Multi-Method Quantification of Acetyl-Coenzyme A and Further Acyl-Coenzyme A Species in Normal and Ischemic Rat Liver.

Thioesters of coenzyme A (CoA) carrying different acyl chains (acyl-CoAs) are central intermediates of many metabolic pathways and donor molecules for protein lysine acylation. Acyl-CoA species largely differ in terms of cellular concentrations and physico-chemical properties, rendering their analysis challenging. Here, we compare several approaches to quantify cellular acyl-CoA concentrations in normal and ischemic rat liver, using HPLC and LC-MS/MS for multi-acyl-CoA analysis, as well as NMR, fluorimetric and spectrophotometric techniques for the quantification of acetyl-CoAs. In particular, we describe a simple LC-MS/MS protocol that is suitable for the relative quantification of short and medium-chain acyl-CoA species. We show that ischemia induces specific changes in the short-chain acyl-CoA relative concentrations, while mild ischemia (1-2 min), although reducing succinyl-CoA, has little effects on acetyl-CoA, and even increases some acyl-CoA species upstream of the tricarboxylic acid cycle. In contrast, advanced ischemia (5-6 min) also reduces acetyl-CoA levels. Our approach provides the keys to accessing the acyl-CoA metabolome for a more in-depth analysis of metabolism, protein acylation and epigenetics.

Nuclear-specific gene expression in heterokaryons of the filamentous ascomycete Neurospora tetrasperma.

Heterokaryosis is a system in which genetically distinct nuclei coexist within the same cytoplasm. While heterokaryosis dominates the life cycle of many fungal species, the transcriptomic changes associated with the transition from homokaryosis to heterokaryosis is not well understood. Here, we analyse gene expression profiles of homokaryons and heterokaryons from three phylogenetically and reproductively isolated lineages of the filamentous ascomycete Neurospora tetrasperma. We show that heterokaryons are transcriptionally distinct from homokaryons in the sexual stage of development, but not in the vegetative stage, suggesting that the phenotypic switch to fertility in heterokaryons is associated with major changes in gene expression. Heterokaryon expression is predominantly defined by additive effects of its two nuclear components. Furthermore, allele-specific expression analysis of heterokaryons with varying nuclear ratios show patterns of expression ratios strongly dependent on nuclear ratios in the vegetative stage. By contrast, in the sexual stage, strong deviations of expression ratios indicate a co-regulation of nuclear gene expression in all three lineages. Taken together, our results show two levels of expression control: additive effects suggest a nuclear level of expression, whereas co-regulation of gene expression indicate a heterokaryon level of control.

Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication.

The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.

Astrocytes are key but indirect contributors to the development of the symptomatology and pathophysiology of Huntington's disease.

Huntington's disease (HD) is a fatal neurodegenerative disease in which an early and selective vulnerability of striatal Spiny Projection Neurons is observed. However, several studies have highlighted the implication of glial cells, and in particular astrocytes, in the pathophysiological mechanisms of this disease. A better understanding of the respective contributions of neurons and astrocytes in HD is needed and would be important for the development of new therapeutic approaches. Today, no comparable in vivo models expressing the mutant HTT selectively in astrocytes or in neurons are available. In this study, we developed comparable cell-type specific mouse models expressing a fragment of Huntingtin specifically in neurons, astrocytes, or in both cell populations of the adult mouse basal ganglia circuit. This approach allowed us to characterize behavioral alterations occurring as soon as 4 weeks postinjection. Interestingly, less severe but significant behavioral alterations were also observed in the two cell-type specific models. We further showed that astrocytes are less affected by mHTT compared to neurons, in particular concerning mHTT aggregation. Additionally, a more indirect contribution of astrocytes compared to neurons was observed in several pathophysiological mechanisms such as astrogliosis and neuronal dysfunction. Finally, we showed that direct and indirect transcriptional alterations within the glial glutamatergic clearing system are caused by astrocytic and neuronal expression of mHTT, respectively. We anticipate that our study will help to better understand the contributions of astrocytes to HD and guide future therapeutic efforts. GLIA 2016;64:1841-1856.

