplementation have raised concerns about the safety of immunoassays. According to current research, biotin is known to cause interference in immunoassays. Since up to 70% of medical decisions are based on laboratory results and the significantly increased intake of biotin supplements in the recent years, the reliability of immunoassays is essential.

Methods: To evaluate this reliability two experiments were conducted. In the first experiment 59 interference suppressed immunoassays of the manufacturer Roche Diagnostics were examined regarding their sensitivity to a biotin interference. In the second experiment the pharmacokinetic of biotin was examined by supplementing volunteers with biotin.

Results: A combination of the results of both experiments suggests that a biotin interference in laboratory findings is probable. Contrary to the current state of research on sandwich immunoassays, falsely elevated test results occur more frequently than falsely low results.

Conclusion: The interference suppressed immunoassays have shown in the experiment that they are susceptible to a biotin interference. Therefore, laboratory institutions, medical staff and patients must be aware of the possibility of a biotin interference. As a result, Roche Diagnostics may consider reviewing the interference suppression and their indications of the tests.
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http://dx.doi.org/10.1002/jcla.23940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8418509PMC
September 2021

Assessing the severity of laparotomy and partial hepatectomy in male rats-A multimodal approach.

PLoS One 2021 2;16(8):e0255175. Epub 2021 Aug 2.

Institute for Laboratory Animal Science & Experimental Surgery, Faculty of Medicine, RWTH Aachen University, Aachen, Germany.

This study assessed the postoperative severity after three different visceral surgical interventions in rats by using objective parameters pertaining to various disciplines. The objective was to evaluate whether the degree of severity increases with the invasiveness of the intervention and whether this is in accordance with the EU Directive 2010/63. 136 adult male WistarHan rats were assigned to three groups: Sham-laparotomy (Sham) [7 days post-surgical survival time]; 50% partial hepatectomy (PH); 70% PH [PH groups with 1, 3, or 7 days post-surgical survival times]. Post-surgical severity assessment was performed via several multimodal assessment tools: I) model-specific score sheet focusing on body weight, general condition, spontaneous behavior, and the animals' willingness to move as well as on wound healing; II) Open Field tests evaluating the total distance and velocity an animal moved within 10 minutes and its rearing behavior during the test; III) telemetric data analyzing heart rate and blood pressure; and IV) analysis of blood (AST, ALT, and hemogram) and fecal samples (fecal corticosterone metabolites). Significant differences among the experimental groups and models were observed. We demonstrated that the Open Field test can detect significant changes in severity levels. Sham-laparotomy and removal of 50% of the liver mass were associated with comparable severity (mild-moderate); the severity parameters returned to baseline levels within seven days. Removal of 70% of the liver tissue seemed to be associated with a moderate severity grade and entailed a longer recovery period (>7 days) for complete regeneration. We recommend the use of Open Field tests as part of multimodal objective severity assessment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0255175PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8328343PMC
November 2021

Quantification of Short Chain Fatty Acids (acetate, butyrate, propionate) in human blood with ion exclusion chromatography.

Pract Lab Med 2021 Aug 10;26:e00244. Epub 2021 Jul 10.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, Aachen, Germany.

Short Chain Fatty Acids (SCFAs), i.e. acetate, propionate and butyrate, are mainly produced by bacterial fermentation of undigested carbohydrates in the human colon. Most important are omega-3, omega-6 and unsaturated fatty acids as being important for a healthy lifestyle. SCFAs are fundamental for proper intestinal flora and they can help to prevent type 2 diabetes. SCFAs such as acetate and propionate show promise as candidates to increase satiety-enhancing properties of food. Here we describe a simple method for determining organic acids in human blood.
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http://dx.doi.org/10.1016/j.plabm.2021.e00244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8318910PMC
August 2021

Macrophage migration inhibitory factor exerts pro-proliferative and anti-apoptotic effects via CD74 in murine hepatocellular carcinoma.

Br J Pharmacol 2021 Nov 24;178(22):4452-4467. Epub 2021 Aug 24.

