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- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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- Groenen, M. A., et al.
(författare)
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Analyses of pig genomes provide insight into porcine demography and evolution
- 2012
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 491:7424, s. 393-398
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Tidskriftsartikel (refereegranskat)abstract
- For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars approximately 1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.
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| 4. |
- Rajewsky, N., et al.
(författare)
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LifeTime and improving European healthcare through cell-based interceptive medicine
- 2020
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Ingår i: Nature. - : Springer Nature. - 0028-0836 .- 1476-4687. ; 587:7834, s. 377-386
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Tidskriftsartikel (refereegranskat)abstract
- LifeTime aims to track, understand and target human cells during the onset and progression of complex diseases and their response to therapy at single-cell resolution. This mission will be implemented through the development and integration of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during progression from health to disease. Analysis of such large molecular and clinical datasets will discover molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. Timely detection and interception of disease embedded in an ethical and patient-centered vision will be achieved through interactions across academia, hospitals, patient-associations, health data management systems and industry. Applying this strategy to key medical challenges in cancer, neurological, infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.
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| 5. |
- De Leoz, M. L. A., et al.
(författare)
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NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods
- 2020
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476. ; 19:1, s. 11-30
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Tidskriftsartikel (refereegranskat)abstract
- A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories. Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
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| 6. |
- Bonham, LW, et al.
(författare)
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Genetic variation across RNA metabolism and cell death gene networks is implicated in the semantic variant of primary progressive aphasia
- 2019
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1, s. 10854-
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Tidskriftsartikel (refereegranskat)abstract
- The semantic variant of primary progressive aphasia (svPPA) is a clinical syndrome characterized by neurodegeneration and progressive loss of semantic knowledge. Unlike many other forms of frontotemporal lobar degeneration (FTLD), svPPA has a highly consistent underlying pathology composed of TDP-43 (a regulator of RNA and DNA transcription metabolism). Previous genetic studies of svPPA are limited by small sample sizes and a paucity of common risk variants. Despite this, svPPA’s relatively homogenous clinicopathologic phenotype makes it an ideal investigative model to examine genetic processes that may drive neurodegenerative disease. In this study, we used GWAS metadata, tissue samples from pathologically confirmed frontotemporal lobar degeneration, and in silico techniques to identify and characterize protein interaction networks associated with svPPA risk. We identified 64 svPPA risk genes that interact at the protein level. The protein pathways represented in this svPPA gene network are critical regulators of RNA metabolism and cell death, such as SMAD proteins and NOTCH1. Many of the genes in this network are involved in TDP-43 metabolism. Contrary to the conventional notion that svPPA is a clinical syndrome with few genetic risk factors, our analyses show that svPPA risk is complex and polygenic in nature. Risk for svPPA is likely driven by multiple common variants in genes interacting with TDP-43, along with cell death,x` working in combination to promote neurodegeneration.
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| 7. |
- Leymarie, N., et al.
(författare)
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Interlaboratory Study on Differential Analysis of Protein Glycosylation by Mass Spectrometry: The ABRF Glycoprotein Research Multi-Institutional Study 2012
- 2013
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476. ; 12:10, s. 2935-2951
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Tidskriftsartikel (refereegranskat)abstract
- One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods. T6G 2G2, Canada. [Cipollo, John F.; An, Yanming] US FDA, Ctr Biol Evaluat & Res, Bethesda, MD 20993 USA. [Desaire, Heather; Go, Eden P.] Univ Kansas, Lawrence, KS 66045 USA. [Goldman, Radoslav; Pompach, Petr; Sanda, Miloslav] Georgetown Univ, Dept Oncol, Washington, DC [Halim, Adnan; Larson, Goran; Nilsson, Jonas] Univ Gothenburg, Sahlgrenska Acad, Dept Clin Chem & [Hensbergen, Paul J.; Wuhrer, Manfred] Leiden Univ, Med Ctr, Biomol Mass Spectrometry Unit, NL- [Jabs, Wolfgang; Marx, Kristina; Resemann, Anja; Schweiger-Hufnagel, Ulrike; Suckau, Detlev] Bruker [Ly, Mellisa; Staples, Gregory O.] Agilent Technol, Agilent Labs, Santa Clara, CA 95051 USA. [Mechref, Yehia; Song, Ehwang] Texas Tech Univ, Dept Chem & Biochem, Lubbock, TX 79409 USA. [Nyalwidhe, Julius O.; Watson, Megan] Eastern Virginia Med Sch, Leroy T Canoles Jr Canc Res Ctr, Dept [Packer, Nicolle H.; Thaysen-Andersen, Morten] Macquarie Univ, Dept Chem & Biomol Sci, Biomol [Sihlbom, Carina] Gothenburg Univ, Prote Core Facil, Gothenburg, Sweden. [Tang, Haixu] Indiana Univ, Sch Informat, Bloomington, IN 47405 USA. [Valmuv, Leena] Finnish Red Cross Blood Serv, Helsinki 00310, Finland. [Wada, Yoshinao] Osaka Med Ctr Maternal & Child Hlth, Res Inst, Izumi Ku, Osaka 5941101, Japan.
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