Agreement of seven 25-hydroxy vitamin D3 immunoassays and three high performance liquid chromatography methods with liquid chromatography tandem mass spectrometry.

BACKGROUND: Several recent studies have shown some discrepancies between 25-hydroxyvitamin D [25(OH)D] assay methods, despite some improvement in the past few years. The accuracy of 25(OH)D assay methods is still a real challenge for clinical laboratories. The aim of this study was to assess the agreement between a large panel of routine assays and a two-dimensional liquid chromatography/tandem mass spectrometry (2D LC-MS/MS) method, selected as the reference method. METHODS: Forty-nine human plasma samples with only endogenous 25(OH)D3 were analyzed with 11 different methods, especially with three LC-UV methods that differed in the extraction step. Seven routine immunoassays were also tested: two manual (RIA and EIA from IDS) and five fully-automated methods. The results of the 25(OH)D3 assays were compared with those of the 2D LC-MS/MS method using weighted Deming regression analysis, Bland-Altman plots and concordance correlation coefficient (CCC). The ability of these methods to properly classify patients was evaluated by sorting results depending on vitamin D status. RESULTS: The CCC was >0.90 for the three LC-UV methods and for most of the automated IA, meaning substantial agreement with 2D LC-MS/MS results. The ability to properly classify patients according to their vitamin D status was overall satisfactory for most of the methods tested (concordance >90%). CONCLUSIONS: The immunoassays available on Liaison, Isys, Architect and Elecsys, together with our in-house LC-UV method preceded by an SLE step met the minimum requirements for the assessment of vitamin D status in clinical laboratories.

[Role and interest of hepcidin in iron homeostasis]. / Rôle et intérêt du dosage de l'hepcidine dans l'homéostasie du fer.

Iron is an essential element to the well functionning of the organism and requires careful maintenance of its homeostasis. This is mainly due to hepcidin, a hormone secreted by the liver that controls the flow of iron within the body. It has a hyposideremic action by reducing the expression of ferroportin, the only protein known to this day, which can export iron into the extracellular environment. This has the effect of decreasing intestinal absorption and increasing intracellular retention, especially in macrophages. Hepcidin is stimulated by elevation of iron and inflammation while activation of erythropoiesis inhibits it. Understanding its regulation allows a better understanding of the pathophysiological mechanisms of overload diseases and iron deficiency. Therefore, hepcidin analysis is interesting for the exploration of iron homeostasis. In combination with other biological parameters of iron status, it is possible to find better instructions for therapeutic managements of iron metabolism diseases. Thus, an assaying method by coupling liquid chromatography and tandem mass spectrometry has been implemented at Grenoble University Hospital and the analytical performances of this assay met the laboratory requirements in terms of reliability of the analysis.

Seed dormancy distribution: explanatory ecological factors.

BACKGROUND AND AIMS: Knowledge of those traits that vary with latitude should be helpful in predicting how they may evolve locally under climate change. In the sea beet Beta vulgaris ssp. maritima, seed dormancy largely controls the timing of germination, is highly heritable and varies geographically; it is therefore thought to be selected by climate. The aim here was to characterize the variation in seed dormancy among sea beet populations across the French distribution area, as well as the ecological factors in situ that are correlated with and that could therefore select for seed dormancy. The relative importance of genetic inheritance vs. non-genetic variation is also evaluated. METHODS: The proportions of dormant seeds from 85 natural populations encompassing different climates over the whole French distribution area were measured under controlled conditions. Germination phenology was observed in a common garden experiment. Dormancy variation of seeds collected in situ was compared with that of seeds collected on plants grown in the greenhouse. KEY RESULTS: The proportions of dormant seeds in the greenhouse were highly variable, covering almost the entire range from 0 to 1, and followed a geographical pattern from lower dormancy at high latitudes to high dormancy at low latitudes. The distribution of dormancy was positively correlated with yearly temperatures, especially summer temperatures. Minimum temperatures in winter did not significantly explain the trait variation. The genetic component of the total variation was significant and is probably completed by an important adjustment to the local conditions brought about by maternal adaptive phenotypic plasticity. CONCLUSIONS: Dormancy in sea beet could be interpreted as a way to limit summer germination and spread germination over the first autumn and spring or following autumns. This highly heritable trait has the potential to evolve in the relatively near future because of climate change.