Department of Medicine III, University Hospital RWTH Aachen, Aachen, Germany.

Background And Purpose: Macrophage migration inhibitory factor (MIF) is an inflammatory and chemokine-like protein expressed in different inflammatory diseases as well as solid tumours. CD74-as the cognate MIF receptor-was identified as an important target of MIF. We here analysed the role of MIF and CD74 in the progression of hepatocellular carcinoma (HCC) in vitro and in vivo.

Experimental Approach: Multilocular HCC was induced using the diethylnitrosamine/carbon tetrachloride (DEN/CCl ) model in hepatocyte-specific Mif knockout (Mif ), Cd74-deficient, and control mice. Tumour burden was compared between the genotypes. MIF, CD74 and Ki67 expression were investigated in tumour and surrounding tissue. In vitro, the effects of the MIF/CD74 axis on the proliferative and apoptotic behaviour of hepatoma cells and respective signalling pathways were assessed after treatment with MIF and anti-CD74 antibodies.

Key Results: DEN/CCl treatment of Mif mice resulted in reduced tumour burden and diminished proliferation capacity within tumour tissue. In vitro, MIF stimulated proliferation of Hepa 1-6 and HepG2 cells, inhibited therapy-induced cell death and induced ERK activation. The investigated effects could be reversed using a neutralizing anti-CD74 antibody, and Cd74 mice developed fewer tumours associated with decreased proliferation rates.

Conclusion And Implications: We identified a pro-tumorigenic role of MIF during proliferation and therapy-induced apoptosis of HCC cells. These effects were mediated via the MIF cognate receptor CD74. Thus, inhibition of the MIF/CD74 axis could represent a promising target with regard to new pharmacological therapies aimed at HCC.
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http://dx.doi.org/10.1111/bph.15622DOI Listing
November 2021

In Vitro Compression Model for Orthodontic Tooth Movement Modulates Human Periodontal Ligament Fibroblast Proliferation, Apoptosis and Cell Cycle.

Biomolecules 2021 06 23;11(7). Epub 2021 Jun 23.

Department of Orthodontics, Dental Clinic, University Hospital RWTH Aachen, 52074 Aachen, Germany.

Human Periodontal Ligament Fibroblasts (hPDLF), as part of the periodontal apparatus, modulate inflammation, regeneration and bone remodeling. Interferences are clinically manifested as attachment loss, tooth loosening and root resorption. During orthodontic tooth movement (OTM), remodeling and adaptation of the periodontium is required in order to enable tooth movement. hPDLF involvement in the early phase-OTM compression side was investigated for a 72-h period through a well-studied in vitro model. Changes in the morphology, cell proliferation and cell death were analyzed. Specific markers of the cell cycle were investigated by RT-qPCR and Western blot. The study showed that the morphology of hPDLF changes towards more unstructured, unsorted filaments under mechanical compression. The total cell numbers were significantly reduced with a higher cell death rate over the whole observation period. hPDLF started to recover to pretreatment conditions after 48 h. Furthermore, key molecules involved in the cell cycle were significantly reduced under compressive force at the gene expression and protein levels. These findings revealed important information for a better understanding of the preservation and remodeling processes within the periodontium through Periodontal Ligament Fibroblasts during orthodontic tooth movement. OTM initially decelerates the hPDLF cell cycle and proliferation. After adapting to environmental changes, human Periodontal Ligament Fibroblasts can regain homeostasis of the periodontium, affecting its reorganization.
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http://dx.doi.org/10.3390/biom11070932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8301966PMC
June 2021

Exosome-Derived MicroRNAs of Human Milk and Their Effects on Infant Health and Development.

Biomolecules 2021 06 7;11(6). Epub 2021 Jun 7.

Institute for Clinical Chemistry and Laboratory Medicine, University Hospital of Regensburg, University of Regensburg, D-93053 Regensburg, Germany.

