Intracellular Glycogen Accumulation Research Articles

Repurposing SGLT2 inhibitors: Treatment of renal proximal tubulopathy in Fanconi-Bickel syndrome with empagliflozin.

Renal proximal tubulopathy in Fanconi-Bickel syndrome is caused by impaired basolateral glucose transport via GLUT2 and consequently, intracellular accumulation of glucose and glycogen. SGLT2 inhibitors act on apical glucose reabsorption of renal proximal tubular cells. The purpose of this study was to retrospectively describe the first experiences with repurposing the SGLT2 inhibitor empagliflozin to treat the generalized tubulopathy in Fanconi-Bickel syndrome. A case series was conducted of seven persons from five families (five males, two females; three children, who were 14y5m, 2y9m, and 1y6m old) with genetically confirmed Fanconi-Bickel syndrome, off-label treated with empagliflozin. Median (range) age at start of empagliflozin was 27 years (1y6m - 61y) and duration of follow-up under empagliflozin treatment was 169 days (57-344). Under empagliflozin (up to 25 mg/d), biochemical parameters of tubular cell integrity (urinary N-acetyl-glucosaminidase) and/or tubular functions (including urinary α1-microglobulin) improved in all persons with Fanconi-Bickel syndrome, albeit to varying degrees. Clinically, supplementations (i.e., phosphate, alkali, carnitine, and alfacalcidol) could be completely discontinued in the three children, whereas results in the four adult patients were more variable and not as significant. Empagliflozin was well-tolerated and no symptomatic hypoglycemia was observed. In conclusion, SGLT2 inhibitors such as empagliflozin shift the metabolic block in Fanconi-Bickel syndrome, that is, they intervene specifically in the underlying pathophysiology and can thus attenuate renal proximal tubulopathy, especially when started in early childhood.

The impact of bed rest on human skeletal muscle metabolism

Insulin sensitivity and metabolic flexibility decrease in response to bed rest, but the temporal and causal adaptations in human skeletal muscle metabolism are not fully defined. Here, we use an integrative approach to assess human skeletal muscle metabolism during bed rest and provide a multi-system analysis of how skeletal muscle and the circulatory system adapt to short- and long-term bed rest (German Clinical Trials: DRKS00015677). We uncover that intracellular glycogen accumulation after short-term bed rest accompanies a rapid reduction in systemic insulin sensitivity and less GLUT4 localization at the muscle cell membrane, preventing further intracellular glycogen deposition after long-term bed rest. We provide evidence of a temporal link between the accumulation of intracellular triglycerides, lipotoxic ceramides, and sphingomyelins and an altered skeletal muscle mitochondrial structure and function after long-term bed rest. An intracellular nutrient overload therefore represents a crucial determinant for rapid skeletal muscle insulin insensitivity and mitochondrial alterations after prolonged bed rest.

Analyzing factors associated with the receipt of surgery in clear cell sarcoma.

447 Background: Clear cell sarcoma (CCS) is a ultrarare soft tissue sarcoma, which is highly aggressive and associated with a poor prognosis. CCS is histologically characterized by clear cells, representative of intracellular glycogen accumulation. Primary tumors commonly arise from tendinous sheaths or aponeuroses in the upper and lower extremities—the most common site being around the ankles. Average age at diagnosis is 25 years old. 5-year survival is between 40 and 60%. CCS is the result of fusion gene EWSR1/ATF1. Multimodal therapy, including wide surgical excision with radiotherapy, is often used to treat CCS. Chemotherapy may be used if patients are not eligible for surgery. No significant study has analyzed the factors which predict the utilization of wide surgical excision. This study aims to describe the factors involved in the receipt of this surgery. Methods: The National Cancer Database (NCDB) was used to identify patients diagnosed with CCS from 2004 to 2020 using histology codes 9044 as assigned by the Commission on Cancer Accreditation program. Kaplan-Meier, ANOVA Chi-Square, and Logistic Regression tests were performed, and data were analyzed using SPSS version 27. Statistical significance was set at α = 0.05. Results: 669 patients with CCS formed the initial sample. Of these patients, 466 patients (69.7%) received a surgical procedure of the primary site. Additionally, 14 patients (2.09%) received radiation and 69 patients (10.31%) received adjuvant chemotherapy. The receipt of surgery was associated with a marked increase in median survival ( 91.5 months vs. 8.525 months; p<0.05). Surgical resection with clear margins was achieved in 409 patients (61.14%), of whom experienced improved survival compared to patients with residual tumor burden. Each additional year of age in a patient conferred a 2.4% decrease in likelihood of the patient receiving surgery. Lower stage disease was significantly more likely to be treated surgically (OR 11.4; p<0.001), while stage IV disease and abdominal primary site tumors were associated with lower rates of surgery. Facility type (academic vs. nonacademic), insurance, or distance travelled for care had no effect on rates of surgery. Conclusions: As a significant portion of CCS patients receive surgical treatment, this study confirms that tumor resection is associated with increased overall survival of patients with CCS. Improved survival with surgery correlates with clear surgical margins and full tumor resection. It appears that patients are more likely to be treated surgically if their disease is of lower stage. Patients with abdominal primary site tumors or stage IV disease are less likely to receive surgery. This research serves as the beginning of developing standardized treatment plans for CCS patients. Further studies are needed to better understand the factors involved in receiving surgery and other treatment modalities as part of CCS treatment.

