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Perianal fistula is a debilitating disease and challenging condition to treat. Recently, the use of stem cells has been shown to improve healing of fistulas.We wish to examine the use of an umbilical cord derived stem cell graft in a pilot study as a novel scaffold/stem inlay implanted into fistula repairs for anovaginal fistula to examine healing rates.This was a pilot study.This study took place in a colorectal surgery practice.Patients with anovaginal fistula were consented to participate. Cryopreserved umbilical cord tissue graft with viable cells was incorporated as an in-lay using a previously reported technique by the authors. Demographic data including history of prior repairs and inflammatory bowel disease were included. All patients were followed for a minimum of 6 weeks.The primary measures were safety and efficacy of novel stem cell graft in the treatment of anovaginal fistula.From 9/2017-9/2019, 15 patients underwent anovaginal fistula repair. Three of these patients underwent a second repair, for a total of 18 repairs. No patient was intentionally diverted while 3 patients presented for repair with a pre-existing stoma. The majority of repairs were previous repair failures (12, 67%) and seven repairs were performed on 5 patients with inflammatory bowel disease (IBD). Median follow-up was 30 weeks (6 - 104). The safety profile for cryopreserved umbilical cord tissue graft was excellent as no adverse events occurred. Overall complete healing rate was 39% and 12 (67%) repairs resulted in improvement of symptoms.This was a small pilot study.This is the largest series using cryopreserved umbilical cord graft for anovaginal fistula repair. The utilization of umbilical cord was safe and effective at closing defects. Randomized studies are necessary to determine added benefits over current standard of care. See Video Abstract at http://links.lww.com/DCR/B896.

Lafora disease (LD) is a fatal childhood-onset dementia characterized by the extensive accumulation of glycogen aggregates—the so-called Lafora Bodies (LBs)—in several organs. The accumulation of LBs in the brain underlies the neurological phenotype of the disease. LBs are composed of abnormal glycogen and various associated proteins, including p62, an autophagy adaptor that participates in the aggregation and clearance of misfolded proteins. To study the role of p62 in the formation of LBs and its participation in the pathology of LD, we generated a mouse model of the disease (malinKO) lacking p62. Deletion of p62 prevented LB accumulation in skeletal muscle and cardiac tissue. In the brain, the absence of p62 altered LB morphology and increased susceptibility to epilepsy. These results demonstrate that p62 participates in the formation of LBs and suggest that the sequestration of abnormal glycogen into LBs is a protective mechanism through which it reduces the deleterious consequences of its accumulation in the brain.

Summary Lafora disease (LD) is a fatal childhood dementia characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either EPM2A, encoding the glycogen phosphatase laforin, or EPM2B, encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many EPM2A variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing the functional consequences of laforin missense mutations in vitro using complementary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes that were supported by clinical cases. For example, F321C and G279C mutations have attenuated functional defects and are associated with slow progression. This pipeline enabled rapid characterization and classification of newly identified EPM2A mutations, providing clinicians and researchers genetic information to guide treatment of LD patients., Graphical abstract, Highlights • Lafora disease (LD) patients present with varying clinical progression • LD missense mutations differentially affect laforin function • An empirical in vitro pipeline is used to classify laforin missense mutations • Patient progression can be predicted based on mutation class, Disease; Biochemistry; Structural biology; Biophysics

BackgroundPerianal Crohn’s disease (pCD) is a debilitating complication affecting up to 30% of Crohn’s disease (CD) population, leading to increased morbidity, mortality and decreased quality of life. Despite the growing armamentarium of medications for luminal CD, their efficacy in pCD remains poorly studied.AimTo determine the efficacy of ustekinumab, a biologic approved for luminal CD, in pCD through a retrospective cohort study and systematic review.MethodsA retrospective cohort study on patients with CD with active perianal fistulae treated with ustekinumab from September 2013 to August 2019 was performed to determine perianal fistula response and remission at 6 and 12 months after ustekinumab induction. A systematic review was performed to further establish rates of fistula response and remission with ustekinumab.ResultsAt 6 months, 48.1% (13/27) patients achieved fistula response with none achieving fistula remission on provider exam, and 59.3% (16/27) achieved patient-reported symptomatic improvement with 3.7% (1/27) achieving symptomatic remission. At 1 year, on provider exam, 55.6% (5/9) had fistula response with none achieving fistula remission, and 100% (9/9) had symptomatic improvement with 22.2% (2/9) achieving symptomatic remission. There were no major safety signals during 1-year follow-up. The systematic review of 25 studies found 44% (92/209) of patients with active perianal fistulas had a clinical response within 6 months of follow-up, and 53.9% (85/152) of patients with 12 months of follow-up achieved clinical response.ConclusionUstekinumab presents a safe and effective therapy for treatment of pCD. Prospective, randomised trials are needed to further elucidate long-term efficacy of ustekinumab for pCD.

