Caloric Vestibular Stimulation Reduces Pain and Somatoparaphrenia in a Severe Chronic Central Post-Stroke Pain Patient: A Case Study

Central post-stroke pain is a neuropathic syndrome characterized by intolerable contralesional pain and, in rare cases, somatic delusions. To date, there is limited evidence for the effective treatments of this disease. Here we used caloric vestibular stimulation to reduce pain and somatoparaphrenia in a 57-year-old woman suffering from central post-stroke pain. Resting-state functional magnetic resonance imaging was used to assess the neurological effects of this treatment. Following vestibular stimulation we observed impressive improvements in motor skills, pain, and somatic delusions. In the functional connectivity study before the vestibular stimulation, we observed differences in the patient’s left thalamus functional connectivity, with respect to the thalamus connectivity of a control group (N = 20), in the bilateral cingulate cortex and left insula. After the caloric stimulation, the left thalamus functional connectivity with these regions, which are known to be involved in the cortical response to pain, disappeared as in the control group. The beneficial use of vestibular stimulation in the reduction of pain and somatic delusion in a CPSP patient is now documented by behavioral and imaging data. This evidence can be applied to theoretical models of pain and body delusions.     

Effects of cadmium on lymphocyte activation

The effects of cadmium (Cd) on phytohemoagglutinin or phorbol myristate acetate-induced lymphocyte activation were investigated and a dose-dependent inhibition of cell proliferation was found. Kinetic studies revealed that the Cd-sensitive step is an early event of T cell stimulation. Failure of IL2 secretion and reduction of IL2 receptor expression in the Cd-treated cells are also reported. Regardless of which mechanism is responsible for Cd effects, our studies show that the inhibition of lymphocyte activation is associated with reduced [3H]phorbol dibutyrate binding to Ca2+-phospholipid-dependent protein kinase and altered breakdown of phosphatidylinositols. Thus, Cd interferes with two biochemical events which play a critical role in lymphocyte signal transduction and activation.     

Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia

Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-α (SAT-α) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-α, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.     

Exploring the functional soil-microbe interface and exoenzymes through soil metaexoproteomics

