Efficacy of HDAC Inhibitors in Driving Peroxisomal ß-Oxidation and Immune Responses in Human Macrophages: Implications for Neuroinflammatory Disorders.

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by ß-oxidation. Impaired VLCFA catabolism due to primary or secondary peroxisomal alterations is featured in neurodegenerative and neuroinflammatory disorders such as X-linked adrenoleukodystrophy and multiple sclerosis (MS). Here, we identified that healthy human macrophages upregulate the peroxisomal genes involved in ß-oxidation during myelin phagocytosis and pro-inflammatory activation, and that this response is impaired in peripheral macrophages and phagocytes in brain white matter lesions in MS patients. The pharmacological targeting of VLCFA metabolism and peroxisomes in innate immune cells could be favorable in the context of neuroinflammation and neurodegeneration. We previously identified the epigenetic histone deacetylase (HDAC) inhibitors entinostat and vorinostat to enhance VLCFA degradation and pro-regenerative macrophage polarization. However, adverse side effects currently limit their use in chronic neuroinflammation. Here, we focused on tefinostat, a monocyte/macrophage-selective HDAC inhibitor that has shown reduced toxicity in clinical trials. By using a gene expression analysis, peroxisomal ß-oxidation assay, and live imaging of primary human macrophages, we assessed the efficacy of tefinostat in modulating VLCFA metabolism, phagocytosis, chemotaxis, and immune function. Our results revealed the significant stimulation of VLCFA degradation with the upregulation of genes involved in peroxisomal ß-oxidation and interference with immune cell recruitment; however, tefinostat was less potent than the class I HDAC-selective inhibitor entinostat in promoting a regenerative macrophage phenotype. Further research is needed to fully explore the potential of class I HDAC inhibition and downstream targets in the context of neuroinflammation.

Biomarker-based risk prediction for the onset of neuroinflammation in X-linked adrenoleukodystrophy.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is highly variable, ranging from slowly progressive adrenomyeloneuropathy to severe brain demyelination and inflammation (cerebral ALD, CALD) affecting males with childhood peak onset. Risk models integrating blood-based biomarkers to indicate CALD onset, enabling timely interventions, are lacking. Therefore, we evaluated the prognostic value of blood biomarkers in addition to current neuroimaging predictors for early detection of CALD. METHODS: We measured blood biomarkers in a retrospective, male CALD risk-assessment cohort consisting of 134 X-ALD patients and 66 controls and in a phenotype-blinded validation set (25 X-ALD boys, 4-13 years) using Simoa®and Luminex® technologies. FINDINGS: Among 25 biomarkers indicating axonal damage, astrocye/microglia activation, or immune-cell recruitment, neurofilament light chain (NfL) had the highest prognostic value for early indication of childhood/adolescent CALD. A plasma NfL cut-off level of 8.33 pg/mL, determined in the assessment cohort, correctly discriminated CALD with an accuracy of 96% [95% CI: 80-100] in the validation group. Multivariable logistic regression models revealed that combining NfL with GFAP or cytokines/chemokines (IL-15, IL-12p40, CXCL8, CCL11, CCL22, and IL-4) that were significantly elevated in CALD vs healthy controls had no additional benefit for detecting neuroinflammation. Some cytokines/chemokines were elevated only in childhood/adolescent CALD and already upregulated in asymptomatic X-ALD children (IL-15, IL-12p40, and CCL7). In adults, NfL levels distinguished CALD but were lower than in childhood/adolescent CALD patients with similar (MRI) lesion severity. Blood GFAP did not differentiate CALD from non-inflammatory X-ALD. INTERPRETATION: Biomarker-based risk prediction with a plasma NfL cut-off value of 8.33 pg/mL, determined by ROC analysis, indicates CALD onset with high sensitivity and specificity in childhood X-ALD patients. A specific pro-inflammatory cytokine/chemokine profile in asymptomatic X-ALD boys may indicate a primed, immanent inflammatory state aligning with peak onset of CALD. Age-related differences in biomarker levels in adult vs childhood CALD patients warrants caution in predicting onset and progression of CALD in adults. Further evaluations are needed to assess clinical utility of the NfL cut-off for risk prognosis of CALD onset. FUNDING: Austrian Science Fund, European Leukodystrophy Association.