Significance Mitochondrial dysfunction has been associated with many age-dependent neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease, yet understanding how a neuron maintains a pool of functional mitochondria in sufficient density and location throughout axons in vivo still remains enigmatic. Through an unbiased in vivo forward genetic screen, we identified an endosomal sorting complexes required for transport component, TSG101, as a modulator of mitochondrial number and size in neurons and provide evidence to place TSG101 as a negative regulator of mitochondrial biogenesis. We further find that TSG101 does not contribute to Parkin/Pink1-mediated mitophagy and discovered that macroautophagy regulators ATG1 and ATG6 are dispensable in axonal mitochondrial number regulation. Through in vivo screening, it is possible to unravel translatable mechanisms of axonal mitochondrial maintenance., There is a tight association between mitochondrial dysfunction and neurodegenerative diseases and axons that are particularly vulnerable to degeneration, but how mitochondria are maintained in axons to support their physiology remains poorly defined. In an in vivo forward genetic screen for mutants altering axonal mitochondria, we identified tsg101. Neurons mutant for tsg101 exhibited an increase in mitochondrial number and decrease in mitochondrial size. TSG101 is best known as a component of the endosomal sorting complexes required for transport (ESCRT) complexes; however, loss of most other ESCRT components did not affect mitochondrial numbers or size, suggesting TSG101 regulates mitochondrial biology in a noncanonical, ESCRT-independent manner. The TSG101-mutant phenotype was not caused by lack of mitophagy, and we found that autophagy blockade was detrimental only to the mitochondria in the cell bodies, arguing mitophagy and autophagy are dispensable for the regulation of mitochondria number in axons. Interestingly, TSG101 mitochondrial phenotypes were instead caused by activation of PGC-1ɑ/Nrf2-dependent mitochondrial biogenesis, which was mTOR independent and TFEB dependent and required the mitochondrial fission–fusion machinery. Our work identifies a role for TSG101 in inhibiting mitochondrial biogenesis, which is essential for the maintenance of mitochondrial numbers and sizes, in the axonal compartment.

Summary Glycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.

Significant progress has been made in elucidating single nucleotide polymorphism diversity in the human population. However, the majority of the variation space in the genome is structural and remains partially elusive. One form of structural variation is tandem repeats (TRs). Expansion of TRs are responsible for over 40 diseases, but we hypothesize these represent only a fraction of the pathogenic repeat expansions that exist. Here we characterize long or expanded TR variation in 1,115 human genomes as well as a replication cohort of 2,504 genomes, identified using ExpansionHunter Denovo. We found that individual genomes typically harbor several rare, large TRs, generally in non-coding regions of the genome. We noticed that these large TRs are enriched in their proximity to Alu elements. The vast majority of these large TRs seem to be expansions of smaller TRs that are already present in the reference genome. We are providing this TR profile as a resource for comparison to undiagnosed rare disease genomes in order to detect novel disease-causing repeat expansions.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ERBB2, the gene encoding HER2, is mutated in 2-4% of breast cancers. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against breast cancers harboring HER2 activating mutations, suggesting these tumors depend on HER2 signaling. Co-occurring HER2 and HER3 (ERBB3) mutations have been reported in patients who respond to neratinib (Hanker et al., Cancer Discov. 2017) suggesting the possibility of cooperativity of both oncogenes. Co-expression of the mutant intracellular domains of HER2 and HER3 in HEK293 cells enhanced phosphorylation of HER3 and ERK compared to expression of either mutant alone, which was blocked by 100 nM neratinib. Interrogation of TCGA, METABRIC, Project GENIE, and Foundation Medicine datasets revealed that gain-of-function mutations in ERBB2 and ERBB3 co-occur with a statistically significant frequency. For example, in GENIE, ERBB2 mutations co-occur with mutations in ERBB3 (8.3% of ERBB2-mutant vs 2.3% of ERBB2 WT; q=1.37x10-10). We hypothesized that co-occurring mutations in HER2 and HER3 cooperate to enhance HER2 signaling and dependence and breast cancer progression. Thirty-four unique breast cancers were found to harbor co-occurring mutations in HER2 and HER3, the most common of which were ERBB2L755S/ERBB3E928G (n=10), ERBB2V777L/ERBB3E928G(n=6), and ERBB2L869R/Q/ERBB3E928G (n=4). Using co-immunoprecipitation assays with HER2 and HER3 antibodies in transfected HEK293 cells, we found that co-expression of HER3E928G with wild type (WT) HER2, or co-expression of HER2L755S or HER2L869R with HER3WT, slightly increased HER2-HER3 dimerization. However, binding was strongest between double mutants. This was accompanied by the highest levels of Y1289 p-HER3 in cells expressing both HER3E928G and each HER2L755S, HER2V777L, or HER2L869R compared to cells expressing each HER2 or HER3 mutant with a respective WT heterodimer partner. Structural modeling of the HER2L869R/HER3E928G double-mutant predicted that the HER3 mutation, located at the dimer interface, may enhance heterodimerization of the kinase domains through decreased bulk and electrostatic repulsion. We also noted that the HER2L755S mutation is predicted to be in close proximity to HER3E928G ( MCF7 “knock-in” cells incorporating HER2L755S, HER2V777L, or HER2L869R (or HER2WT) were stably transduced with HER3E928G or HER3WT. Co-expression of double mutants strongly enhanced estrogen-independent growth in 3D Matrigel over cells expressing either mutant alone. We are currently testing inhibitors of HER2/HER3 signaling, including neratinib ± trastuzumab, trastuzumab + pertuzumab, and the ERBB1-3 antibody mixture Sym013, to determine therapeutic strategies to block the cooperative growth induced by co-occurring HER2 and HER2 mutations. Conclusions: Co-expression of mutant HER2 and mutant HER3 promotes HER2/HER binding, HER3 phosphorylation, and breast tumor cell proliferation. We aim to identify therapeutic vulnerabilities for patients with co-occurring HER2 and HER3 mutations. Citation Format: Hanker AB, Koch JP, Ye D, Sliwoski G, Sheehan J, Kinch LN, Red Brewer M, He J, Miller VA, Lalani AS, Cutler, Jr. RE, Croessmann S, Zabransky DJ, Meiler J, Arteaga CL. Co-occurring gain-of-function mutations in HER2 and HER3 cooperate to enhance HER2/HER3 binding, HER-dependent signaling, and breast cancer growth [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD3-05.