Functionally important proteins at the interface of cell and soil are of potentially low abundance when compared with commonly recovered intracellular proteins. A novel approach was developed and used to extract the metaexoproteome, the subset of proteins found outside the cell, in the context of a soil enriched with the nitrogen-containing recalcitrant polymer chitin. The majority of proteins recovered was of bacterial origin and localized to the outer membrane or extracellular milieu. A wide variety of transporter proteins were identified, particularly those associated with amino-acid and phosphate uptake. The metaexoproteome extract retained chitinolytic activity and we were successful in detecting Nocardiopsis-like chitinases that correlated with the glycoside hydrolase family 18 (GH18) chi gene data and metataxonomic analysis. Nocardiopsis-like chitinases appeared to be solely responsible for chitinolytic activity in soil. This is the first study to detect and sequence bacterial exoenzymes with proven activity in the soil enzyme pool.Metaproteomics is an emerging technique for directly assessing cellular function and interactions within an environment. In complex environments such as soil, there is a vast dynamic range of microbial species abundance and protein expression levels. Data acquisition is biased towards high-abundance proteins, for example, chaperonins, ribosomal proteins, elongation factors and ATP synthases (Benndorf et al., 2007; Dill et al., 2010). Removal of these intracellular proteins could allow access to functionally important low-abundance proteins in the soil enzyme pool and at the interface of cell and soil, the soil metaexoproteome.Chitin provides one of the dominant sources of organic nitrogen in soil (Gooday, 1990) and chitinases are implicated in its mineralization in a wide range of contexts (Rhazi et al., 2000; Muzzarelli, 2011), especially in nitrogen-poor soils (Olander and Vitousek, 2000). The molecular diversity of chitinases in soil microbial communities has been studied (Williamson et al., 2000; Metcalfe et al., 2002; Hjort et al., 2010) but very few have focused on the functional contributions of members of the chitinolytic bacterial community. We report here the first attempt to recover and analyse extracellular proteins in soil adopting a novel approach to extract the metaexoproteome. Our data indicate that one actinobacterial group was disproportionately responsible for chitin breakdown.Soil was sampled from an island off the north coast of Cuba known for its high biodiversity and wide range of chitinolytic bacteria (Williamson et al., 2000; Williamson, 2001). Microcosms were constructed and amended with 1% crude crab shell (α-chitin) or squid pen (β-chitin) to enrich the microbial community, an unamended control was included for the 16S rRNA gene metataxonomic analysis (Supplementary Method S1). Community DNA was extracted and sequenced on a 454 GS FLX instrument with titanium reagents (Roche, Basel, Switzerland) using eubacterial primers Gray28F and Gray519R (Dowd et al., 2008) and GH18 Group A chi primers GASQF and GASQR (Williamson et al., 2000); the data were analysed with the bioinformatics package QIIME (Caporaso et al., 2010) (Supplementary Method S2). The metaexoproteome extraction is a modification of Masciandaro et al. (2008). In brief, 100 g soil was gently agitated with a K₂SO₄-based extraction solution (1:3 w/v) then the solid fraction and cells removed by centrifugation and filter sterilization before dilution (3:1 v/v) with 18.2 MΩ cm water and dialysis overnight. The retentate was concentrated to a final volume of ∼1 ml by ultrafiltration and using a centrifugal concentrator for direct loading onto a one-dimensional SDS–polyacrylamide gel electrophoresis gel. Gel-dependent nanoflow liquid chromatography-tandem MS (nanoLC-MS/MS) analysis was performed and the resultant Micromass peak list files interrogated with the NCBInr database using the MASCOT search engine (Matrix Science, London, UK). The full list of proteins was filtered to remove the few eukaryotic proteins and hits with <2 significant unique peptides (Supplementary Methods S3 and S4).To successfully target the metaexoproteome, cell integrity must be maintained. Minimal cell lysis during the extraction was demonstrated experimentally by spiking soil with Escherichia coli overexpressing His-tagged phosphoribosyl isomerase A in the cytoplasm and attempting to detect the His-tag in the extract by western blot (Supplementary Method S5), as no protein was detected we believe the method did not lyse cells. The majority of 52 recovered proteins were Gram-negative in origin and attributed to the extracellular fraction or outer membrane (Supplementary Tables S1 and S2). Across both amendments, 73% of proteins were predicted to have a signal peptide (Nielsen et al., 1997), 13% to have transmembrane helices (Sonnhammer et al., 1998; Krogh et al., 2001), 17% to be TRAP transporters and 52% to be ABC transporters. These features are suggestive of export or being membrane bound and indicate that the metaexoproteome is representing the functional interface between cell and environment. In vitro secretomes commonly feature a similar range of TRAP and ABC transporters in addition to selected extracellular enzymes depending on the enrichment (Adav et al., 2010; Christie-Oleza and Armengaud, 2010; Christie-Oleza et al., 2012). The only extracellular enzymes identified were chitinases.Recovered proteins were affiliated with three phyla, Proteobacteria, Actinobacteria and Bacteroidetes, this correlated well with the 16S rRNA gene data set (Figure 1). Only two genera dominated the metaexoproteome, both in terms of number of proteins recovered and protein abundance measured by emPAI (Ishihama et al., 2005), the actinomycete Nocardiopsis and the rhizobiale Nitratireductor. Approximately 17% of the identified proteins were matched to Nocardiopsis. The family Nocardiopsaceae was undetected in the unamended 16S rRNA gene data set but was one of the few actinobacterial groups to increase in abundance with α-chitin amendment, accounting for 3.7% of the community.Open in a separate windowFigure 1A visual summary of the assigned bacterial community structure, recovered metaexoproteome community and GH18 chi gene taxonomic matches for the combined α- and β-chitin-amended soil. For clarity, low-abundance taxa have been grouped under ‘Other'' and for the GH18 chi gene pie chart Stenotrophomonas, Amycolatopsis and Verrucosispora are not labelled as each account for <0.06% of their respective class segment.The majority of proteins were related to the transport and metabolism of amino acids, carbohydrates and inorganic ions, namely phosphate and phosphonate. Two GH18 chitinases were identified by peptides from within their catalytic domains, ChiA from Nocardiopsis lucentensis and N. dassonvillei (Supplementary Table S1). Corresponding Nocardiopsis chiA-like sequences were identified in the GH18 chi gene pyrosequencing data set (Figure 1). Nocardiopsis chitinases have been shown to have chitinolytic activity against α- and β-chitin (Tsujibo et al., 2003) and to be capable of fast and complete degradation of crystalline chitin in liquid media (Sorokin et al., 2012).A fluorogenic chitinase assay (Sigma-Aldrich, St Louis, MO, USA) was performed on the extracts from α-chitin-amended microcosm soil and metaexoproteome (Supplementary Method S6). Both extracts showed activity against the monomeric substrate but the metaexoproteome extract had proportionally higher activity against the more representative di-NAG and tri-NAG substrates. It is probable that the chitinase activity detected in the metaexoproteome extract is attributable to the Nocardiopsis chiA-like chitinases detected in the sequenced aliquot of the extract and represents the first example of an active exoenzyme extracted, assayed and sequenced from a soil.The efficiency of mass spectrometry via in-gel digestion would preclude recovery of low-abundance peptides. Nocardiopsis-like proteins must therefore contribute disproportionately to the functional activity of the soil and thus the degradation of chitin. This is in marked contrast to the prevalence data for 16S rRNA gene analysis and GH18 chi gene analysis. Despite numerous attempts it was not possible to cultivate Nocardiopsis-like strains directly from the soil.     

