Expression, Purification and Characterization of Soluble Red Rooster Laforin as a Fusion Protein in Escherichia Coli

BACKGROUND: The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue. RESULTS: In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin. CONCLUSIONS: Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease

Adolescents' Responses to Pictorial Warnings on Their Parents' Cigarette Packs

Pictorial cigarette pack warnings are a promising policy solution to increase smoking cessation among adults. However, little is known regarding adolescents' responses to pictorial warnings, particularly in real-world settings

Polyglucosan Body Structure in Lafora Disease

Abnormal carbohydrate structures known as polyglucosan bodies (PGBs) are associated with neurodegenerative disorders, glycogen storage diseases (GSDs), and aging. A hallmark of the GSD Lafora disease (LD), a fatal childhood epilepsy caused by recessive mutations in the EPM2A or EPM2B genes, are cytoplasmic PGBs known as Lafora bodies (LBs). LBs result from aberrant glycogen metabolism and drive disease progression. They are abundant in brain, muscle and heart of LD patients and Epm2a-/- and Epm2b-/- mice. LBs and PGBs are histologically reminiscent of starch, semicrystalline carbohydrates synthesized for glucose storage in plants. In this study, we define LB architecture, tissue-specific differences, and dynamics. We propose a model for how small polyglucosans aggregate to form LBs. LBs are very similar to PGBs of aging and other neurological disorders, and so these studies have direct relevance to the general understanding of PGB structure and formation

Testing warning messages on smokers’ cigarette packages: a standardised protocol

Lab experiments on cigarette warnings typically use a brief one-time exposure that is not paired with the cigarette packs smokers use every day, leaving open the question of how repeated warning exposure over several weeks may affect smokers. This proof of principle study sought to develop a new protocol for testing cigarette warnings that better reflects real-world exposure by presenting them on cigarette smokers’ own packs

Phenotypic characterization of a new EPM2A mutation (N163D)

2nd Biennial International Lafora Workshop. La Jolla, California, 23-24 de junio de 2016.Lafora disease (LD, OMIM 254780) is a fatal rare disorder characterized by epilepsy and neurodegeneration. In the vast majority of cases LD is related to mutations in either the EPM2A gene (encoding the glucan phosphatase laforin) or the EPM2B gene (encoding the E3-ubiquitin ligase malin). Alterations in these genes consist of deletions or missense and nonsense mutations. These alterations are spread all over the laforin and malin protein sequences and it remains to be shown whether they correlate with the severity of the disease. We have recently gained access to a primary fibroblast sample form a compound heterozygous EPM2A patient (Y112X/N163D), who shows a slow progression of the disease. As the Y112X mutation is related to regular progression of the disease and to our knowledge the laforin N 163D mutation is novel, here we have carried out the phenotypic characterization of the laforin N163D mutation. We have expressed the laforin-N 163D mutant in bacteria and purified the corresponding protein. Using OMFP as substrate we have observed no major changes in phosphatase activity. The mutant protein was also as stable as wild type when expressed either in bacteria or in mammalian cells. However, it showed a severe impairment in the interaction with regular laforin partners, as laforin itself, malin, R5/PTG and R6 (by yeast two-hybrid assays). Probably, this lack of interaction is the cause of the pathogenic profile of this novel mutation. We have recently reported that human primary fibroblasts from LD patients have an impairment of mitochondrial function with increased production of reactive oxygen species (ROS). In agreement with these observations we found that primary fibroblasts from EPM2A Y112X/N 163D patient presented higher levels of superoxide and lower levels of superoxide dismutase activity. The levels of thioredoxin1 (Trx1) were also decreased in the LD samples. All these results indicate that primary fibroblasts from EPM2A Y112X/N163D patient suffer from oxidative stress.This work was supported by grants from the Spanish Ministry of Education and Science (SAF2014-54604-C3-1-R) and from CIBERER to PS.Peer reviewe

“My First Thought was Croutons”: Perceptions of Cigarettes and Cigarette Smoke Constituents Among Adult Smokers and Nonsmokers

Understanding what people think about harmful and potentially harmful constituents in cigarettes and cigarette smoke has new urgency given legislation requiring US Food and Drug Administration (FDA) to disclose constituent information. Our study sought to obtain qualitative information on what people think about these constituents and the language they use to talk about them

The Hedgehog Signaling Pathway is Expressed in the Adult Mouse Hypothalamus and Modulated by Fasting