Here we demonstrate biallelic mutations in sorbitol dehydrogenase (SORD) as the most frequent recessive form of hereditary neuropathies. We identified 45 cases from 38 families across multiple ethnicities, carrying a particular nonsense mutation in SORD, c.753delG; p.Ala253GlnfsTer27, either in homozygous or compound heterozygous state with a second variant. With an allele frequency of 0.004 in healthy controls, the p.Ala253GlnfsTer27 variant represents one of the most common pathogenic alleles in humans. SORD is an enzyme that converts sorbitol into fructose, in the two-step polyol pathway that has been implicated in diabetic neuropathy. In patient-derived fibroblasts, we find a complete loss of SORD protein as well as increased intracellular sorbitol. Also, serum fasting sorbitol level was over 100 times higher in patients homozygous for the p.Ala253GlnfsTer27 mutation compared to healthy individuals. In Drosophila, we show that loss of SORD orthologues causes synaptic degeneration and progressive motor impairment. Reducing the polyol influx by treatment with aldose reductase inhibitors normalized intracellular sorbitol levels in patient fibroblasts and in Drosophila, and also dramatically ameliorated motor and eye phenotypes. Together, these findings establish a potentially treatable cause in a significant fraction of patients with inherited neuropathies and may contribute to a better understanding of the pathophysiology of diabetic neuropathy.

Summary Mitochondria are essential in long axons to provide metabolic support and sustain neuron integrity. A healthy mitochondrial pool is maintained by biogenesis, transport, mitophagy, fission, and fusion, but how these events are regulated in axons is not well defined. Here, we show that the Drosophila glutathione S-transferase (GST) Gfzf prevents mitochondrial hyperfusion in axons. Gfzf loss altered redox balance between glutathione (GSH) and oxidized glutathione (GSSG) and initiated mitochondrial fusion through the coordinated action of Mfn and Opa1. Gfzf functioned epistatically with the thioredoxin peroxidase Jafrac1 and the thioredoxin reductase 1 TrxR-1 to regulate mitochondrial dynamics. Altering GSH:GSSG ratios in mouse primary neurons in vitro also induced hyperfusion. Mitochondrial changes caused deficits in trafficking, the metabolome, and neuronal physiology. Changes in GSH and oxidative state are associated with neurodegenerative diseases like Alzheimer’s. Our demonstration that GSTs are key in vivo regulators of axonal mitochondrial length and number provides a potential mechanistic link., Highlights • A glutathione S-transferase (GST), Gfzf, decreases mitochondrial fusion in axons • Gfzf loss causes hyperfusion through enhanced oxidation of glutathione • GST loss or enhanced oxidation of glutathione increases the function of mitofusin • Hyperfusion was associated with functional changes but not neurodegeneration, Smith and Lin et al. identify a glutathione S-transferase that decreases mitochondrial fusion in axons of neurons through unbiased genetic screening using Drosophila. gfzf mutants induce sustained hyperfusion through an enhanced oxidation of glutathione that increases the function of mitofusin.