Glycogen storage disease type I: Genetic etiology, clinical manifestations, and conventional and gene therapies.

Glycogen storage disease type I (GSDI) is an inherited metabolic disorder characterized by a deficiency of enzymes or proteins involved in glycogenolysis and gluconeogenesis, resulting in excessive intracellular glycogen accumulation. While GSDI is classified into four different subtypes based on molecular genetic variants, GSDIa accounts for approximately 80%. GSDIa and GSDIb are autosomal recessive disorders caused by deficiencies in glucose-6-phosphatase (G6Pase-α) and glucose-6-phosphate-transporter (G6PT), respectively. For the past 50 years, the care of patients with GSDI has been improved following elaborate dietary managements. GSDI patients currently receive dietary therapies that enable patients to improve hypoglycemia and alleviate early symptomatic signs of the disease. However, dietary therapies have many limitations with a risk of calcium, vitamin D, and iron deficiency and cannot prevent long-term complications, such as progressive liver and renal failure. With the deepening understanding of the pathogenesis of GSDI and the development of gene therapy technology, there is great progress in the treatment of GSDI. Here, we review the underlying molecular genetics and the current clinical management strategies of GSDI patients with an emphasis on promising experimental gene therapies.

Low aerobic capacity in McArdle disease: A role for mitochondrial network impairment?

BackgroundMcArdle disease is caused by myophosphorylase deficiency and results in complete inability for muscle glycogen breakdown. A hallmark of this condition is muscle oxidation impairment (e.g., low peak oxygen uptake (VO2peak)), a phenomenon traditionally attributed to reduced glycolytic flux and Krebs cycle anaplerosis. Here we hypothesized an additional role for muscle mitochondrial network alterations associated with massive intracellular glycogen accumulation. MethodsWe analyzed in depth mitochondrial characteristics-content, biogenesis, ultrastructure-and network integrity in skeletal-muscle from McArdle/control mice and two patients. We also determined VO2peak in patients (both sexes, N = 145) and healthy controls (N = 133). ResultsBesides corroborating very poor VO2peak values in patients and impairment in muscle glycolytic flux, we found that, in McArdle muscle: (a) damaged fibers are likely those with a higher mitochondrial and glycogen content, which show major disruption of the three main cytoskeleton components-actin microfilaments, microtubules and intermediate filaments-thereby contributing to mitochondrial network disruption in skeletal muscle fibers; (b) there was an altered subcellular localization of mitochondrial fission/fusion proteins and of the sarcoplasmic reticulum protein calsequestrin-with subsequent alteration in mitochondrial dynamics/function; impairment in mitochondrial content/biogenesis; and (c) several OXPHOS-related complex proteins/activities were also affected. ConclusionsIn McArdle disease, severe muscle oxidative capacity impairment could also be explained by a disruption of the mitochondrial network, at least in those fibers with a higher capacity for glycogen accumulation. Our findings might pave the way for future research addressing the potential involvement of mitochondrial network alterations in the pathophysiology of other glycogenoses.

Atrial Flutter in PRKAG2 Syndrome: Clinical and Electrophysiological Characteristics.

