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| 2. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 3. |
- 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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| 4. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 5. |
- Abbafati, Cristiana, et al.
(författare)
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- 2020
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Hokfelt, Tomas, et al.
(författare)
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Four decades of research on Viktor Mutt's neuropeptides with focus on galanin
- 2024
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Ingår i: Proceedings of the Estonian Academy of Sciences. - : ESTONIAN ACAD PUBLISHERS. - 1736-6046 .- 1736-7530. ; 73:3, s. 170-192
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Tidskriftsartikel (refereegranskat)abstract
- The aim of the article is to describe our work on peptides discovered in Dr. Mutt's laboratory, particularly galanin. Some personal recollections of meetings with Viktor Mutt and a brief overview of early neuropeptide research at Karolinska Institutet are provided. General aspects on neuropeptide signalling and neuropeptide-neurotransmitter coexistence are followed by the presentation of a possible involvement of the galanin system in pain and depression. Special emphasis is on the role of galanin in the rat and human locus coeruleus. Additional analyses of the human postmortem brains have given results on galanin and other peptides both in the normal prefrontal cortex as well as in different brain regions of depressed patients who have committed suicide and in control subjects. Possible options for developing treatment strategies for pain and depression based on galaninergic mechanisms are discussed. Finally, some recent drugs approved by the FDA for the treatment of conditions such as migraine, which target the signalling of other peptides, are highlighted. In conclusion, the aim of the article is to highlight the potential of the large group of neuropeptides as targets for the development of drugs that may further help patients with illnesses afflicting the nervous system.
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| 7. |
- Hokfelt, Tomas, et al.
(författare)
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Neuropeptide and Small Transmitter Coexistence: Fundamental Studies and Relevance to Mental Illness
- 2018
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Ingår i: Frontiers in Neural Circuits. - : FRONTIERS MEDIA SA. - 1662-5110. ; 12
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Forskningsöversikt (refereegranskat)abstract
- Neuropeptides are auxiliary messenger molecules that always co-exist in nerve cells with one or more small molecule (classic) neurotransmitters. Neuropeptides act both as transmitters and trophic factors, and play a role particularly when the nervous system is challenged, as by injury, pain or stress. Here neuropeptides and coexistence in mammals are reviewed, but with special focus on the 29/30 amino acid galanin and its three receptors GalR1, -R2 and -R3. In particular, galanins role as a co-transmitter in both rodent and human noradrenergic locus coeruleus (LC) neurons is addressed. Extensive experimental animal data strongly suggest a role for the galanin system in depression-like behavior. The translational potential of these results was tested by studying the galanin system in postmortem human brains, first in normal brains, and then in a comparison of five regions of brains obtained from depressed people who committed suicide, and from matched controls. The distribution of galanin and the four galanin system transcripts in the normal human brain was determined, and selective and parallel changes in levels of transcripts and DNA methylation for galanin and its three receptors were assessed in depressed patients who committed suicide: upregulation of transcripts, e.g., for galanin and GalR3 in LC, paralleled by a decrease in DNA methylation, suggesting involvement of epigenetic mechanisms. It is hypothesized that, when exposed to severe stress, the noradrenergic LC neurons fire in bursts and release galanin from their soma/dendrites. Galanin then acts on somato-dendritic, inhibitory galanin autoreceptors, opening potassium channels and inhibiting firing. The purpose of these autoreceptors is to act as a brake to prevent overexcitation, a brake that is also part of resilience to stress that protects against depression. Depression then arises when the inhibition is too strong and long lasting - a maladaption, allostatic load, leading to depletion of NA levels in the forebrain. It is suggested that disinhibition by a galanin antagonist may have antidepressant activity by restoring forebrain NA levels. A role of galanin in depression is also supported by a recent candidate gene study, showing that variants in genes for galanin and its three receptors confer increased risk of depression and anxiety in people who experienced childhood adversity or recent negative life events. In summary, galanin, a neuropeptide coexisting in LC neurons, may participate in the mechanism underlying resilience against a serious and common disorder, MDD. Existing and further results may lead to an increased understanding of how this illness develops, which in turn could provide a basis for its treatment.
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| 8. |
- Wang, Peng, et al.
(författare)
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Depression-like behavior in rat: Involvement of galanin receptor subtype 1 in the ventral periaqueductal gray
- 2016
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 113:32, s. E4726-E4735
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Tidskriftsartikel (refereegranskat)abstract
- The neuropeptide galanin coexists in rat brain with serotonin in the dorsal raphe nucleus and with noradrenaline in the locus coeruleus (LC), and it has been suggested to be involved in depression. We studied rats exposed to chronic mild stress (CMS), a rodent model of depression. As expected, these rats showed several endophenotypes relevant to depression-like behavior compared with controls. All these endophenotypes were normalized after administration of a selective serotonin reuptake inhibitor. The transcripts for galanin and two of its receptors, galanin receptor 1 (GALR1) and GALR2, were analyzed with quantitative real-time PCR using laser capture microdissection in the following brain regions: the hippocampal formation, LC, and ventral periaqueductal gray (vPAG). Only Galr1 mRNA levels were significantly increased, and only in the latter region. After knocking down Galr1 in the vPAG with an siRNA technique, all parameters of the depressive behavioral phenotype were similar to controls. Thus, the depression-like behavior in rats exposed to CMS is likely related to an elevated expression of Galr1 in the vPAG, suggesting that a GALR1 antagonist could have antidepressant effects.