Lafora disease (LD) is a fatal childhood epilepsy and a non-classical glycogen storage disorder with no effective therapy or cure. LD is caused by recessive mutations in theEPM2AorEPM2Bgenes that encode the glycogen phosphatase laforin and an E3 ubiquitin ligase malin, respectively. A hallmark of LD is the intracellular accumulation of abnormal and insoluble α-linked polysaccharide deposits known as Lafora bodies (LBs) in several tissues, including most regions of the brain. In mouse models of LD, genetic reduction of glycogen synthesis eliminates LB formation and rescues the neurological phenotype. Since multiple groups have confirmed that neurodegeneration and epilepsy result from LB accumulation, a major focus in the field has shifted toward the development of therapies that reduce glycogen synthesis or target LBs for degradation with the goal of treating LD. Herein, we identify the optimal enzymes for degrading LBs, and we develop a novel therapeutic agent by fusing human pancreatic α-amylase to a cellpenetrating antibody fragment. This antibody-enzyme fusion (VAL-0417) degrades LBsin vitro, shows robust cellular uptake, and significantly reduces the LB loadin vivoinEpm2a-/- mice. VAL-0417 is a promising therapeutic for the treatment of LD and a putative precision therapy for an intractable epilepsy. Antibody-enzyme fusions represent a new class of antibody-based drugs that could be utilized to treat glycogen storage disorders and other diseases.One Sentence SummaryAn antibody-enzyme fusion delivering an amylase degrades the toxic polyglucosan bodies that cause Lafora disease, a fatal childhood epilepsy.

Inherited axonopathies represent a spectrum of disorders unified by the common pathological mechanism of length-dependent axonal degeneration. Progressive axonal degeneration can lead to both Charcot-Marie-Tooth type 2 (CMT2) and Hereditary Spastic Paraplegia (HSP) depending on the affected neurons: peripheral motor and sensory nerves or central nervous system axons of the corticospinal tract and dorsal columns, respectively. Inherited axonopathies display an extreme degree of genetic heterogeneity of Mendelian high-penetrance genes. High locus heterogeneity is potentially advantageous to deciphering disease etiology by providing avenues to explore biological pathways in an unbiased fashion. Here, we investigate ‘gene modules’ in inherited axonopathies through a network-based analysis of the Human Integrated Protein-Protein Interaction rEference (HIPPIE) database. We demonstrate that CMT2 and HSP disease proteins are significantly more connected than randomly expected. We define these connected disease proteins as ‘proto-modules’ and show the topological relationship of these proto-modules by evaluating their overlap through a shortest-path based measurement. In particular, we observe that the CMT2 and HSP proto-modules significantly overlapped, demonstrating a shared genetic etiology. Comparison of both modules with other diseases revealed an overlapping relationship between HSP and hereditary ataxia and between CMT2 + HSP and hereditary ataxia. We then use the DIseAse Module Detection (DIAMOnD) algorithm to expand the proto-modules into comprehensive disease modules. Analysis of disease modules thus obtained reveals an enrichment of ribosomal proteins and pathways likely central to inherited axonopathy pathogenesis, including protein processing in the endoplasmic reticulum, spliceosome, and mRNA processing. Furthermore, we determine pathways specific to each axonopathy by analyzing the difference of the axonopathy modules. CMT2-specific pathways include glycolysis and gluconeogenesis-related processes, while HSP-specific pathways include processes involved in viral infection response. Unbiased characterization of inherited axonopathy disease modules will provide novel candidate disease genes, improve interpretation of candidate genes identified through patient data, and guide therapy development.

Cosmic microwave background experiments have experienced an exponential increase in complexity, data size and sensitivity. One of the goals of current and future experiments is to characterize the B-mode power spectrum, which would be considered a strong evidence supporting inflation. The signal associated with inflationary B-modes is very weak, and so a successful detection requires exquisite control over systematic effects, several of which might arise due to the interaction between the electromagnetic properties of the telescope beam, the scanning strategy and the sky model. In this work, we present the Pixel Space COnvolver (PISCO), a new software tool capable of producing mock data streams for a general CMB experiment. PISCO uses a fully polarized representation of the electromagnetic properties of the telescope. PISCO also exploits the massively parallel architecture of Graphic Processing Units to accelerate the main calculation. This work shows the results of applying PISCO in several scenarios, included a realistic simulation of an ongoing experiment, the Cosmology Large Angular Scale Surveyor.

This chapter reviews the history of glycogen-related research and discusses in detail the structure, regulation, chemical properties and subcellular distribution of glycogen and its associated proteins, with particular focus on these aspects in brain tissue.