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.Supplementary key words: frataxin, ceramides, fatty acids oxidation, triglycerides, phospholipids, lipidomics, lipid remodeling, neurodegenerative disorders, triplet repeat expansion, stable isotope tracing

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder with an incidence of 1 in 29,000 (1). Currently it has no approved treatment (1). The main clinical features in FRDA include gait and limb ataxia, dysarthria, sensory loss, and cardiomyopathy (2). Heart failure from cardiomyopathy is the primary cause of death in the majority of patients with FRDA (3). FRDA is caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene that leads to gene silencing and decreased levels of the mitochondrial protein frataxin (4). The number of GAA repeats inversely correlates with frataxin protein level and age of disease onset, both of which determine disease severity (5, 6). The tissues most affected are the heart, dorsal root ganglia, posterior columns of the spinal cord, dentate nucleus, and corticospinal tracts. The exact mechanism by which frataxin deficiency leads to neuro- and cardiodegeneration is not completely understood.One function of frataxin is in the formation of the iron-sulfur (Fe-S) cluster prosthetic groups that are critical for enzymes in the Krebs cycle (aconitase), oxidative phosphorylation (electron transport chain components of complexes I–III), and fatty acid breakdown (β-oxidation) (7, 8). Frataxin localization in the mitochondria (9) further suggests that mitochondrial dysfunction plays a role in FRDA. Decreased conversion of labeled glucose to acetyl-CoA in platelets from patients with FRDA (10) is consistent with studies that show diminished pyruvate oxidation in FRDA (10). Increased incorporation of labeled palmitate into HMG-CoA, an important intermediate in ketogenesis and sterol synthesis, in patients with FRDA suggests increased fatty acid metabolism through β-oxidation (11). Increased β-oxidation produces FADH₂ and NADH that can be utilized to maintain the electrochemical gradient across the inner mitochondrial membrane needed for ATP synthesis. Therefore, increased lipid metabolism observed in FRDA could be important to maintain cellular homeostasis during mitochondrial dysfunction.A recent study found reactive oxygen species-independent accumulation of iron in the nervous system of an FRDA fly model with a mutant frataxin homolog, associated with enhanced sphingolipid synthesis (12). Sphingolipids are linked to increased inflammation (13) and activate 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2) to trigger neurodegeneration (12). The findings in the fly model were replicated in a frataxin knockdown mouse model suggesting that the mechanism is evolutionarily conserved (14). PDK1 activity and sphingolipid levels were also elevated in heart tissues of patients with FRDA compared with healthy controls suggesting that a similar pathway may be activated in humans with FRDA (14).Ceramides are central intermediates in sphingolipid metabolism and have been implicated in several cellular processes including apoptosis (15). Dysregulated ceramides have been the focus of study in a variety of cardiac diseases. High ceramide ratios of Cer 16:0 and 18:0 to Cer 24:0 in plasma are strongly associated with increased risk for major adverse cardiac events (16). Furthermore, increased ceramide levels have been associated with diabetic cardiomyopathy (17) and increased de novo ceramide synthesis has been linked to advanced heart failure (18). The observation of elevated ceramides in FRDA heart tissue raises the question of whether sphingolipids will be dysregulated in other affected and nonaffected tissues.Ideally, metabolic and lipidomic abnormalities should be studied in the most affected tissues, but frataxin deficiency is present in all tissues to different extents (19). Since it is difficult to sample human cardiac tissue from living individuals, peripheral tissues, such as fibroblasts, can be used as models to study metabolic profiles of FRDA. Fibroblasts in culture have the additional advantage of not being influenced by diet or environment, thus providing a stable system for comparing metabolic flux between patients and controls. Recently, RNA sequencing and gene ontology analysis was used to identify differentially expressed genes between FRDA and healthy control fibroblasts and indicated that fibroblasts are an accessible system to study dysregulated pathways in FRDA (20). In the present study, we used highly sensitive and specific liquid chromatography-high resolution mass spectrometry (LC-HRMS) assays to perform metabolomic and lipidomic profiles in fibroblast cells from patients with FRDA with different disease severities. This study complements the RNA sequencing data and gives new insights into the disease mechanism.     