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| 9. |
- Xu, Zhi-Qing David
(författare)
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Neuropeptides, 'gaseous' messengers and classic transmitters : electrophysiological and histochemical studies on coexistence and interactions in the nervous system
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The distribution and functional role of neuropeptides were studied, with special reference to galanin (GAL) and the free radical nitric oxide (NO). In developing systems GAL expression was observed already at E14 in trigeminal and dorsal root ganglion (DRG) neurons and at E15 in sensory epithelia in ear, eye and nose, as well as at E19 during bone formation. Also GAL-R1 receptor mRNA was expressed in the sensory ganglia of embryos but was detected later than the ligand. Thus GAL may have a developmental role in several sensory systems and during bone formation. The GAL-R1 receptor was present in CGRP neurons and was down-regulated by peripheral nerve injury and inflammation. GAL did not change the membrane current in DRG neurons from normal rats, but caused an inward current in DRG neurons from sciatic nerve transected (SNT) rats. Similar results were obtained with CCK-8S. In normal rats neurotensin (NT) receptor mRNA was expressed in about 25% of the small DRG neuron profiles, and NT evoked an outward current in neuropeptide Y (NPY)-insensitive C-type (small) neurons, while NPY induced an outward current in NT-insensitive C-type neurons. In axotomized rats NT only induced an inward current in C-type neurons. These results give evidence for functional peptide receptors in the DRG neurons and that nerve injury causes distinct changes in receptor phenotype, perhaps reflecting attempts of the DRG neurons to cope with, and to counteract the consequences of nerve injury. In the central nervous system, GAL/GMAP-positive fibers in the dorsal and many in the ventral hippocampal formation were noradrenergic. GAL-R2 receptor mRNA was expressed in the granule cell layer in the dentate gyrus. However, especially in the ventral hippocampus some GAL/GMAP fibers were DBH-negative, and did not disappear after 6-OH-DA treatment. This suggests that GAL/GMAP are present in multiple hippocampal systems and that most GALergic fibers are noradrenergic arising from locus coeruleus(LC). GAL-evoked hyperpolarization of LC neurons, and this was accompanied by a decrease in membrane resistance as recorded in a slice preparation. Binding studies revealed GAL binding sites in the LC, but GAL-R1 and GAL-R2 mRNAs were not detectable. Ultrastructural immunocytochemistry showed GAL in many neuronal somata and dendritic processes within the nucleus. These findings suggest that GAL exerts its inhibitory effect in the LC, probably via an increase in K+ conductance, and that endogenous GAL, possibly released from the LC cell bodies and dendrites, may act on autoreceptors or receptors on adjacent neurons in LC. In the dorsal raphe (DR), 5-HT/GAL/NO synthase (NOS)-coexistence neurons were found mainly in the midline. All GAL neuron profiles contained 5-HT, and the proportion of 5-HT/GAL neurons was high while that of 5-HT/NOS neurons was low. Some 5-HT/GAL/NOS neurons were found to project to the striatum. However, only in a few cases could GAL-LI be shown to coexist with 5-HT in striatal fibers with no NOS/5-HT coexistence. The present findings strongly suggest that many DR 5-HT neurons can synthesize and release two additional messenger molecules, GAL and NO. However, little GAL and NOS seem to be transported anterogradely to the nerve terminals in the striatum, supporting the concept of release at the soma/dendrite level. GAL caused a hyperpolarization of 5-HT of DR neurons accompanied by a decrease in membrane resistance. The 5-HT-induced outward current was enhanced and prolonged by preincubation with low concentrations of GAL. The dose-response curve to 5-HT was changed by GAL with a shift to the left. These results suggest that GAL exerts its effect in the DR directly by acting at receptors on the membrane of 5-HT-sensitive neurons via an increase in K+ conductance and indirectly by enhancing the 5-HT action. A small number of NOS-positive, non-noradrenergic neurons were observed within the LC. NOS inhibitors enhanced the EPSP in LC, an effect that was reversed by coadministration of L-arginine. Application of NO-donors increased levels of cGMP as seen with immunohistochemistry as well as induced a hyperpolarization and reduced the EPSP/EPSC in LC neurons. Prior application of hemoglobin prevented the action of NO-donors and enhanced the EPSP/EPSC. Application of the membrane permeable cGMP analog 8-bromo-cGMP mimicked the action of NO-donors. Preincubation with the guanylate cyclase inhibitor ODQ reduced NO-donor-induced hyperpolarization. These results suggest a role for NO in synaptic transmission in the LC, which may act through the NO-cGMP pathway by stimulating guanylyl cyclase and increasing endogenous levels of cGMP. Even if only few NOS-positive neurons are present in the LC, NO may after release from cell bodies, dendrites and axonal processes via diffusion influence many noradrenergic LC neurons.
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