Glucan phosphatases are functionally conserved at the enzymatic level, dephosphorylating glycogen in animals and starch in plants. The human glucan phosphatase laforin is the founding member of the family and it is comprised of a carbohydrate binding module (CBM) domain followed by a dual specificity phosphatase (DSP) domain. Plants encode for two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a DSP domain followed by a CBM domain, while LSF2 contains a DSP domain and lacks a CBM. This review demonstrates how glucan phosphatase function is conserved and highlights how each family member employs a unique mechanism to bind and dephosphorylate glucan substrates.

Charcot-Marie-Tooth (CMT) neuropathies are amongst the most common inherited diseases in neurology. While great strides have been made to identify the genesis of these diseases, a diagnostic gap of 30–60% remains. Classic models of genetic causation may be limited to fully close this gap and, thus, we review the current state and future role of alternative, non-Mendelian forms of genetics in CMT. Promising synergies exist to further define the full genetic architecture of inherited neuropathies, including affordable whole-genome sequencing, increased data aggregation and clinical collaboration, improved bioinformatics and statistical methodology, and vastly improved computational resources. Given the recent advances in genetic therapies for rare diseases, it becomes a matter of urgency to diagnose CMT patients with great fidelity. Otherwise, they will not be able to benefit from such therapeutic options, or worse, suffer harm when pathogenicity of genetic variation is falsely evaluated. In addition, the newly identified modifier and risk genes may offer alternative targets for pharmacotherapy of inherited and, potentially, even acquired forms of neuropathies.

9 páginas, 4 figuras. contiene 2 figuras en material suplementario, Lafora disease (LD, OMIM 254780) is a rare disorder characterized by epilepsy and neurodegeneration leading patients to a vegetative state and death, usually within the first decade from the onset of the first symptoms. 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). In this work, we characterize the mutations present in the EPM2A gene in a patient displaying a slow progression form of the disease. The patient is compound heterozygous with Y112X and N163D mutations in the corresponding alleles. In primary fibroblasts obtained from the patient, we analyzed the expression of the mutated alleles by quantitative real time PCR and found slightly lower levels of expression of the EPM2A gene respect to control cells. However, by Western blotting we were unable to detect endogenous levels of the protein in crude extracts from patient fibroblasts. The Y112X mutation would render a truncated protein lacking the phosphatase domain and likely degraded. Since minute amounts of laforin-N163D might still play a role in cell physiology, we analyzed the biochemical characteristics of the N163D mutation. We found that recombinant laforin N163D protein was as stable as wild type and exhibited near wild type phosphatase activity towards biologically relevant substrates. On the contrary, it showed a severe impairment in the interaction profile with previously identified laforin binding partners. These results lead us to conclude that the slow progression of the disease present in this patient could be either due to the specific biochemical properties of laforin N163D or to the presence of alternative genetic modifying factors separate from pathogenicity., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness SAF2014-54604-C3-1-R and a grant from Generalitat Valenciana (PrometeoII/2014/029); and National Institute of Health grants R01NS070899 and P01NS097197, which established the Lafora Epilepsy Cure Initiative (LECI).

Antioxidant effects of natural antioxidants were evaluated by direct analysis and three high-lipid model systems. The direct analysis assays compared three antioxidant extracts (pomegranate, grape seed and selenium), a synthetic antioxidant (butylated hydroxyanisole) and three natural antioxidant controls (d,l-α-tocopherol, ascorbic acid and gallic acid). Evaluation compared reducing power determined by Folin–Ciocalteu reagent and ferricyanide method, free radical scavenging of 2,2-diphenyl-1-picrylhydrazyl and oxygen radical absorbance capacity. Results indicated that pomegranate and grape seed extracts had better antioxidant efficacy than the synthetic antioxidant. Model systems were constructed in triplicate of three commercial lipid (canola oil, lard and ground beef); samples were subject to an antioxidant treatment (pomegranate extract, grape seed extract, dietary selenium from yeast, butylated hydroxyanisole or no treatment) and chemically evaluated (thiobarbituric acid-reactive substances and diene conjugation) over 9 days. Butylated hydroxyanisole was the most potent antioxidant. The canola oil matrix found no difference in antioxidant treatment under descriptive analysis by a trained panel. Practical Applications Antioxidant food additives are of great importance to the food industry, with many consumers showing a preference for “natural” food additives in the ingredient line. Phenolic compounds, such as those found in pomegranates and grapes, may be particularly useful in the food industry through retarding lipid oxidation. The use of ground beef, lard and oil model systems to study lipid oxidation (or antioxidant capacity by retarding lipid oxidation) is common, but time consuming. Direct analysis and chemical assays that measure oxidative end products can be fast, reliable and less costly that descriptive analysis panels. However, Qualitative Descriptive Analysis (QDA) is commonly used to denote the complete experience that consumer may undergo. Furthermore, evaluating antioxidant efficacy on sight and smell is integral when dealing with products that contain a large amount of polyunsaturated fatty acids, as consumers interact with both aspects during point of purchase.

Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled, due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.

The diagnostic gap for rare neurodegenerative diseases is still considerable, despite continuous advances in gene identification. Many novel Mendelian genes have only been identified in a few families worldwide. Here we report the identification of an autosomal-dominant gene for hereditary spastic paraplegia (HSP) in 10 families that are of diverse geographic origin and whose affected members all carry unique truncating changes in a circumscript region of UBAP1 (ubiquitin-associated protein 1). HSP is a neurodegenerative disease characterized by progressive lower-limb spasticity and weakness, as well as frequent bladder dysfunction. At least 40% of affected persons are currently undiagnosed after exome sequencing. We identified pathological truncating variants in UBAP1 in affected persons from Iran, USA, Germany, Canada, Spain, and Bulgarian Roma. The genetic support ranges from linkage in the largest family (LOD = 8.3) to three confirmed de novo mutations. We show that mRNA in the fibroblasts of affected individuals escapes nonsense-mediated decay and thus leads to the expression of truncated proteins; in addition, concentrations of the full-length protein are reduced in comparison to those in controls. This suggests either a dominant-negative effect or haploinsufficiency. UBAP1 links endosomal trafficking to the ubiquitination machinery pathways that have been previously implicated in HSPs, and UBAP1 provides a bridge toward a more unified pathophysiology.

Antioxidant effects of natural antioxidants in 73%-lean ground beef patties during refrigeration were studied. Samples were subject to an antioxidant treatment (pomegranate extract, grape seed extract, butylated hydroxyanisole, or no treatment) and an irradiation treatment (0 or 1.5 kGy) before being evaluated for both uncooked and cooked indicators of lipid oxidation. Chemical evaluation of antioxidant activity were determined over 9 d through measurement of thiobarbituric reactive substances values. Physical evaluation of antioxidant activity was monitored by changes in physical color and sensory odor analysis on patties before and after cooking. Butylated hydroxyanisole extended the induction period of oxidation in all unirradiated beef patties better than the natural antioxidants. Panelists were able to distinguish a difference (in terms of oxidation attributes) between patty samples of different antioxidant and irradiation treatments while samples were uncooked, but unable to distinguish a difference between patties after cooking.

Soil is a highly transferable source of trace physical material that is both persistent in the environment and varied in composition. This inherent variability can provide useful information to determine the geographical origin of a questioned sample or when comparing and excluding samples, since the composition of soil is dependent on geographical factors such as climate, bedrock geology and land use. Previous studies have limited forensic relevance due to the requirement for large sample amounts and unrealistic differences between the land use and geographical location of the sample sites. In addition the philosophical differences between the disciplines of earth sciences, for which most analytical techniques have been designed, and forensic sciences, particularly with regard to sample preparation and data interpretation have not been fully considered. This study presents an enhanced technique for the analysis of organic components of geoforensic samples by improving the sample preparation and data analysis strategies used in previous research into the analysis of soil samples by high performance liquid chromatography (HPLC). This study provides two alternative sets of marker peaks to generate HPLC profiles which allow both easy visual comparison of samples and the correct assignment of 100% of the samples to their location of origin when discriminating between locations of interest in multivariate statistical analyses. This technique thereby offers an independent form of analysis that is complementary to inorganic geoforensic techniques and offers an easily accessible method for discriminating between close proximity forensically relevant locations.

Glucan phosphatases are a family of enzymes that are functionally conserved at the enzymatic level in animals and plants. These enzymes bind and dephosphorylate glycogen in animals and starch in plants. While the enzymatic function is conserved, the glucan phosphatases employ distinct mechanisms to bind and dephosphorylate glycogen or starch. The founding member of the family is a bimodular human protein called laforin that is comprised of a carbohydrate binding module 20 (CBM20) followed by a dual specificity phosphatase domain. Plants contain two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a chloroplast targeting peptide, dual specificity phosphatase (DSP) domain, a CBM45, and a carboxy-terminal motif. LSF2 is comprised of simply a chloroplast targeting peptide, DSP domain, and carboxy-terminal motif. SEX4 employs an integrated DSP-CBM glucan-binding platform to engage and dephosphorylate starch. LSF2 lacks a CBM and instead utilizes two surface binding sites to bind and dephosphorylate starch. Laforin is a dimeric protein in solution and it utilizes a tetramodular architecture and cooperativity to bind and dephosphorylate glycogen. This chapter describes the biological role of glucan phosphatases in glycogen and starch metabolism and compares and contrasts their ability to bind and dephosphorylate glucans.