Multiple biologically active components of human milk support infant growth, health and development. Milk provides a wide spectrum of mammary epithelial cell-derived extracellular vesicles (MEVs) for the infant. Although the whole spectrum of MEVs appears to be of functional importance for the growing infant, the majority of recent studies report on the MEV subfraction of milk exosomes (MEX) and their miRNA cargo, which are in the focus of this review. MEX and the dominant miRNA-148a play a key role in intestinal maturation, barrier function and suppression of nuclear factor-κB (NF-κB) signaling and may thus be helpful for the prevention and treatment of necrotizing enterocolitis. MEX and their miRNAs reach the systemic circulation and may impact epigenetic programming of various organs including the liver, thymus, brain, pancreatic islets, beige, brown and white adipose tissue as well as bones. Translational evidence indicates that MEX and their miRNAs control the expression of global cellular regulators such as DNA methyltransferase 1-which is important for the up-regulation of developmental genes including insulin, insulin-like growth factor-1, α-synuclein and forkhead box P3-and receptor-interacting protein 140, which is important for the regulation of multiple nuclear receptors. MEX-derived miRNA-148a and miRNA-30b may stimulate the expression of uncoupling protein 1, the key inducer of thermogenesis converting white into beige/brown adipose tissue. MEX have to be considered as signalosomes derived from the maternal lactation genome emitted to promote growth, maturation, immunological and metabolic programming of the offspring. Deeper insights into milk's molecular biology allow the conclusion that infants are both "breast-fed" and "breast-programmed". In this regard, MEX miRNA-deficient artificial formula is not an adequate substitute for breastfeeding, the birthright of all mammals.
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http://dx.doi.org/10.3390/biom11060851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228670PMC
June 2021

Iron metabolism: pathophysiology and pharmacology.

Trends Pharmacol Sci 2021 08 2;42(8):640-656. Epub 2021 Jun 2.

Department of Nanomedicine and Theranostics, Institute for Experimental Molecular Imaging, RWTH University Hospital Aachen, Aachen 52074, Germany. Electronic address:

Iron is essential in many physiological processes, including DNA metabolism, oxygen transport, and cellular energy generation. Deregulated iron metabolism, which results in iron overload or iron deficiency, is observed in many different diseases. We here summarize recent progress in the pathophysiology and pharmacology of iron-overload diseases, such as hereditary hemochromatosis, as well as iron-deficiency disorders, which are typically associated with anemia. The role of iron in immunity and the connection between iron and cancer are also addressed. We finally summarize and discuss the current (pre-) clinical landscape of pharmacotherapies targeting key players involved in iron metabolism.
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http://dx.doi.org/10.1016/j.tips.2021.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611894PMC
August 2021

Understanding the Role of Perilipin 5 in Non-Alcoholic Fatty Liver Disease and Its Role in Hepatocellular Carcinoma: A Review of Novel Insights.

Int J Mol Sci 2021 May 17;22(10). Epub 2021 May 17.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, D-52074 Aachen, Germany.

Consumption of high-calorie foods, such as diets rich in fats, is an important factor leading to the development of steatohepatitis. Several studies have suggested how lipid accumulation creates a lipotoxic microenvironment for cells, leading cells to deregulate their transcriptional and translational activity. This deregulation induces the development of liver diseases such as non-alcoholic fatty liver disease (NAFLD) and subsequently also the appearance of hepatocellular carcinoma (HCC) which is one of the deadliest types of cancers worldwide. Understanding its pathology and studying new biomarkers with better specificity in predicting disease prognosis can help in the personalized treatment of the disease. In this setting, understanding the link between NAFLD and HCC progression, the differentiation of each stage in between as well as the mechanisms underlying this process, are vital for development of new treatments and in exploring new therapeutic targets. Perilipins are a family of five closely related proteins expressed on the surface of lipid droplets (LD) in several tissues acting in several pathways involved in lipid metabolism. Recent studies have shown that depletion acts protectively in the pathogenesis of liver injury underpinning the importance of pathways associated with PLIN5. PLIN5 expression is involved in pro-inflammatory cytokine regulation and mitochondrial damage, as well as endoplasmic reticulum (ER) stress, making it critical target of the NAFLD-HCC studies. The aim of this review is to dissect the recent findings and functions of PLIN5 in lipid metabolism, metabolic disorders, and NAFLD as well as the progression of NAFLD to HCC.
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http://dx.doi.org/10.3390/ijms22105284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156377PMC
May 2021

Cellular Mechanisms of Liver Fibrosis.