PRKAG2 syndrome is a rare autosomal dominant disease, a phenocopy of hypertrophic cardiomyopathy characterized by intracellular glycogen accumulation. Clinical manifestations include ventricular preexcitation, cardiac conduction disorder, ventricular hypertrophy, and atrial arrhythmias. To compare the clinical and electrophysiological characteristics observed in patients with atrial flutter, with and without PRKAG2 syndrome. An observational study comparing patients with atrial flutter: group A consisted of five patients with PRKAG2 syndrome from a family, and group B consisted of 25 patients without phenotype of PRKAG2 syndrome. The level of significance was 5%. All patients in group A had ventricular preexcitation and right branch block, and four had pacemakers (80%). Patients in group A were younger (39±5.4 vs 58.6±17.6 years, p=0.021), had greater interventricular septum (median=18 vs 10 mm; p<0.001) and posterior wall thickness (median=14 vs 10 mm; p=0.001). In group A, four patients were submitted to an electrophysiological study, showing a fasciculoventricular pathway, and atrial flutter ablation was performed in tree. All patients in group B were submitted to ablation of atrial flutter, with no evidence of accessory pathway. Group B had a higher prevalence of hypertension, diabetes mellitus, coronary artery disease and sleep apnea, with no statistically significant difference. Patients with PRKAG2 syndrome presented atrial flutter at an earlier age and had fewer comorbidities when compared to patients with atrial flutter without mutation phenotype. The occurrence of atrial flutter in young individuals, especially in the presence of ventricular preexcitation and familial ventricular hypertrophy, should raise the suspicion of PRKAG2 syndrome.

Altered electrical properties in skeletal muscle of mice with glycogen storage disease type II

Electrical impedance methods, including electrical impedance myography, are increasingly being used as biomarkers of muscle health since they measure passive electrical properties of muscle that alter in disease. One disorder, Pompe Disease (Glycogen storage disease type II (GSDII)), remains relatively unstudied. This disease is marked by dramatic accumulation of intracellular myofiber glycogen. Here we assessed the electrical properties of skeletal muscle in a model of GSDII, the Pompe6neo/6neo (Pompe) mouse. Ex vivo impedance measurements of gastrocnemius (GA) were obtained using a dielectric measuring cell in 30-week-old female Pompe (N = 10) and WT (N = 10) mice. Longitudinal and transverse conductivity, σ, and the relative permittivity, εr, and Cole–Cole complex resistivity parameters at 0 Hz and infinite frequency, ρo and ρ∞, respectively, and the intracellular resistivity, ρintracellular were determined from the impedance data. Glycogen content (GC) was visualized histologically and quantified biochemically. At frequencies > 1 MHz, Pompe mice demonstrated significantly decreased longitudinal and transverse conductivity, increased Cole–Cole parameters, ρo and ρo-ρ∞, and decreased ρintracellular. Changes in longitudinal conductivity and ρintracellular correlated with increased GC in Pompe animals. Ex vivo high frequency impedance measures are sensitive to alterations in intracellular myofiber features considered characteristic of GSDII, making them potentially useful measures of disease status.

Loss of Autophagy Causes Increased Apoptosis of Tibial Plateau Chondrocytes in Guinea Pigs with Spontaneous Osteoarthritis.

The goal of the present study was to observe the effect of autophagy in tibial plateau chondrocytes on apoptosis in spontaneous knee osteoarthritis (OA) in guinea pigs. Fifty 2-month-old female Hartley guinea pigs were divided into a normal group (10 animals, all euthanized after 7 months) and an OA group (40 animals, 10 of which were euthanized after 10 months). Immunohistochemistry, RT-qPCR and Western blotting were used to evaluate autophagy levels, intracellular glycogen accumulation and apoptosis in tibial plateau chondrocytes in vivo and in vitro. The remaining 30 guinea pigs in the OA group were divided into 3 groups: a rapamycin group, a normal saline group, and a 3-methyladenine (3-MA) group. Intracellular glycogen accumulation and chondrocyte apoptosis were assessed by altering the level of autophagy in chondrocytes in vivo. When spontaneous OA occurred in guinea pigs, autophagy levels in tibial plateau chondrocytes decreased, while intracellular glycogen accumulation and the rate of chondrocyte apoptosis increased. After enhancing the level of autophagy in tibial plateau chondrocytes in guinea pigs with OA, intracellular glycogen accumulation and the rate of chondrocyte apoptosis decreased, while inhibiting autophagy had the opposite effects. The results indicate that the function of autophagy in chondrocytes may at least partly involve the catabolism of glycogen. In guinea pigs with OA, the level of autophagy in tibial plateau chondrocytes decreased, and chondrocytes were unable to degrade intracellular glycogen into glucose, leading to less energy for chondrocytes and increased apoptosis.