Untargeted metabolomics approach to discriminate mistletoe commercial products.

Mistletoe (Viscum album L.) is used in German-speaking European countries in the field of integrative oncology linking conventional and complementary medicine therapies to improve quality of life. Various companies sell extracts, fermented or not, for injection by subcutaneous or intra-tumoral route with a regulatory status of anthroposophic medicinal products (European Medicinal Agency (EMA) assessment status). These companies as well as anthroposophical physicians argue that complex matrices composed of many molecules in mixture are necessary for activity and that the host tree of the mistletoe parasitic plant is the main determining factor for this matrix composition. The critical point is that parenteral devices of European mistletoe extracts do not have a standard chemical composition regulated by EMA quality guidelines, because they are not drugs, regulatory speaking. However, the mechanism of mistletoe's anticancer activity and its effectiveness in treating and supporting cancer patients are not fully understood. Because of this lack of transparency and knowledge regarding the matrix chemical composition, we undertook an untargeted metabolomics study of several mistletoe extracts to explore and compare their fingerprints by LC-(HR)MS(/MS) and 1H-NMR. Unexpectedly, we showed that the composition was primarily driven by the manufacturer/preparation method rather than the different host trees. This differential composition may cause differences in immunostimulating and anti-cancer activities of the different commercially available mistletoe extracts as illustrated by structure-activity relationships based on LC-MS/MS and 1H-NMR identifications completed by docking experiments. In conclusion, in order to move towards an evidence-based medicine use of mistletoe, it is a priority to bring rigor and quality, chemically speaking.

Comparative analysis of genome-wide DNA methylation in Neurospora.

DNA methylation is an epigenetic mark that plays an important role in genetic regulation in eukaryotes. Major progress has been made in dissecting the molecular pathways that regulate DNA methylation. Yet, little is known about DNA methylation variation over evolutionary time. Here we present an investigation of the variation of DNA methylation and transposable element (TE) content in species of the filamentous ascomycetes Neurospora. We generated genome-wide DNA methylation data at single-base resolution, together with genomic TE content and gene expression data, of 10 individuals representing five closely related Neurospora species. We found that the methylation levels were low (ranging from 1.3% to 2.5%) and varied among the genomes in a species-specific way. Furthermore, we found that the TEs over 400 bp long were targeted by DNA methylation, and in all genomes, high methylation correlated with low GC, confirming a conserved link between DNA methylation and Repeat Induced Point (RIP) mutations in this group of fungi. Both TE content and DNA methylation pattern showed phylogenetic signal, and the species with the highest TE load (N. crassa) also exhibited the highest methylation level per TE. Our results suggest that DNA methylation is an evolvable trait and indicate that the genomes of Neurospora are shaped by an evolutionary arms race between TEs and host defence.

Cell-Type-Specific Gene Expression Profiling in Adult Mouse Brain Reveals Normal and Disease-State Signatures.