Front Pharmacol 2021 6;12:671640. Epub 2021 May 6.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH University Hospital Aachen, Aachen, Germany.

The liver is a central organ in the human body, coordinating several key metabolic roles. The structure of the liver which consists of the distinctive arrangement of hepatocytes, hepatic sinusoids, the hepatic artery, portal vein and the central vein, is critical for its function. Due to its unique position in the human body, the liver interacts with components of circulation targeted for the rest of the body and in the process, it is exposed to a vast array of external agents such as dietary metabolites and compounds absorbed through the intestine, including alcohol and drugs, as well as pathogens. Some of these agents may result in injury to the cellular components of liver leading to the activation of the natural wound healing response of the body or fibrogenesis. Long-term injury to liver cells and consistent activation of the fibrogenic response can lead to liver fibrosis such as that seen in chronic alcoholics or clinically obese individuals. Unidentified fibrosis can evolve into more severe consequences over a period of time such as cirrhosis and hepatocellular carcinoma. It is well recognized now that in addition to external agents, genetic predisposition also plays a role in the development of liver fibrosis. An improved understanding of the cellular pathways of fibrosis can illuminate our understanding of this process, and uncover potential therapeutic targets. Here we summarized recent aspects in the understanding of relevant pathways, cellular and molecular drivers of hepatic fibrosis and discuss how this knowledge impact the therapy of respective disease.
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http://dx.doi.org/10.3389/fphar.2021.671640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8134740PMC
May 2021

The neglected biliary mucus and its phosphatidylcholine content: a putative player in pathogenesis of primary cholangitis-a narrative review article.

Ann Transl Med 2021 Apr;9(8):738

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH University Hospital Aachen, Aachen, Germany.

Primary sclerosing cholangitis (PSC) is a rare progressive cholangitis resulting in cirrhosis and cholangiocellular carcinoma. The pathogenesis is unclear and an effective medical therapy is not available. It is highly associated to ulcerative colitis for which recently a disturbance of the tight junction (TJ) barrier has been claimed as etiologic feature. Genetic mouse models with intestinal TJ disruption showed a defective transport of phosphatidylcholine (PC) to intestinal mucus. Consequently, an ulcerative colitis phenotype developed. In the present study we evaluate whether there is also a paracellular transport of PC through TJ to the apical side of cholangiocytes. As in ulcerative colitis, a TJ defect could lead to deficient PC in biliary mucus. It would impair the protective barrier against aggressive bile acids in bile. Indeed with polarized biliary tumor cells a vectorial transport of PC from basal to luminal side was demonstrated using a transwell culture system. PC was not taken up by the cells but moved paracellularly via TJ to the apical side driven by luminal HCO- generated by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the anion exchange protein 2 (AE2). If such a TJ-mediated PC translocation to the apical surface of cholangiocytes could be disrupted in a genetic mouse model, a PSC phenotype would be expected. With such an experimental model functional operative therapies can be evaluated. We propose that disruption of TJ mediated paracellular transport of PC to the apical side of cholangiocytes could lead to biliary mucus PC depletion. This may be a pathogenetic factor for development of PSC.
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http://dx.doi.org/10.21037/atm-20-3591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106090PMC
April 2021

Therapeutic strategies in Wilson disease: pathophysiology and mode of action.

Ann Transl Med 2021 Apr;9(8):732

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, RWTH University Hospital Aachen, Aachen, Germany.