Creatine Kinase Pathway Inhibition Alters GSK3 and WNT Signaling in EVI1-Positive Acute Myeloid Leukemia

Targeting cellular metabolism has emerged as a promising therapeutic strategy in acute myeloid leukemia (AML). Several inhibitors of key metabolic dependencies are currently being tested in clinical trials. The impact of inhibition of these metabolic enzymes on downstream signaling pathways, however, remains incompletely understood. We recently identified the mitochondrial creatine kinase, CKMT1, involved in arginine-creatine metabolism, as a new metabolic vulnerability in a subset of AML driven by the oncogene EVI1 (Fenouille et al., Nature Medicine, 2017). We determined that suppression of arginine-creatine metabolism by CKMT1-directed shRNAs, or by the small molecule cyclocreatine, selectively altered mitochondrial-dependent ATP production in EVI1 -positive cells, thereby reducing their growth.In an effort to identify downstream signaling pathways altered with CKMT1 inhibition, we interrogated RNA sequencing data from three EVI1 -positive cell lines, TF-1, UT-7, and UCSD-AML1, after 24 hours of cyclocreatine treatment. This analysis revealed that gene sets related to the GSK3 and WNT pathways were among those most enriched in genes whose expression was suppressed by cyclocreatine. We then established that creatine kinase (CK) pathway inhibition impaired the expression of GSK3 and WNT target genes, including genes upstream in the pathway, such as DVL1, DVL2, DVL3, FZD3, FZD6, FZD7, and FZD8, and more downstream targets, such as CTNNB1, MYC, PPARD, CCND1, and JUN. In line with these transcriptional effects, the level of GSK3 phosphorylation at the inhibitory serine sites of the two GSK3 isoforms (S21 on GSK3A and S9 on GSK3B), was strongly enhanced upon cyclocreatine treatment, confirming the inhibition of GSK3. Intracellular glycogen accumulation, a direct phenotypic consequence of GSK3 inhibition, was also observed in cyclocreatine-treated AML cells.To investigate whether inhibition of GSK3 signaling was important for the cyclocreatine-mediated differentiation and growth effects, we generated constitutively active GSK3 mutants by substituting serines 21 and 9 with non-phosphorylable alanines (S21A and S9A) on GSK3A and GSK3B, respectively. Co-expression of active mutants GSK3A/B S21A/S9A completely abrogated cyclocreatine-induced CD117 downregulation, a marker of stemness. We then compared the effects of CK pathway inhibition in two mouse models: NrasG12D + Evi1 -driven AML, containing a clonal Evi1 integration and expressing high transcript levels of Evi1, and NrasG12D -driven AML that does not express Evi1. We previously established that the NrasG12D + Evi1 -driven disease burden was selectively altered by CK pathway inhibition. In this context, we have now demonstrated that overexpression of Gsk3b S9A in NrasG12D + Evi1 cells attenuated the anti-leukemic effect of cyclocreatine leading to altered survival.Inhibition of both the alpha and beta isoforms of GSK3 is well reported to induce β-catenin stabilization, an undesirable effect in AML therapy. Surprisingly, however, in addition to its inhibitory effect on GSK3, cyclocreatine treatment also repressed mRNA and protein levels of CTNNB1 . This effect was rescued by the reactivation of arginine-creatine metabolism upon phospho-creatine supplementation. Despite GSK3 pathway inhibition, cyclocreatine treatment prevented nuclear CTNNB1 accumulation in EVI1-positive cells and thereby did not activate the CTNNB1 transcriptional program.Taken together, our results demonstrate that the blockade of the creatine kinase pathway inhibits GSK3 and silences the WNT pathway, impeding canonical control of GSK3 on WNT signaling. We thus identified a therapeutic strategy to promote the anti-leukemic and pro-maturating effects of GSK3 inhibition without inducing potentially pro-leukemogenic β-catenin signaling. DisclosuresHermine:Novartis: Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; Celgene: Research Funding; Hybrigenics: Research Funding; INatherys: Equity Ownership, Research Funding. Stegmaier:Novartis: Consultancy, Research Funding.