The role of brain cell-type-specific functions and profiles in pathological and non-pathological contexts is still poorly defined. Such cell-type-specific gene expression profiles in solid, adult tissues would benefit from approaches that avoid cellular stress during isolation. Here, we developed such an approach and identified highly selective transcriptomic signatures in adult mouse striatal direct and indirect spiny projection neurons, astrocytes, and microglia. Integrating transcriptomic and epigenetic data, we obtained a comprehensive model for cell-type-specific regulation of gene expression in the mouse striatum. A cross-analysis with transcriptomic and epigenomic data generated from mouse and human Huntington's disease (HD) brains shows that opposite epigenetic mechanisms govern the transcriptional regulation of striatal neurons and glial cells and may contribute to pathogenic and compensatory mechanisms. Overall, these data validate this less stressful method for the investigation of cellular specificity in the adult mouse brain and demonstrate the potential of integrative studies using multiple databases.

Human Induced Pluripotent Stem Cell-Derived Astrocytes Are Differentially Activated by Multiple Sclerosis-Associated Cytokines.

Recent studies highlighted the importance of astrocytes in neuroinflammatory diseases, interacting closely with other CNS cells but also with the immune system. However, due to the difficulty in obtaining human astrocytes, their role in these pathologies is still poorly characterized. Here, we develop a serum-free protocol to differentiate human induced pluripotent stem cells (hiPSCs) into astrocytes. Gene expression and functional assays show that our protocol consistently yields a highly enriched population of resting mature astrocytes across the 13 hiPSC lines differentiated. Using this model, we first highlight the importance of serum-free media for astrocyte culture to generate resting astrocytes. Second, we assess the astrocytic response to IL-1ß, TNF-α, and IL-6, all cytokines important in neuroinflammation, such as multiple sclerosis. Our study reveals very specific profiles of reactive astrocytes depending on the triggering stimulus. This model provides ideal conditions for in-depth and unbiased characterization of astrocyte reactivity in neuroinflammatory conditions.

The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes.

Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases.

Hemangiosarcomas, medulloblastomas, and other tumors in Ink4c/p53-null mice.

Ink4 proteins inhibit the enzymatic activities of cyclin D-dependent kinases, thereby governing transcriptional programs that depend on the activities of the retinoblastoma protein and other retinoblastoma family members (p107 and p130). Mice lacking Ink4c and p53 spontaneously develop a broad spectrum of neoplasms, usually presenting with multiple tumors of different histological types and dying of cancer by 6 months of age. Whereas thymic lymphomas or pituitary tumors predominate in mice lacking p53 or Ink4c, respectively, animals lacking both genes develop many vascular tumors and also present with medulloblastomas not observed in the parental strains. Unlike p53, loss of the Arf tumor suppressor did not contribute to the appearance of vascular or cerebellar tumors. Vascular tumors ranged in severity from angiomas to hemangiosarcomas, some of which could be transplanted into immunocompromised mice. Intriguingly, loss of Ink4c but maintenance of at least one Ink4d allele was required for formation of medulloblastomas in p53-null mice. In situ hybridization revealed that, in newborn mice, Ink4c is detected in the pia mater and in an adjacent layer of rapidly dividing cells within the cerebellar external granule layer (EGL), whereas Ink4d is primarily expressed in Purkinje neurons. Because the pia mater and Purkinje cells sandwich the cerebellar EGL from which medulloblastomas are presumed to arise, Ink4 proteins might function in a cell-autonomous manner in governing neuronal cell cycle exit as well as in a non-cell-autonomous manner in controlling the production of diffusible mitogens and chemokines that influence postnatal development of the cerebellar EGL.

Four years of LC-MS/MS method for quantification of 25-hydroxyvitamin D (D2+D3) for clinical practice.