Biological effects of the dual phenotypic Janus mutation of ret cosegregating with both multiple endocrine neoplasia type 2 and Hirschsprung's disease

Gain-of-function mutations of ret receptor tyrosine kinase, the signaling receptor for glial cell line-derived neurotrophic factor, cause sporadic thyroid and adrenal malignancies as well as endocrine cancer syndromes, such as multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma. Loss-of-function mutations of ret cause Hirschsprung's disease (HSCR) or colonic aganglionosis. In 20-30% of families with a mutation at residues 609, 611, 618, or 620 of RET, MEN 2A and familial medullary thyroid carcinoma cosegregate with HSCR. These mutations constitutively activate RET due to aberrant disulfide homodimerization and diminish the level of RET at the plasma membrane. It is not known how these mutations simultaneously lead to both gain- and loss-of-function RET-associated diseases. We provide an explanation for the dual phenotypic Janus mutation at Cys620 of RET. In Madin-Darby canine kidney (MDCK) cells, the Janus mutation impairs the glial cell line-derived neurotrophic factor-induced effects of RET on cell migration, differentiation, and survival but simultaneously promotes rapid cell proliferation.     

Red wine consumption may affect sperm biology: The effects of different concentrations of the phytoestrogen Myricetin on human male gamete function

Myricetin is a natural flavonoid, particularly enriched in red wines, whose occurrence is widespread among plants. Despite extensive research, the beneficial effects of Myricetin on human health are still controversial. Here, we tested the estrogen‐like effect of the phytoestrogen Myricetin on human ejaculated sperm biology. To this aim, human normozoospermic samples were exposed to increasing concentrations (10 nM, 100 nM, and 1 µM) of Myricetin. Motility, viability, capacitation‐associated biochemical changes (i.e., cholesterol efflux and tyrosine phosphorylation), acrosin activity, as well as glucose utilization and fatty‐acid oxidation (i.e., glucose and lipid metabolism) were all significantly increased by low doses of Myricetin. Importantly, both estrogen receptors α and β (ERs) and phosphatidylinositol‐3‐OH kinase (PI3K)/AKT signaling are activated in the presence of Myricetin since these were both abrogated by specific inhibitors of each pathway. Our results show how Myricetin, through ERs and PI3K/AKT signalings, potentiates sperm function. This effect is dose‐dependent at low concentrations of Myricetin (up to 100 nM), whereas higher amounts do not seem to improve any further sperm motility, viability, or other tested features, and, in some cases, they reduced or even abrogated the efficacy exerted by lower doses. Further studies are needed to elucidate if high levels of Myricetin, which could be attained even with moderate wine consumption, could synergize with endogenous estrogens in the female reproductive tract, interfering with the physiological sperm fertilization process. Mol. Reprod. Dev. © 2012 Wiley Periodicals, Inc.