Targeting Glycogen Metabolism as a Novel Therapeutic Approach in Anaplastic Thyroid Cancer

Effective treatment options for well-differentiated papillary (PTC) and follicular (FTC) thyroid cancers afford positive patient prognoses. The absence of effective interventions for the stem-like, dedifferentiated anaplastic thyroid cancer (ATC) results in poor patient outcomes with a mortality rate higher than all other endocrine cancers combined (1). While receptor tyrosine kinase inhibitors such as sorafenib can extend ATC patient survival, drug resistance and tumor reoccurrence often develop (2). Therefore, there is a critical need for more effective targeted therapies for ATC. Although the cell signaling landscape of ATC tumors is well described, very little is known about tumorigenic adaptations in ATC cellular metabolism. Tumors exhibit an increased consumption of glucose compared to normal tissues to fuel tumor progression. Some cancers meet this high glucose requirement by storing and breaking down glycogen. In our studies here, we show for the first time that normal thyroid, PTC, FTC, and ATC cells express genes necessary for glycogen metabolism. We confirm these observations in patient samples in normal thyroid and thyroid cancer patient samples via immunofluorescence in tissue microarrays. Furthermore, we detect intracellular glycogen stores in cell lines representing normal thyroid, PTC, FTC, and ATC cells. Importantly, we demonstrate that glycogen phosphorylase inhibitors result in accumulation of intracellular glycogen and induce subsequent apoptosis in ATC cells. We further show that glycogen phosphorylase inhibitors synergize with kinase inhibitors such as sorafenib and buparlisib to decrease ATC cell viability. Our work establishes glycogen metabolism as a novel metabolic process in thyroid cells that is associated with thyroid cancer dedifferentiation and provides insight to the effectiveness of inhibiting glycogen metabolism as a therapeutic strategy in ATC.

Decreased Autophagy Level in Tibial Plateau Chondrocytes Causes Increased Apoptosis of Chondrocytes in Spontaneous Osteoarthritis of Guinea Pigs

Background: To observe the effect of autophagy in tibial plateau chondrocytes on apoptosis in spontaneous knee osteoarthritis (OA) in guinea pigs. Methods: Fifty 2-month-old female Hartley guinea pigs were divided into a normal group, which was euthanized after 7 months, and an OA group, ten of which were euthanized after 10 months. Immunohistochemistry, PCR and western blotting were used to evaluate the level of autophagy, intracellular glycogen accumulation and apoptosis in tibial plateau chondrocytes in vivo and in vitro. The remaining 30 guinea pigs in the OA group were divided into 3 groups: a rapamycin group, a normal saline group and a 3-MA group. Intracellular glycogen accumulation and chondrocyte apoptosis were observed by changing the level of autophagy in chondrocytes in vivo. Findings: When spontaneous OA occurred in the guinea pigs, the level of autophagy in tibial plateau chondrocytes decreased,and intracellular glycogen accumulation and the rate of chondrocyte apoptosis increased. After enhancing the autophagy level of tibial plateau chondrocytes in OA guinea pigs, the intracellular glycogen accumulation and the rate of chondrocyte apoptosis decreased. When the autophagy level of the chondrocytes was weakened, intracellular glycogen accumulation was further increased, and the apoptosis rate was higher. Interpretation: The autophagy function of chondrocytes may be at least partly involved in the catabolism of glycogen in chondrocytes. In OA guinea pigs, the autophagy level in tibial plateau chondrocytes decreased, and the chondrocytes were unable to degrade intracellular glycogen into glucose, leading to less energy for the chondrocytes and increased apoptosis. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (no. 81572098), Natural Science Foundation of Shanxi Province of China (no.201701D121161) and Key Research and Development Projects of Shanxi Province (no.201803D31160). Declaration of Interests: None declared. Ethics Approval Statement: This study was approved by the Ethics Committee of Shanxi Medical University (approval number: SXMUE2019004). All experiments were performed in accordance with the Public Health Service Policy and the Guide to the Care and Use of Laboratory Animals.

Glycogen Metabolism Impairment via Single Gene Mutation in the glgBXCAP Operon Alters the Survival Rate of Escherichia coli Under Various Environmental Stresses.