Many methods for routine total plasma/serum 25-hydroxyvitamin D (25OHD) measurements are available from automated immunoassays to the most specific LC-MS/MS techniques. These last ones are nowadays numerous, still perfectible but more powerful than immunoassays in specific illnesses. We presented a robust method for simple quantification of 25-hydroxyvitamin D(2) (25OHD(2)) and 25-hydroxyvitamin D(3) (25OHD(3)) in human plasma by LC-APCI-MS/MS. This method is reliable and easy to perform for clinical measurements as we report a 4-year of clinical laboratory use. A brief off-line sample pretreatment (protein precipitation with addition of the internal standard) was realized then the supernatant was loaded into 96-well plates and analyzed by an online SPE-LC/MS/MS method on an APCI mode. 25OHD(2) and 25OHD(3) were both measured. The chromatographic system was thought and optimized for providing a dedicated line for this measurement on a shared instrument. The linearity was tested up to 380 nmol/L for both 25OHD(2) and 25OHD(3). The limit of quantification (LOQ) was 7 and 8 nmol/L for 25OHD3 and 25OHD(2), respectively. Routine imprecision and bias were found in agreement with recommended limits for routine testing, CV≤10% and bias≤5%. Since July 2010, our participation to DEQAS was successfully validated. This simple robust online SPE-LC/MS/MS method is suitable for routine measurement of 25OHD(2) and 25OHD(3) in human adult plasma. The assay operates for 4 years and has performed more than 40,000 patient samples on a shared instrument.

Development and validation of a method using supported liquid extraction for aldosterone determination in human plasma by LC-MS/MS.

BACKGROUND: Accurate quantitation of aldosterone is essential for screening, diagnosis and subtype classification in primary aldosteronism. A simple, sensitive method for aldosterone in human plasma using supported liquid extraction (SLE) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and validated. METHODS: Plasma samples were diluted with water containing d7-aldosterone as internal standard. The samples were extracted with methyl-tert-butyl-ether (MTBE) on SLE cartridges. Separation was carried out on a Luna C18 (2) column using a methanol-water gradient. Detection was performed in the negative electrospray multiple reaction monitoring (MRM) quantitation. The use of water-based calibrators was evaluated against calibrators prepared in steroid-free serum. RESULTS: The assay was linear up to 3265pmol/L with an LOQ of approximately 40pmol/L. Within-run and between-run precision for plasma aldosterone were less than 10% except at low level near LOQ but were still less than 14.7% (Westgard's desirable specification). The mean recovery of the analyte added to plasma was greater than 97.7% and matrix effects were less than 4%. Comparison with another LC-MS/MS method was performed on a more sensitive instrument (ABSciex TQ 5500) and gave the equation API 3000=0.957×TQ 5500+12.6, linear regression r(2)=0.974 (n=43). An estimation of the reference interval for adults was established on a group of healthy volunteers (n=53). Calibration with water-based calibrators was validated and can be used for measurement of aldosterone by LC-MS/MS. CONCLUSIONS: This method is reliable, easy to perform on plasma specimens in a clinical environment and is attractive because of its simplicity.

Sex biased spatial strategies relying on the integration of multimodal cues in a rat model of schizophrenia: impairment in predicting future context?

Male and female Wistar rats were treated postnatally (PND 5-16) with BSO (l-buthionine-(S,R)-sulfoximine) to provide a rat model of schizophrenia based on transient glutathione deficit. In the watermaze, BSO-treated male rats perform very efficiently in conditions where a diversity of visual information is continuously available during orientation trajectories [1]. Our hypothesis is that the treatment impairs proactive strategies anticipating future sensory information, while supporting a tight visual adjustment on memorized snapshots, i.e. compensatory reactive strategies. To test this hypothesis, BSO rats' performance was assessed in two conditions using an 8-arm radial maze task: a semi-transparent maze with no available view on the environment from maze centre [2], and a modified 2-parallel maze known to induce a neglect of the parallel pair in normal rats [3-5]. Male rats, but not females, were affected by the BSO treatment. In the semi-transparent maze, BSO males expressed a higher error rate, especially in completing the maze after an interruption. In the 2-parallel maze shape, BSO males, unlike controls, expressed no neglect of the parallel arms. This second result was in accord with a reactive strategy using accurate memory images of the contextual environment instead of a representation based on integrating relative directions. These results are coherent with a treatment-induced deficit in proactive decision strategy based on multimodal cognitive maps, compensated by accurate reactive adaptations based on the memory of local configurations. Control females did not express an efficient proactive capacity in the semi-transparent maze, neither did they show the significant neglect of the parallel arms, which might have masked the BSO induced effect. Their reduced sensitivity to BSO treatment is discussed with regard to a sex biased basal cognitive style.