Wilson disease is a copper overload disease treatable with the chelators D-penicillamine and trientine to enhance urinary excretion or with zinc which predominantly inhibits absorption. By lifelong treatment a normal life expectancy and significant improvement of hepatic injury as well as neurologic manifestation is achievable. Here we evaluate the mode of action for effective therapy of Wilson disease. We postulate that there is no quantitative removal of copper from the liver possible. The therapeutic goal is the removal of toxic free copper (non-ceruloplasmin, but albumin bound copper). This is achievable by the induction of metallothionein which is accomplished by chelators and in particular by zinc. For control of therapy the option of a direct measurement of free copper would be preferable over the less reliable calculation of this fraction. A therapeutic challenge is still the full restoration of neurological deficits which can hardly be reached by the available chelators. Whether bis-choline-tetrathiomolybdate as intracellular copper chelator is an option has to be awaited. It is concluded that the goal of actual drug therapy in Wilson disease is the normalization of free copper in serum.
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http://dx.doi.org/10.21037/atm-20-3090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106045PMC
April 2021

Non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH)-related liver fibrosis: mechanisms, treatment and prevention.

Ann Transl Med 2021 Apr;9(8):729

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, Aachen, Germany.

Liver fibrosis is the excessive expression and accumulation of extracellular matrix proteins in the liver. Fibrotic scarring occurs as the consequence of chronic injury and inflammation. While the successful treatment of hepatitis B and C reduced the burden of liver disease related to viral hepatitis, non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) are nowadays the leading causes of hepatic fibrosis worldwide. Although basic research activities have significantly advanced our understanding of the molecular disease pathogenesis, the present therapeutic options for fibrosis are still limited. In advanced disease stages, liver transplantation often remains the only curative treatment. This highlights the necessity of preventive strategies to avoid complications of fibrosis, particularly cirrhosis, portal hypertension and liver cancer. Lifestyle modifications (weight loss, exercise, healthy diet) are the basis for prevention and treatment of NAFLD-associated fibrosis. In the present review, we discuss recent advances in antifibrotic prevention and therapy. In particular, we review the current concepts for antifibrotic drug candidates in the treatment of NAFLD and NASH. While some compounds aim at reverting pathogenic liver metabolism, an alternative approach is to disconnect the injury (e.g., NAFLD) from inflammation and/or fibrosis. Investigational drugs typically target metabolic pathways, insulin resistance, hepatocyte death, inflammatory cell recruitment or activation, the gut-liver axis, matrix expression or matrix turnover. While several promising drug candidates failed in phase 2 or 3 clinical trials (including elafibranor, emricasan and selonsertib), promising results with the farnesoid X receptor agonist obeticholic acid, the pan-PPAR agonist lanifibranor and the chemokine receptor CCR2/CCR5 inhibitor cenicriviroc support the expectation of an effective pharmacological therapy for liver fibrosis in the near future. Tackling NAFLD-associated fibrosis from different directions by combinatorial drug treatment and effective lifestyle changes hold the greatest prospects.
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http://dx.doi.org/10.21037/atm-20-4354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106094PMC
April 2021

It's all about the spaces between cells: role of extracellular matrix in liver fibrosis.

Ann Transl Med 2021 Apr;9(8):728

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, Aachen, Germany.

Liver fibrosis is one of the leading complications of a variety of chronic liver disorders, including the nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, liver cirrhosis and liver failure. The progression of liver fibrosis is driven by chronic inflammation, which activates the secretory fibroblasts to the myofibroblast phenotype. These specialized liver cells are called as hepatic stellate cells (HSCs). The excessive extracellular matrix (ECM) secretion creates a large number of complications. Fibrosis is the result of imbalance between the matrix synthesizing and matrix degrading factors. The major ECM proteins include the matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), lysyl oxidases (LOX), lysyl oxidase-like (LOXLs) enzymes, tenascins and others. These ECM proteins present novel avenues for the therapeutics of liver fibrosis. The current review highlights the major role played by these critical matrix proteins in liver fibrosis. Further, some of the targeted formulations used against these proteins are discussed and suggestions are provided to select the course of research for successful clinical translation of basic research findings for the amelioration of liver fibrosis.
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http://dx.doi.org/10.21037/atm-20-2948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106070PMC
April 2021

Unresolved basic issues in hepatology.