Glycogen is a highly branched polysaccharide that is widely present in all life domains. It has been identified in many bacterial species and functions as an important energy storage compound. In addition, it plays important roles in bacterial transmission, pathogenicity, and environmental viability. There are five essential enzymes (coding genes) directly involved in bacterial glycogen metabolism, which forms a single operon glgBXCAP with a suboperonic promoter in glgC gene in Escherichia coli. Currently, there is no comparative study of how the disruptions of the five glycogen metabolism genes influence bacterial phenotypes, such as growth rate, biofilm formation, and environmental survival, etc. In this study, we systematically and comparatively studied five E. coli single-gene mutants (ΔglgC, ΔglgA, ΔglgB, ΔglgP, ΔglgX) in terms of glycogen metabolism and explored their phenotype changes with a focus on environmental stress endurance, such as nutrient deprivation, low temperature, desiccation, and oxidation, etc. Biofilm formation in wild-type and mutant strains was also compared. E. coli wild-type stores the highest glycogen content after around 20-h culture while disruption of degradation genes (glgP, glgX) leads to continuous accumulation of glycogen. However, glycogen primary structure was abnormally changed in ΔglgP and ΔglgX. Meanwhile, increased accumulation of glycogen facilitates the growth of E. coli mutants but reduces glucose consumption in liquid culture and vice versa. Glycogen metabolism disruption also significantly and consistently increases biofilm formation in all the mutants. As for environmental stress endurance, glycogen over-accumulating mutants have enhanced starvation viability and reduced desiccation viability while all mutants showed decreased survival rate at low temperature. No consistent results were found for oxidative stress resistance in terms of glycogen metabolism disruptions, though ΔglgA shows highest resistance toward oxidation with unknown mechanisms. In sum, single gene disruptions in glgBXCAP operon significantly influence bacterial growth and glucose consumption during culture. Accumulation and structure of intracellular glycogen were also significantly altered. In addition, we observed significant changes in E. coli environmental viabilities due to the deletions of certain genes in the operon. Further investigations shall be focused on the molecular mechanisms behind these phenotype changes.

Cytological intracellular glycogen evaluation using PAS and PAS-D stains to correlate plasma glucose in diabetics.

Oral cytology studies have claimed that cytoplasmic Periodic Acid Schiff (PAS) positivity in type-2 diabetics is due to glycogen content. But, it can also be due to mucin and glycoconjugates. 1. To confirm that cytoplasmic PAS positivity in type-2 diabetics is due to glycogen using diastase. 2. To know the effect of diabetes by determining the number of glycogen-containing cells in the smear. 3. To assess the impact of duration of diabetes based on PAS staining of cells. 4. To correlate between random blood glucose level and the number of PAS-positive cells. Study population comprised 45 individuals with 30 type-2 diabetics as case group (Group I < 5 years duration; Group II > 5 years duration) and 15 healthy volunteers (age and gender-matched) as control. For all subjects, random blood glucose was estimated and two cytosmears were obtained. The smears were stained with PAS and PAS-diastase stains (PAS-D). Staining intensity was documented as score 1 (mild-to-moderate) and score 2 (moderate-to-intense) and data obtained were statistically analyzed in SPSS version 16.0. Mann-Whitney U test revealed that in diabetics cytoplasmic PAS positivity is because of glycogen (P < 0.05). There is an increase in the number of glycogen-containing cells (P < 0.05) in diabetics. The duration of diabetes had less impact on intracellular glycogen accumulation (P > 0.05). Spearman's correlation test revealed no significant correlation (P > 0.05) between random blood glucose and a number of PAS-positive cells. PAS positivity is because of intracellular glycogen accumulation in type-2 diabetics. It can convey the glycaemic status of an individual in the recent past, thus a beneficial role in screening and therapeutic monitoring.

Muscle diffusion tensor imaging in glycogen storage disease V (McArdle disease).

To evaluate differences in diffusion parameters in thigh muscles in patients with glycogen storage disease type V (McArdle disease) using muscle diffusion tensor imaging (mDTI) compared to healthy controls METHODS: In this prospective study, we evaluated thigh muscles from hip to knee of 10 McArdle patients (5 female, mean age 33.7 ± 14.4years) and 10 healthy age- and gender-matched volunteers. MRI scans were performed at 3T and comprised mDTI, T1-weighted and T2-weighted imaging between May 2015 and May 2017. Needle biopsy of the vastus lateralis muscle was performed in three McArdle patients. The muscle tissue was analyzed by using histochemical and enzyme-histochemical techniques for glycogen content and histopathological changes. Mean values of the eigenvalues (λ1-λ3), fractional anisotropy (FA), and mean diffusivity (MD) were obtained for the vastus lateralis, vastus medialis, rectus femoris, biceps femoris, semitendinosus, and semimembranosus and compared between groups using Student's t tests, as well as ANCOVA; significance level was set at p < 0.05. Needle biopsy showed intracellular glycogen accumulation in skeletal muscle fibers of three McArdle patients. Extracellular histopathological changes were not found. Muscle DTI analysis did not show statistically significant differences between patients and controls for any of the muscles. Despite intracellular glycogen accumulation in the three biopsy samples, mDTI parameters were not altered in McArdle patients compared to controls. We conclude that the currently used mDTI acquisition and processing lack the sensitivity to detect intracellular changes due to accumulated glycogen in this cohort of McArdle patients. • Despite intracellular glycogen accumulation in three examined biopsy samples, mDTI parameters were not altered in McArdle patients compared to controls. • In its current form, diffusion MR does not provide additional information in quantifying intracellular glycogen accumulations within skeletal muscle fibers in McArdle patients.