Ultra-fast cyclosporin A quantitation in whole blood by Laser Diode Thermal Desorption-tandem mass spectrometry; comparison with High Performance Liquid Chromatography-tandem mass spectrometry.

In the last decade the quantitation of immunosuppressive drugs has seen vast improvements in analytical methods, optimizing time, accuracy of analysis and cost. Laser Diode Thermal Desorption (LDTD) coupled to Atmospheric Pressure Chemical ionization-tandem mass spectrometry (APCI-MS/MS) represents a technological breakthrough that removes the chromatographic separation step and thereby significantly increases the analytical throughput for the quantitation of cyclosporin A (CsA) in whole blood for therapeutic drug monitoring (TDM). A simple protein precipitation step was used prior to depositing 5 µL of the extract on a 96-well LazWell™ plate and CsA was quantified by LDTD-APCI-MS/MS. The laser pattern was set to ramp from 0 to 45% laser power within 2 s. The APCI parameters were set to negative needle voltage (-2 µA), carrier gas temperature (30°C) and air flow rate (3 L min(-1)). The negative ion single reaction monitoring transitions for CsA and its internal standard cyclosporin D (CsD) were respectively m/z 1201.1/1088.9 and m/z 1214.8/1102.8; obtained with a collision energy of -40 V. The analysis was achieved within 9 s from sample to sample. The extraction procedure yielded high recovery (92%; RSD=9.4%, n=6). The lower limit of quantitation was fixed at the first level of calibration: 23.5 ng mL(-1) (accuracy=112.3%; RSD=9.6%; n=6) and a blank+6 point linear regression up to 965 ng mL(-1) was used. Using 4 levels of quality control (QC), intra-day assays (n=6) ranged from 93.5 to 95.7% (bias) and from 3.4 to 13.1% (RSD) while inter-day assays (n=6) ranged from 92.9 to 105.3% (bias) and from 4.9 to 7.5% (RSD). An inter-sample contamination of CsA of 2.3% was calculated that was considered negligible with respect to the range of CsA concentrations. Whole blood samples (120) from patients under CsA treatment were analyzed by LDTD-APCI-MS/MS and HPLC-ESI-MS/MS, the gold standard reference method for CsA quantification. Both methods agreed (P≥0.99), with a coefficient of correlation of 0.99 (95% confidence interval 0.982-0.991). The Passing-Bablok regression revealed no significant deviation from linearity (Cusum test, P=0.11). This method seems suitable for use in TDM of CsA.

Star fruit poisoning is potentially life-threatening in patients with moderate chronic renal failure.

PURPOSE: Star fruit intoxications have been reported mainly in uremic patients, leading to various degrees of neurological symptoms and potentially fatal outcomes. Nephrotoxicity has been reported in few patients with normal renal function or moderate chronic renal impairment (CRI). The present report describes clinical course, management, and outcome of six patients with moderate CRI admitted to ICU for severe star fruit intoxication. METHODS: Over a 1-year period we observed six cases of star fruit intoxication. All but two patients were prospectively monitored. For each case we collected clinical characteristics, management, and outcome. RESULTS: On admission, all patients presented acute renal failure with underlying moderate CRI and required intubation for coma. The most common symptoms were hiccups, mental confusion, seizures, and coma. Status epilepticus was authenticated in three patients. Management consisted of several methods of renal replacement therapy (RRT) and supportive measures. Four patients survived without sequelae and two patients died. CONCLUSIONS: Severe star fruit intoxication can occur in patients with moderate CRI with a potentially fatal outcome. Prompt continuous RRT should be instituted.