Sciatic nerve fractional anisotropy and neurofilament light chain protein are related to sensorimotor deficit of the upper and lower limbs in patients with type 2 diabetes.

Background: Diabetic sensorimotor polyneuropathy (DSPN) is one of the most prevalent and poorly understood diabetic microvascular complications. Recent studies have found that fractional anisotropy (FA), a marker for microstructural nerve integrity, is a sensitive parameter for the structural and functional nerve damage in DSPN. The aim of this study was to investigate the significance of proximal sciatic nerve's FA on different distal nerve fiber deficits of the upper and lower limbs and its correlation with the neuroaxonal biomarker, neurofilament light chain protein (NfL). Materials and methods: Sixty-nine patients with type 2 diabetes (T2DM) and 30 healthy controls underwent detailed clinical and electrophysiological assessments, complete quantitative sensory testing (QST), and diffusion-weighted magnetic resonance neurography of the sciatic nerve. NfL was measured in the serum of healthy controls and patients with T2DM. Multivariate models were used to adjust for confounders of microvascular damage. Results: Patients with DSPN showed a 17% lower sciatic microstructural integrity compared to healthy controls (p<0.001). FA correlated with tibial and peroneal motor nerve conduction velocity (NCV) (r=0.6; p<0.001 and r=0.6; p<0.001) and sural sensory NCV (r=0.50; p<0.001). Participants with reduced sciatic nerve´s FA showed a loss of function of mechanical and thermal sensation of upper (r=0.3; p<0.01 and r=0.3; p<0.01) and lower (r=0.5; p<0.001 and r=0.3; p=<0.01) limbs and reduced functional performance of upper limbs (Purdue Pegboard Test for dominant hand; r=0.4; p<0.001). Increased levels of NfL and urinary albumin-creatinine ratio (ACR) were associated with loss of sciatic nerve´s FA (r=-0.5; p<0.001 and r= -0.3, p= 0.001). Of note, there was no correlation between sciatic FA and neuropathic symptoms or pain. Conclusion: This is the first study showing that microstructural nerve integrity is associated with damage of different nerve fiber types and a neuroaxonal biomarker in DSPN. Furthermore, these findings show that proximal nerve damage is related to distal nerve function even before clinical symptoms occur. The microstructure of the proximal sciatic nerve and is also associated with functional nerve fiber deficits of the upper and lower limbs, suggesting that diabetic neuropathy involves structural changes of peripheral nerves of upper limbs too.

Serum IFNα2 levels are associated with disease activity and outperform IFN-I gene signature in a longitudinal childhood-onset SLE cohort.

OBJECTIVE: To study the association of serum IFNα2 levels measured by ultrasensitive single-molecule array (Simoa) and the IFN-I gene signature (IGS) with disease activity and determine whether these assays can mark disease activity states in a longitudinal cohort of childhood-onset SLE (cSLE) patients. METHODS: Serum IFNα2 levels were measured in 338 samples from 48 cSLE patients and 67 healthy controls using an IFNα Simoa assay. Five-gene IGS was measured by RT-PCR in paired whole blood samples. Disease activity was measured by clinical SELENA-SLEDAI and BILAG-2004. Low disease activity was defined by Low Lupus Disease Activity State (LLDAS) and flares were characterized by SELENA-SLEDAI flare index. Analysis was performed using linear mixed models. RESULTS: A clear positive correlation was present between serum IFNα2 levels and the IGS (r = 0.78, P < 0.0001). Serum IFNα2 levels and IGS showed the same significant negative trend in the first 3 years after diagnosis. In this timeframe, mean baseline serum IFNα2 levels decreased by 55.1% (Δ 201 fg/ml, P < 0.001) to a mean value of 164 fg/ml, which was below the calculated threshold of 219.4 fg/ml that discriminated between patients and healthy controls. In the linear mixed model, serum IFNα2 levels were significantly associated with both cSELENA-SLEDAI and BILAG-2004, while the IGS did not show this association. Both IFN-I assays were able to characterize LLDAS and disease flare in receiver operating characteristic analysis. CONCLUSIONS: Serum IFNα2 levels measured by Simoa technology are associated with disease activity scores and characterize disease activity states in cSLE.