BENEFICIAL EFFECTS OF MAGNETIC FIELDS ON THE HUMAN HEART A REVIEW OF CURRENT ADVANCES AND PROPOSED MECHANISMS

The main purpose of this manuscript is to introduce the reader to the present “state of the art” in experimental Low Level Magnetic Fields nerve stimulation approaches to improve chaotic cardiac arrhythmias and muscle contractions. The area addressed in this paper is the transcutaneous application of Low Level Magnetic Fields to a branch of the vagus nerve. Cardiac Arrhythmias such as Atrial Fibrillation, as well as and muscle contractility were reversed by Low Level Transcutaneous Stimulation (LL-TS) of the Auricular Branch of the Vagus Nerve (ABVN). This treatment has been successfully reported in humans as well as in awaken and anesthetized animals such as rodents and dogs. The functional improvement has been attributed to a reversal of cardiac myocytes remodeling (reversal of fibrosis) resulting from experimentally provoking induced rapid heartbeats. The prolonged duration of a tachycardia state induces intracellular accumulation of glycogen leading into fibrosis, this in turn is hypothesized to impede the intercellular electrical communications of the specialized cardiac cells. It also reduces the functional ability of the main pumping chamber (Left Ventricle).

Mutation analysis of CHCHD2 and CHCHD10 in Italian patients with mitochondrial myopathy

Mutations in CHCHD2 and CHCHD10 were recently reported in a broad spectrum of neurodegenerative diseases, for example, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, or mitochondrial myopathy (MM). The aim of the study was to evaluate the prevalence of CHCHD2 and CHCHD10 mutations in Italian MM patients without mitochondrial DNA mutations. The coding regions of CHCHD2 and CHCHD10 were sequenced in 62 MM patients. None of the patients showed CHCHD2 mutations, whereas 1 sporadic MM patient carried a homozygous Pro96Thr substitution in CHCHD10. Muscle biopsy of this patient showed intracellular glycogen accumulation with cytochrome c oxidase negative and ragged red fibers. Our study suggests that the homozygous Pro96Thr mutation in CHCHD10 might be pathogenic but does not support a major role for CHCHD2 in MM pathogenesis.

Lafora disease in miniature Wirehaired Dachshunds

Lafora disease (LD) is an autosomal recessive late onset, progressive myoclonic epilepsy with a high prevalence in the miniature Wirehaired Dachshund. The disease is due to a mutation in the Epm2b gene which results in intracellular accumulation of abnormal glycogen (Lafora bodies). Recent breed-wide testing suggests that the carrier plus affected rate may be as high as 20%. A characteristic feature of the disease is spontaneous and reflex myoclonus; however clinical signs and disease progression are not well described. A survey was submitted to owners of MWHD which were homozygous for Epm2b mutation (breed club testing program) or had late onset reflex myoclonus and clinical diagnosis of LD. There were 27 dogs (11 male; 16 female) for analysis after young mutation-positive dogs that had yet to develop disease were excluded. Average age of onset of clinical signs was 6.94 years (3.5–12). The most common initial presenting sign was reflex and spontaneous myoclonus (77.8%). Other presenting signs included hypnic myoclonus (51.9%) and generalized seizures (40.7%). Less common presenting signs include focal seizures, “jaw smacking”, “fly catching”, “panic attacks”, impaired vision, aggression and urinary incontinence. All these clinical signs may appear, and then increase in frequency and intensity over time. The myoclonus in particular becomes more severe and more refractory to treatment. Signs that developed later in the disease include dementia (51.9%), blindness (48.1%), aggression to people (25.9%) and dogs (33.3%), deafness (29.6%) and fecal (29.6%) and urinary (37.0%) incontinence as a result of loss of house training (disinhibited type behavior). Further prospective study is needed to further characterize the canine disease and to allow more specific therapeutic strategies and to tailor therapy as the disease progresses.

