| 1. |
|
|
| 2. |
- Klionsky, Daniel J., et al.
(författare)
-
Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
-
Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
-
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.
|
|
| 3. |
|
|
| 4. |
- Beecham, Ashley H, et al.
(författare)
-
Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis.
- 2013
-
Ingår i: Nature genetics. - : Springer Science and Business Media LLC. - 1546-1718 .- 1061-4036. ; 45:11, s. 1353-60
-
Tidskriftsartikel (refereegranskat)abstract
- Using the ImmunoChip custom genotyping array, we analyzed 14,498 subjects with multiple sclerosis and 24,091 healthy controls for 161,311 autosomal variants and identified 135 potentially associated regions (P < 1.0 × 10(-4)). In a replication phase, we combined these data with previous genome-wide association study (GWAS) data from an independent 14,802 subjects with multiple sclerosis and 26,703 healthy controls. In these 80,094 individuals of European ancestry, we identified 48 new susceptibility variants (P < 5.0 × 10(-8)), 3 of which we found after conditioning on previously identified variants. Thus, there are now 110 established multiple sclerosis risk variants at 103 discrete loci outside of the major histocompatibility complex. With high-resolution Bayesian fine mapping, we identified five regions where one variant accounted for more than 50% of the posterior probability of association. This study enhances the catalog of multiple sclerosis risk variants and illustrates the value of fine mapping in the resolution of GWAS signals.
|
|
| 5. |
|
|
| 6. |
- Klionsky, Daniel J., et al.
(författare)
-
Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes
- 2008
-
Ingår i: Autophagy. - : Landes Bioscience. - 1554-8627 .- 1554-8635. ; 4:2, s. 151-175
-
Forskningsöversikt (refereegranskat)abstract
- Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.
|
|
| 7. |
- Baniulis, Danas, et al.
(författare)
-
Monoclonal antibody CL5 recognizes the amino terminal domain of human phagocyte flavocytochrome b558 large subunit, gp91phox
- 2005
-
Ingår i: European Journal of Haematology. - : Wiley. - 0902-4441 .- 1600-0609. ; 74:4, s. 337-347
-
Tidskriftsartikel (refereegranskat)abstract
- Human phagocyte flavocytochrome b558 (Cytb) is a heterodimeric integral membrane protein that serves as the electron transferase of the β-nicotinamide adenine dinucleotidephosphate, reduced (NADPH)-oxidase, an enzyme complex important in the host defense function of phagocytic cells. In this study, we report the characterization of monoclonal antibody (mAb) CL5 that is specific for the large subunit, gp91phox, of the oxidase protein. This antibody recognizes gp91phox by immunoblot analysis of membrane extracts and samples of the immunopurified gp91phox/p22 phox heterodimer, prepared on anti-p22phox affinity matrices. Phage display analysis confirmed this specificity, indicating that the CL5 epitope contains the region 135-DPYSVALSELGDR of gp91phox. The antibody was used to probe for the presence of gp91phox in membrane preparations from neutrophils of patients with nine genetically distinct forms of X-linked chronic granulomatous disease (CGD). The causative mutations included missense errors as well as nonsense errors that result in premature termination of gp91phox synthesis. Analysis of the CGD samples by immunoblotting indicated that CL5 recognizes only the full-length wild-type and two missense mutations, consistent with the absence of stable short gp91 phox peptide expression in CGD neutrophils. Interestingly, CL5 was also shown to be cross-reactive with cytosolic and membrane-bound gelsolin, identified by purification, mass spectrometry and immunoblot analysis. CL5 probably cross-reacts with the sequence 771-DPLDRAMAEL in the C-terminus of gelsolin. We conclude that mAb CL5 is a useful probe for detection of full length and possibly truncated N-terminal fragments of gp91phox from membranes of Cytb-producing cells. © Blackwell Munksgaard 2005.
|
|
| 8. |
- Baniulis, Danas, et al.
(författare)
-
Unusual polyclonal anti-gp91phox peptide antibody interactions with X-linked chronic granulomatous disease-derived human neutrophils are not from compensatory expression of Nox proteins 1, 3, or 4
- 2005
-
Ingår i: European Journal of Haematology. - : Wiley. - 0902-4441 .- 1600-0609. ; 74:3, s. 241-249
-
Tidskriftsartikel (refereegranskat)abstract
- To obtain topological information about human phagocyte flavocytochrome b558 (Cytb), rabbit anti-peptide antibodies were raised against synthetic peptides mimicking gp91phox regions: 1-9 (MGN), 30-44 (YRV), 150-159 (ESY), 156-166 (ARK), 247-257 (KIS-1, KIS-2). Following affinity purification on immobilized peptide matrices, all antibodies but not prebleed controls recognized purified detergent-solubilized Cytb by enzyme-linked immunosorbent assay (ELISA). Affinity-purified antibodies recognizing KIS, ARK and ESY but not YRV, MGN or prebleed IgG specifically detected gp91phox in immunoblot analysis. Antibodies recognizing MGN, ESY, ARK and KIS but not YRV or the prebleed IgG fraction labeled intact normal neutrophils. Surprisingly, all antibodies, with the exception of YRV and pre-immune IgG controls, bound both normal and Cytb-negative neutrophils from the obligate heterozygous mother of a patient with X-linked chronic granulomatous disease (X-CGD) and all neutrophils from another patient lacking the gp91phox gene. Further immunochemical examination of membrane fractions derived from nine genetically unrelated patients with X-CGD, using an antibody that recognizes other Nox protein family members, suggests that the unusual reactivity observed does not reflect the compensatory expression of gp91phox homologs Nox1, 3 or 4. These results suggest that an unusual surface reactivity exists on neutrophils derived from X-linked chronic granulomatous disease patients that most likely extends to normal neutrophils as well. The study highlights the need for caution in interpreting the binding of rabbit polyclonal antipeptide antibodies to human neutrophils in general and, in the specific case of antibodies directed against Cytb, the need for Cytb-negative controls.
|
|
| 9. |
|
|
| 10. |
- Khatri, B., et al.
(författare)
-
Genome-wide association study identifies Sjogren's risk loci with functional implications in immune and glandular cells
- 2022
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
-
Tidskriftsartikel (refereegranskat)abstract
- Sjogren's disease is a complex autoimmune disease with twelve established susceptibility loci. This genome-wide association study (GWAS) identifies ten novel genome-wide significant (GWS) regions in Sjogren's cases of European ancestry: CD247, NAB1, PTTG1-MIR146A, PRDM1-ATG5, TNFAIP3, XKR6, MAPT-CRHR1, RPTOR-CHMP6-BAIAP6, TYK2, SYNGR1. Polygenic risk scores yield predictability (AUROC = 0.71) and relative risk of 12.08. Interrogation of bioinformatics databases refine the associations, define local regulatory networks of GWS SNPs from the 95% credible set, and expand the implicated gene list to >40. Many GWS SNPs are eQTLs for genes within topologically associated domains in immune cells and/or eQTLs in the main target tissue, salivary glands. The genetic architecture underlying Sjogren's syndrome is not fully understood. Here, the authors perform a genome-wide association study to identify 10 new genetic risk regions, implicating genes involved in immune and salivary gland function.
|
|
