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      Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila

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          Abstract

          Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.

            Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a phiC31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the phiC31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the phiC31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.
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              Biological roles of glycans

              Ajit Varki (2016)
              Abstract Simple and complex carbohydrates (glycans) have long been known to play major metabolic, structural and physical roles in biological systems. Targeted microbial binding to host glycans has also been studied for decades. But such biological roles can only explain some of the remarkable complexity and organismal diversity of glycans in nature. Reviewing the subject about two decades ago, one could find very few clear-cut instances of glycan-recognition-specific biological roles of glycans that were of intrinsic value to the organism expressing them. In striking contrast there is now a profusion of examples, such that this updated review cannot be comprehensive. Instead, a historical overview is presented, broad principles outlined and a few examples cited, representing diverse types of roles, mediated by various glycan classes, in different evolutionary lineages. What remains unchanged is the fact that while all theories regarding biological roles of glycans are supported by compelling evidence, exceptions to each can be found. In retrospect, this is not surprising. Complex and diverse glycans appear to be ubiquitous to all cells in nature, and essential to all life forms. Thus, >3 billion years of evolution consistently generated organisms that use these molecules for many key biological roles, even while sometimes coopting them for minor functions. In this respect, glycans are no different from other major macromolecular building blocks of life (nucleic acids, proteins and lipids), simply more rapidly evolving and complex. It is time for the diverse functional roles of glycans to be fully incorporated into the mainstream of biological sciences.
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                Author and article information

                Contributors
                Role: Reviewing Editor
                Role: Senior Editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                22 March 2023
                2023
                : 12
                : e78280
                Affiliations
                [1 ] Department of Biochemistry and Biophysics, Texas A&M University ( https://ror.org/01f5ytq51) College Station United States
                [2 ] Departments of Molecular and Human Genetics and Neuroscience, Baylor College of Medicine, and Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital ( https://ror.org/02pttbw34) Houston United States
                [3 ] Complex Carbohydrate Research Center, University of Georgia ( https://ror.org/00te3t702) Athens United States
                [4 ] Translational Metabolic Laboratory, Department of Neurology, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center ( https://ror.org/05wg1m734) Nijmegen Netherlands
                University College London ( https://ror.org/02jx3x895) United Kingdom
                New York University ( https://ror.org/0190ak572) United States
                University College London ( https://ror.org/02jx3x895) United Kingdom
                University College London ( https://ror.org/02jx3x895) United Kingdom
                The Scripps Research Institute ( https://ror.org/02dxx6824) United States
                Author notes
                [†]

                These authors contributed equally to this work.

                [‡]

                Ocular Genomics Institute, Massachusetts Eye and Ear, Boston, United States.

                [§]

                Departments of Neuroscience and Cell Biology, Yale University School of Medicine, New Haven, United States.

                [#]

                Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia.

                Author information
                https://orcid.org/0009-0007-0458-8445
                https://orcid.org/0000-0003-4806-8891
                https://orcid.org/0000-0001-5992-5989
                https://orcid.org/0000-0001-9126-1481
                Article
                78280
                10.7554/eLife.78280
                10110239
                36946697
                8a07c1c5-8cb2-4a86-8b78-816ac8fdfcb9
                © 2023, Scott, Novikov et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 01 March 2022
                : 16 March 2023
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: NS099409
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: NS075534
                Award Recipient :
                Funded by: TAMU-COANCYT;
                Award ID: 2012-037(S)
                Award Recipient :
                Funded by: TAMU AgriLife IHA;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: GM103490
                Award Recipient :
                Funded by: Radboud Consortium for Glycoscience;
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Biochemistry and Chemical Biology
                Neuroscience
                Custom metadata
                The sialylation pathway is uniquely partitioned in Drosophila between glia and neurons and mediates a novel mechanism of glia-neuron coupling that regulates neural functions, promotes tolerance to heat and oxidative stress, and maintains the normal level of voltage-gated sodium channels.

                Life sciences
                neuron-glia interactions,sialylation,glycosylation,oxidative stress,voltage-gated sodium channel,sialyltransferase,d. melanogaster

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