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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)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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- Bennett, N. S., et al.
(author)
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Enhanced n-type dopant solubility in tensile-strained Si
- 2008
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In: Thin Solid Films. - : Elsevier BV. - 0040-6090 .- 1879-2731. ; 517:1, s. 331-333
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Journal article (peer-reviewed)abstract
- The creation of highly conductive ultrashallow-doped regions in strained Si is a key requirement for future Si based devices. It is shown that in the presence of tensile strain, Sb becomes a contender to replace As in strain-engineered CMOS devices due to advantages in sheet resistance. While strain reduces resistance for both As and Sb; a result of enhanced electron mobility, the reduction is significantly larger for Sb due to an increase in donor activation. Differential Hall measurements suggest this is a consequence of a strain-induced Sb solubility enhancement following solid-phase epitaxial regrowth, increasing Sb solubility in Si to levels approaching 10(21) cm(-3). Experiments highlight the importance of maintaining substrate strain during thermal annealing to maintain this high Sb activation.
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- Powlowski, J, et al.
(author)
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On the role of DmpK, an auxiliary protein associated with multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.
- 1997
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 272:2
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Journal article (peer-reviewed)abstract
- DmpK from Pseudomonas sp. strain CF600 represents a group of proteins required by phenol-degrading bacteria that utilize a multicomponent iron-containing phenol hydroxylase. DmpK has been overexpressed in Escherichia coli and purified to homogeneity; it lacks redox cofactors and was found to strongly inhibit phenol hydroxylase in vitro. Chemical cross-linking experiments established that DmpK binds to the two largest subunits of the oxygenase component of the hydroxylase; this may interfere with binding of the hydroxylase activator protein, DmpM, causing inhibition. Since expression of DmpK normally appears to be much lower than that of the components of the oxygenase, inhibition may not occur in vivo. Hence, the interaction between DmpK and the oxygenase manifested in the inhibition and cross-linking results prompted construction of E. coli strains in which the oxygenase component was expressed in the presence and absence of a low molar ratio of DmpK. Active oxygenase was detected only when expressed in the presence of DmpK. Furthermore, inactive oxygenase could be activated in vitro by adding ferrous iron, in a process that was dependent on the presence of DmpK. These results indicate that DmpK plays a role in assembly of the active form of the oxygenase component of phenol hydroxylase.
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| 6. |
- Kikuta, H, et al.
(author)
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Genomic regulatory blocks encompass multiple neighboring genes and maintain conserved synteny in vertebrates
- 2007
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In: Genome research. - : Cold Spring Harbor Laboratory. - 1088-9051. ; 17:5, s. 545-555
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Journal article (peer-reviewed)abstract
- We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated “bystander” genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.
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- Miao Jonasson, Junmei, 1972, et al.
(author)
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Social Support, Social Network Size, Social Strain, Stressful Life Events, and Coronary Heart Disease in Women With Type 2 Diabetes: A Cohort Study Based on the Women's Health Initiative
- 2020
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In: Diabetes care. - : American Diabetes Association. - 0149-5992 .- 1935-5548. ; 43:8, s. 1759-1766
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Journal article (peer-reviewed)abstract
- OBJECTIVE We studied associations between social support, social network size, social strain, or stressful life events and risk of coronary heart disease (CHD) in postmenopausal women with type 2 diabetes. RESEARCH DESIGN AND METHODS From the Women's Health Initiative, 5,262 postmenopausal women with type 2 diabetes at baseline were included. Cox proportional hazards regression models adjusted for demographics, depressive symptoms, anthropometric variables, and lifestyle factors were used to examine associations between social factors and CHD. RESULTS A total of 672 case subjects with CHD were observed during an average 12.79 (SD 6.29) years of follow-up. There was a significant linear trend toward higher risk of CHD as the number of stressful life events increased (Pfor trend = 0.01; hazard ratio [HR] [95% CI] for the third and fourth quartiles compared with first quartile: 1.27 [1.03-1.56] and 1.30 [1.04-1.64]). Being married or in an intimate relationship was related to decreased risk of CHD (HR 0.82 [95% CI 0.69-0.97]). CONCLUSIONS Among postmenopausal women with type 2 diabetes, higher levels of stressful life events were associated with higher risk of CHD. Experience of stressful life events might be considered as a risk factor for CHD among women with type 2 diabetes.
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