Ann Transl Med 2021 Apr;9(8):725

Practice for Gastroenterology, Medical Center Baden-Baden, Baden-Baden, Germany. (Email:

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http://dx.doi.org/10.21037/atm-20-8208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105994PMC
April 2021

CT-based determination of excessive visceral adipose tissue is associated with an impaired survival in critically ill patients.

PLoS One 2021 16;16(4):e0250321. Epub 2021 Apr 16.

Department of Medicine III, University Hospital RWTH Aachen, Aachen, Germany.

Objective: Obesity is a negative prognostic factor for various clinical conditions. In this observational cohort study, we evaluated a CT-based assessment of the adipose tissue distribution as a potential non-invasive prognostic parameter in critical illness.

Methods: Routine CT-scans upon admission to the intensive care unit (ICU) were used to analyze the visceral and subcutaneous adipose tissue areas at the 3rd lumbar vertebra in 155 patients. Results were correlated with various prognostic markers and both short-term- and overall survival. Multiple statistical tools were used for data analysis.

Results: We observed a significantly larger visceral adipose tissue area in septic patients compared to non-sepsis patients. Interestingly, patients requiring mechanical ventilation had a significantly higher amount of visceral adipose tissue correlating with the duration of mechanical ventilation. Moreover, both visceral and subcutaneous adipose tissue area significantly correlated with several laboratory markers. While neither the visceral nor the subcutaneous adipose tissue area was predictive for short-term ICU survival, patients with a visceral adipose tissue area above the optimal cut-off (241.4 cm2) had a significantly impaired overall survival compared to patients with a lower visceral adipose tissue area.

Conclusions: Our study supports a prognostic role of the individual adipose tissue distribution in critically ill patients. However, additional investigations need to confirm our suggestion that routine CT-based assessment of adipose tissue distribution can be used to yield further information on the patients' clinical course. Moreover, future studies should address functional and metabolic analysis of different adipose tissue compartments in critical illness.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0250321PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8051769PMC
September 2021

Elevation of autoantibodies to cerebral proteins in hepatic encephalopathy: Another pathogenic factor?

Dig Dis 2021 Apr 9. Epub 2021 Apr 9.

Background: The pathophysiology of hepatic encephalopathy is incompletely understood. It remains illusive how the contributing factors of neuronal ammonia accumulation, cell swelling and inflammation interact.

Objective: Correlation of neuronal autoantibody levels to the degree of hepatic encephalopathy as first indication of immune mediated pathogenesis.

Methods: We investigated serum autoantibody levels of representative brain proteins in patients with hepatic encephalopathy as well as in an experimental rat model with cirrhosis and hepatic encephalopathy after carbon tetrachloride exposure. They were examined in relation to presence of hepatic encephalopathy and the degree of neurological impaiment evaluated by quantitative scores.

Results: In hepatic encephalopathy an increase of all of the examined antibodies was observed in serum. The grade of antibody elevation correlated to the degree of encephalopathy registered by quantitative evaluation of brain dysfunction.

Conclusion: The degree of hepatic encephalopathy parallels neuronal autoantibody elevation. In case a causal relationship could finally be established, it adds to the understanding of hepatic encephalopathy and may open a new perspective for treatment of this handicaping condition by immunosuppressive strategies.
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http://dx.doi.org/10.1159/000516412DOI Listing
April 2021

Role of nanotechnology behind the success of mRNA vaccines for COVID-19.

Nano Today 2021 Jun 26;38:101142. Epub 2021 Mar 26.

Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc) PVNRTVU, Mamnoor, Warangal 506166, Telangana, India.