Americans are becoming more health conscious in their food choices and many are interested in reducing dietary fat intake. Fat replacers can affect meat flavor both by adding flavors of their own, by reducing the original aroma-generating substrate (fat) and by altering release of aroma compounds. When fat is removed from meat, water is generally added to replace it. Water-binding compounds can be added to prevent the added water from cooking out or evaporating and to prevent patty shrinkage. Fat replacers are generally classified by their composition: protein-based replacers including whey, soy and collagen, lipid-based substances such as soy lecithin which function as emulsifiers maintaining the fat that is retained distributed in the product, and carbohydrate-based substances including flours (wheat, soy, oat), starches (potato, modified corn starch, tapioca) and gums (carrageenan, xanthin). Duplication of the characteristics contributed by fat often requires a combination of replacers to address juiciness and texture (firmness) without negatively impacting flavor.

Five combinations of baby food (puree; puree + pink salmon roe; puree + bone; puree + salmon oil; puree + oil + bone) were formulated from retort-processed sockeye (red) salmon puree base, freeze-dried; then evaluated for color, odor, ability to rehydrate, and storage stability. Baby food was retort-processed, freeze-dried, vacuum-packaged, then stored at 22°C for 4 months. Rehydration was best for the puree only (71.7%), about 53% for puree + roe and puree + oil, and about 48.5% for that containing puree + oil + bone. Adding roe to freeze-dried salmon baby food puree increased fishy, rancid, metallic, and sweaty odor; and lightened and decreased redness, yellowness, and visual color intensity. Adding salmon oil increased salmon odor slightly, yellowness, and visual color intensity and decreased color saturation during storage. Adding salmon bone decreased salmon odor and true red color and increased rancid and buttery odor intensities, yellowness, and visual color intensity. Increasing hue angle indicate...

PG 1351+489 is one of the 20 DBVs ― pulsating helium-atmosphere white dwarf stars ― known and has the simplest power spectrum for this class of star, making it a good candidate to study cooling rates. We report accurate period determinations for the main peak at 489.334 48 s and two other normal modes using data from the Whole Earth Telescope (WET) observations of 1995 and 2009. In 2009, we detected a new pulsation mode and the main pulsation mode exhibited substantial change in its amplitude compared to all previous observations. We were able to estimate the star's rotation period, of 8.9 h, and discuss a possible determination of the rate of period change of (2.0 ± 0.9) x 10 ―13 s s ―1 , the first such estimate for a DBV.

BACKGROUND: The effect of selected antioxidants (grape seed extract (GS), oleoresin rosemary (OR), water-soluble oregano extract (WO), propyl gallate (PG), butylated hydroxyanisole (BHA)), butylated hydroxytoluene (BHT)) on sensory, color and oxidative stability of cooked, frozen, reheated ground beef patties was evaluated. Beef lean and trim were ground; antioxidants and salt were added. Patties were cooked (71 °C), overwrapped in commercial polyvinyl chloride film, and stored frozen (−18 °C), then evaluated monthly for 6 months. Flavor, odor and color were determined using a descriptive panel. Instrumental color was determined by a spectrocolorimeter. Lipid oxidation was determined using thiobarbituric acid reactive substances (TBARS). RESULTS: After 6 months of storage, PG and GS samples had lower rancid odor scores and TBARS than controls. Control samples and those containing BHT did not differ statistically in sensory grassy or rancid odor, indicating that they were the most oxidized. TBARS correlated with grassy, rancid, cardboard and beef odors during the 6-month storage period. CONCLUSIONS: Based on TBARS, the order of effectiveness of the antioxidants was PG and GS > OR > BHA > WO and BHT > control. TBARS were well correlated with sensory evaluations of odor and flavor. Antioxidants also protected a* values during storage. Copyright © 2011 Society of Chemical Industry

Professional precepted immersion courses (capstone) have become the standard as a means to prepare senior nursing students to enter the workforce. Preceptors have a significant role in developing the student nurse, yet exactly how to prepare preceptors for this role has been an ongoing discussion. This qualitative inquiry explored the educational needs of clinical registered nurse (RN) preceptors who work directly with senior nursing students in a professional precepted immersion (capstone) course. A descriptive qualitative design was used to examine preceptors responses to a prepared set of questions about their educational needs. Results showed that preceptors have three distinct sets of learning needs: the need to know the expectations of their role, wanting to know how best to role model for the student, and knowing how to socialize the student into the profession of nursing. Overall, preceptors communicated their desire and commitment to doing the best job possible. They also clearly stated their expectation of faculty to have a physical presence on the nursing unit that included being proactive in resolving mismatches and exposing the student to the roles of provider of care, leader and manager of care, and member of profession.