Inhibition of the Rumen Ciliate Entodinium caudatum by Antibiotics

Axenic cultures of free-living aerobic ciliates, such as Tetrahymena thermophila and Paramecium aurelia, have been established and routinely used in laboratory research, greatly facilitating, or enabling characterization of their metabolism, physiology, and ecology. Ruminal protozoa are anaerobic ciliates, and they play important roles in feed digestion and fermentation. Although, repeatedly attempted, no laboratory-maintainable axenic culture of ruminal ciliates has been established. When axenic ciliate cultures are developed, antibiotics are required to eliminate the accompanying bacteria. Ruminal ciliates gradually lose viability upon antibiotic treatments, and the resultant axenic cultures can only last for short periods of time. The objective of this study was to evaluate eight antibiotics that have been evaluated in developing axenic cultures of ruminal ciliates, for their toxicity to Entodinium caudatum, which is the most predominant ruminal ciliate species. Scanning and transmission electron microscopy (TEM) showed that the antibiotics damaged both the cell surface and nuclei of E. caudatum and increased accumulation of intracellular glycogen. Combinations of the three least toxic antibiotics failed to eliminate the bacteria that are present in the E. caudatum culture. The combination of ampicillin, carbenicillin, streptomycin, and oxytetracycline was able to eliminate all the bacteria, but the resultant axenic E. caudatum culture gradually lost viability. Adding the bacterial fraction (live) separated from an untreated E. caudatum culture reversed the viability decline and recovered the growth of the treated E. caudatum culture, whereas feeding nine strains of live bacteria isolated from E. caudatum cells, either individually or in combination, could not. Nutritional and metabolic dependence on its associated bacteria, accompanied with direct and indirect inhibition by antibiotics, makes it difficult to establish an axenic culture of E. caudatum. Monoxenic or polyxenic cultures of E. caudatum could be developed if the essential symbiotic partner(s) can be identified.

Intracellular accumulation of trehalose and glycogen in an extreme oligotroph, Rhodococcus erythropolis N9T-4.

An extreme oligotroph, Rhodococcus erythropolis N9T-4, showed intracellular accumulation of trehalose and glycogen under oligotrophic conditions. No trehalose accumulation was observed in cells grown on the rich medium. Deletion of the polyphosphate kinase genes enhanced the trehalose accumulation and decreases the intracellular glycogen contents, suggesting an oligotrophic relationship between among the metabolic pathways of trehalose, glycogen, and inorganic polyphosphate biosyntheses.

Osmium tetroxide post-fixation and periodic acid-Schiff dual-staining technique to demonstrate intracellular lipid and glycogen in the mouse liver section – a novel method for co-visualization of intracellular contents in paraffin-embedded tissue

Intracellular accumulation of excessive glycogen or lipid in the liver occurs as a manifestation of metabolic dysfunction or drug-induced toxicity in humans or animals. The identification of individual components (lipids, glycogen, or water) involved is essential to the diagnosis of disease and pathogenesis. The aqueous fixatives and processing solvents used in paraffin embedding for routine hematoxylin and eosin stains remove both lipid and glycogen, leaving clear vacuoles of which the precise content is unknown in routine hematoxylin and eosin-stained sections. Traditional methods for detecting intracellular lipids and glycogen require two separate techniques, such as periodic acid-Schiff (PAS) reaction, with or without diastase digestion for glycogen, and osmium tetroxide (OsO4) post-fixation for lipid and utilization of different tissue sections. We describe here, for the first time, a novel OsO4/PAS dual-staining technique that permits a simple color-coded detection and direct visualization of both lipids and glycogen in the same paraffin-embedded tissue section. In this study, liver samples were collected from diet-induced obese (C57BL/6) or db/db mice and fixed in 10% neutral buffered formalin for 24 h. The formalin-fixed samples were post-fixed with OsO4, processed, sectioned, stained with PAS reaction, and evaluated microscopically for lipid and glycogen contents. Both lipid and glycogen were clearly identifiable with subjectively quantifiable severity and distribution in the same liver section. In conclusion, OsO4/PAS dual-staining, in our opinion, could be used in routine practice and provides a resource-saving innovation in differentiating glycogen from lipids in the same section of paraffin-embedded tissue sections.