The emergency use authorization (EUA) by the US-FDA for two mRNA-based vaccines BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) has brought hope of addressing the COVID-19 pandemic which has killed more than two million people globally. Nanotechnology has played a significant role in the success of these vaccines. Nanoparticles (NPs) aid in improving stability by protecting the encapsulated mRNA from ribonucleases and facilitate delivery of intact mRNA to the target site. The overwhelming success of these two mRNA based vaccines with ~95% efficacy in phase III clinical trials can be attributed to their unique nanocarrier, the "lipid nanoparticles" (LNPs). LNPs are unique compared with bilayered liposomes and provide improved stability of the cargo, possess rigid morphology, and aid in better cellular penetration. This EUA is a major milestone and showcases the immense potential of nanotechnology for vaccine delivery and for fighting against future pandemics. Currently, these two vaccines are aiding in the alleviation of the COVID-19 health crisis and demonstrate the potential utility of nanomedicine for tackling health problems at the global level.
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http://dx.doi.org/10.1016/j.nantod.2021.101142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997390PMC
June 2021

A Scoping Review on Lipocalin-2 and Its Role in Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma.

Int J Mol Sci 2021 Mar 11;22(6). Epub 2021 Mar 11.

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, 52074 Aachen, Germany.

Excess calorie intake and a sedentary lifestyle have made non-alcoholic fatty liver disease (NAFLD) one of the fastest growing forms of liver disease of the modern world. It is characterized by abnormal accumulation of fat in the liver and can range from simple steatosis and non-alcoholic steatohepatitis (NASH) to cirrhosis as well as development of hepatocellular carcinoma (HCC). Biopsy is the golden standard for the diagnosis and differentiation of all NAFLD stages, but its invasiveness poses a risk for patients, which is why new, non-invasive ways of diagnostics ought to be discovered. Lipocalin-2 (LCN2), which is a part of the lipocalin transport protein family, is a protein formally known for its role in iron transport and in inflammatory response. However, in recent years, its implication in the pathogenesis of NAFLD has become apparent. LCN2 shows significant upregulation in several benign and malignant liver diseases, making it a good candidate for the NAFLD biomarker or even a therapeutic target. What makes LCN2 more interesting to study is the fact that it is overexpressed in HCC development induced by chronic NASH, which is one of the primary causes of cancer-related deaths. However, to this day, neither its role as a biomarker for NAFLD nor the molecular mechanisms of its implication in NAFLD pathogenesis have been completely elucidated. This review aims to gather and closely dissect the current knowledge about, sometimes conflicting, evidence on LCN2 as a biomarker for NAFLD, its involvement in NAFLD, and NAFLD-HCC related pathogenesis, while comparing it to the findings in similar pathologies.
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http://dx.doi.org/10.3390/ijms22062865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000927PMC
March 2021

Wilson disease - the impact of hyperimmunity on disease activity: A case report.

World J Clin Cases 2021 Feb;9(6):1386-1393

Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry, Rheinisch-Westfaelische Technische Hochschule University Hospital Aachen, Aachen D-52074, Germany.

Background: In Wilson disease lack of biliary copper excretion causes hepatocellular injury by accumulation of free toxic copper. Its overspill to serum accounts for neuronal damage as second common manifestation. Therapy with copper chelators or zinc targets the removal of this free copper. However, in some patients liver disease persists for unknown reason despite normalized free copper. The discovery of a hyperimmunity as a contributing pathogenetic factor was discovered in this case report with implication also for other liver diseases.

Case Summary: A 9-year-old girl was diagnosed in August 2009 by family screening of having asymptomatic Wilson disease with elevated transaminases. Already at time of diagnosis antinuclear antibodies (ANA) were elevated without hyperimmunoglobulinemia (immunoglobulin G, IgG). After one year of therapy with D-penicillamine transaminases normalized together with free serum copper. Under continuous therapy with copper chelators free copper remained normal until today, whereas transaminases raised to alanine aminotransferase values of 571 U/L in December 2019. For hyperimmunity a tentative steroid course on top of D-penicillamine improved transaminases. Thus hyperimmunity may have impact on liver inflammation after control of the metabolic disturbance. A retrospective cohort study confirmed the common association of elevated transaminases with ANA, but no IgG elevation.

Conclusion: This hyperimmune-triggered condition may represent a new entity which per se or on top of other liver diseases induces liver inflammation responsive to steroids.
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http://dx.doi.org/10.12998/wjcc.v9.i6.1386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896689PMC
February 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021
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