Fresh beef was dipped into ascorbic acid (AA; 500 ppm), butylated hydroxyanisole (BHA, 150 ppm), butylated hydroxytoluene (BHT, 100 ppm) or propyl gallate (PG; 100 ppm); control samples were dipped in deionized water. Samples were vacuum packaged, then irradiated (0, 1.25 or 2.50 kGy). After irradiation, samples were stored (0, 7 or 14 days) at 4C. Color was assessed instrumentally (L*, a*, b*values, hue angles, chroma) and visually. All antioxidants increased L*value by two units (lighter); however, irradiation dose had no effect. Irradiation increased wet dog and rancid odors of beef; however, it decreased sour odor, a*value, chroma and hue angle, and increased visual green and brown colors, indicating that it reduced redness and color intensity. Antioxidants reduced L*(decreased lightening) and a*values (redness) compared to controls. BHA had the greatest effect on a*value. AA decreased visual green color the most; however, BHA and BHT were also effective. Antioxidants had inconsistent effects on b*value, chroma and hue angle. Storage decreased L*and a*values, and chroma. BHA and PG were most effective at maintaining low thiobarbituric acid-reactive substances when samples were irradiated. PRACTICAL APPLICATIONS Irradiation has the potential to reduce microbial load, increasing both the safety and shelf life of red meat products; however, it has the potential to damage fresh meat color, resulting in unacceptable discoloration. Irradiation can increase off-odors (wet dog, rancid) of beef; however, it can decrease sour odor, instrumental measures of red color, and increase visual green and brown colors. Use of antioxidants including ascorbic acid, butylated hydroxytoluene, butylated hydroxyanisole and propyl gallate can help maintain the color of irradiated beef held in refrigerated storage for up to 2 weeks.

To investigate the extent to which macrophages respond to Salmonella infection, we infected RAW 264.7 macrophages with Salmonella enterica serotype Typhimurium and analyzed macrophage proteins at various time points following infection by using a global proteomic approach. A total of 1,006 macrophage and 115 Salmonella proteins were identified with high confidence. Most of the Salmonella proteins were observed in the late stage of the infection time course, which is consistent with the fact that the bacterial cells proliferate inside RAW 264.7 macrophages. The peptide abundances of most of the identified macrophage proteins remained relatively constant over the time course of infection. Compared to those of the control, the peptide abundances of 244 macrophage proteins (i.e., 24% of the total identified macrophage proteins) changed significantly after infection. The functions of these Salmonella -affected macrophage proteins were diverse, including production of antibacterial nitric oxide (i.e., inducible nitric oxide synthase), production of prostaglandin H 2 (i.e., cyclooxygenase 2), and regulation of intracellular traffic (e.g., sorting nexin 5 [SNX5], SNX6, and SNX9). Diverse functions of the Salmonella -affected macrophage proteins demonstrate a global macrophage response to Salmonella infection. Western blot analysis not only confirmed the proteomic results for a selected set of proteins but also revealed that (i) the protein abundance of mitochondrial superoxide dismutase increased following macrophage infection, indicating an infection-induced oxidative stress in mitochondria, and (ii) in contrast to infection of macrophages by wild-type Salmonella , infection by the sopB deletion mutant had no negative impact on the abundance of SNX6, suggesting a role for SopB in regulating the abundance of SNX6.

Background: Recent data reported by the American Association of Colleges of Nursing suggest that nursing education is experiencing a continued increase in applicants. However, because of a faculty shortage, many qualified students are denied enrollment. Strategies that would encourage individuals to consider a career in nursing education would somewhat alleviate applicant restrictions. Methods: A descriptive phenomenological study was conducted exploring experienced baccalaureate educators' perspectives. Questions were posed about their role experiences. Analysis was conducted with van Manen's procedural steps for examination. Results: Emergent themes were identified. These themes, reflective of the educators' experience, offer insight and guidance to novice educators striving for success. Conclusion: To alleviate the nursing shortage, increased efforts should be directed toward encouraging qualified individuals to pursue a career as a nurse educator and mentoring those who are novices. The wisdom gleaned from experienced nurse educators may help achieve these goals. J Contin Educ Nurs 2009;40(8):367–370.

Penile rehabilitation therapy following radical prostatectomy is a much debated topic. Erectile dysfunction is still a significant contributor to postoperative morbidity following radical prostatectomy, despite meticulous nerve-sparing technique. Secondary smooth muscle changes in the penis have been identified as the underlying causes of penile atrophy, veno-occlusive dysfunction, and fibrosis. Initial observations that intracavernous injection therapies used on a regular basis postoperatively resulted in improvements in the return of spontaneous erectile function led to the development of penile rehabilitation protocols. Chronic dosing of PDE-V inhibitors is now commonly used by urologists after radical prostatectomy. Despite the current enthusiasm of penile rehabilitation therapy, current scientific evidence with clinical trials is still limited.