The hedgehog signaling pathway is best known for its role in developmental patterning of the neural tube and limb bud. More recently, hedgehog signaling has been recognized for its roles in growth of adult tissues and maintenance of progenitor cell niches. However, the role of hedgehog signaling in fully differentiated cells like neurons in the adult brain is less clear. In mammals, coordination of hedgehog pathway activity relies on primary cilia and patients with ciliopathies such as Bardet-Biedl and Alström syndrome exhibit clinical features clearly attributable to errant hedgehog such as polydactyly. However, these ciliopathies also present with features not clearly associated with hedgehog signaling such as hyperphagia-associated obesity. How hedgehog signaling may contribute to feeding behavior is complex and unclear, but cilia are critical for proper energy homeostasis. Here, we provide a detailed analysis of the expression of core components of the hedgehog signaling pathway in the adult mouse hypothalamus with an emphasis on feeding centers. We show that hedgehog pathway genes continue to be expressed in differentiated neurons important for the regulation of feeding behavior. Furthermore, we demonstrate for the first time that pathway activity is regulated at the transcriptional level by fasting. These data suggest that hedgehog signaling is involved in the proper functioning of brain regions that regulate feeding behavior and that hedgehog pathway dysfunction may play a role in the obesity observed in certain ciliopathies

Lack of astrocytic glycogen alters synaptic plasticity but not seizure susceptibility

Brain glycogen is mainly stored in astrocytes. However, recent studies both in vitro and in vivo indicate that glycogen also plays important roles in neurons. By conditional deletion of glycogen synthase (GYS1), we previously developed a mouse model entirely devoid of glycogen in the central nervous system (GYS1Nestin-KO). These mice displayed altered electrophysiological properties in the hippocampus and increased susceptibility to kainate-induced seizures. To understand which of these functions are related to astrocytic glycogen, in the present study, we generated a mouse model in which glycogen synthesis is eliminated specifically in astrocytes (GYS1Gfap-KO). Electrophysiological recordings of awake behaving mice revealed alterations in input/output curves and impaired long-term potentiation, similar, but to a lesser extent, to those obtained with GYS1Nestin-KO mice. Surprisingly, GYS1Gfap-KO mice displayed no change in susceptibility to kainate-induced seizures as determined by fEPSP recordings and video monitoring. These results confirm the importance of astrocytic glycogen in synaptic plasticity

“That’s probably what my mama’s lungs look like”: how adolescent children react to pictorial warnings on their parents’ cigarette packs

Abstract Background Pictorial cigarette pack warnings discourage smoking, but most evidence comes from studies of adults. Our qualitative study explored adolescents’ reactions to pictorial warnings on their parents’ cigarette packs. Methods We interviewed 24 adolescents whose parents received pictorial warnings on their cigarette packs as part of a randomized clinical trial. We conducted a thematic content analysis of the interview transcripts. Results Pictorial cigarette pack warnings led adolescents to imagine the depicted health effects happening to their parents, which elicited negative emotions. The warnings inspired adolescents to initiate conversations with their parents and others about quitting smoking. Adolescents believed the warnings would help smokers quit and prevent youth from starting smoking. Some current smokers said the warnings made them consider quitting. Conclusions Conversations about the pictorial warnings may amplify their effectiveness for smokers, their adolescent children, and friends of the adolescent children. Cigarette pack warnings may reach a broad audience that includes adolescent children of smokers

A comparative genomics screen identifies a Sinorhizobium meliloti 1021 sodM-like gene strongly expressed within host plant nodules

Background We have used the genomic data in the Integrated Microbial Genomes system of the Department of Energy’s Joint Genome Institute to make predictions about rhizobial open reading frames that play a role in nodulation of host plants. The genomic data was screened by searching for ORFs conserved in α-proteobacterial rhizobia, but not conserved in closely-related non-nitrogen-fixing α-proteobacteria. Results Using this approach, we identified many genes known to be involved in nodulation or nitrogen fixation, as well as several new candidate genes. We knocked out selected new genes and assayed for the presence of nodulation phenotypes and/or nodule-specific expression. One of these genes, SMc00911, is strongly expressed by bacterial cells within host plant nodules, but is expressed minimally by free-living bacterial cells. A strain carrying an insertion mutation in SMc00911 is not defective in the symbiosis with host plants, but in contrast to expectations, this mutant strain is able to out-compete the S. meliloti 1021 wild type strain for nodule occupancy in co-inoculation experiments. The SMc00911 ORF is predicted to encode a “SodM-like” (superoxide dismutase-like) protein containing a rhodanese sulfurtransferase domain at the N-terminus and a chromate-resistance superfamily domain at the C-terminus. Several other ORFs (SMb20360, SMc01562, SMc01266, SMc03964, and the SMc01424-22 operon) identified in the screen are expressed at a moderate level by bacteria within nodules, but not by free-living bacteria. Conclusions Based on the analysis of ORFs identified in this study, we conclude that this comparative genomics approach can identify rhizobial genes involved in the nitrogen-fixing symbiosis with host plants, although none of the newly identified genes were found to be essential for this process