Cerebrospinal fluid concentration of complement component 4A is increased in first episode schizophrenia.

Postsynaptic density is reduced in schizophrenia, and risk variants increasing complement component 4A (C4A) gene expression are linked to excessive synapse elimination. In two independent cohorts, we show that cerebrospinal fluid (CSF) C4A concentration is elevated in patients with first-episode psychosis (FEP) who develop schizophrenia (FEP-SCZ: median 0.41 fmol/ul [CI = 0.34-0.45], FEP-non-SCZ: median 0.29 fmol/ul [CI = 0.22-0.35], healthy controls: median 0.28 [CI = 0.24-0.33]). We show that the CSF elevation of C4A in FEP-SCZ exceeds what can be expected from genetic risk variance in the C4 locus, and in patient-derived cellular models we identify a mechanism dependent on the disease-associated cytokines interleukin (IL)-1beta and IL-6 to selectively increase neuronal C4A mRNA expression. In patient-derived CSF, we confirm that IL-1beta correlates with C4A controlled for genetically predicted C4A RNA expression (r = 0.39; CI: 0.01-0.68). These results suggest a role of C4A in early schizophrenia pathophysiology.

Neurofilament proteins as a potential biomarker in chemotherapy-induced polyneuropathy.

BACKGROUNDPaclitaxel chemotherapy frequently induces dose-limiting sensory axonal polyneuropathy. Given that sensory symptoms are challenging to assess objectively in clinical practice, an easily accessible biomarker for chemotherapy-induced polyneuropathy (CIPN) holds the potential to improve early diagnosis. Here, we describe neurofilament light chain (NFL), a marker for neuroaxonal damage, as a translational surrogate marker for CIPN.METHODSNFL concentrations were measured in an in vitro model of CIPN, exposing induced pluripotent stem cell-derived sensory neurons (iPSC-DSNs) to paclitaxel. Patients with breast or ovarian cancer undergoing paclitaxel chemotherapy, breast cancer control patients without chemotherapy, and healthy controls were recruited in a cohort study and examined before chemotherapy (V1) and after 28 weeks (V2, after chemotherapy). CIPN was assessed by the validated Total Neuropathy Score reduced (TNSr), which combines patient-reported symptoms with data from clinical examinations. Serum NFL (NFLs) concentrations were measured at both visits with single-molecule array technology.RESULTSNFL was released from iPSC-DSNs upon paclitaxel incubation in a dose- and time-dependent manner and was inversely correlated with iPSC-DSN viability. NFLs strongly increased in paclitaxel-treated patients with CIPN, but not in patients receiving chemotherapy without CIPN or controls, resulting in an 86% sensitivity and 87% specificity. An NFLs increase of +36 pg/mL from baseline was associated with a predicted CIPN probability of more than 0.5.CONCLUSIONNFLs was correlated with CIPN development and severity, which may guide neurotoxic chemotherapy in the future.TRIAL REGISTRATIONClinicalTrials.gov NCT02753036.FUNDINGDeutsche Forschungsgemeinschaft (EXC 257 NeuroCure), BMBF (Center for Stroke Research Berlin, 01 EO 0801), Animalfree Research, EU Horizon 2020 Innovative Medicines Initiative 2 Joint Undertaking (TransBioLine, 821283), Charité 3R - Replace - Reduce - Refine.

Serum interferon-α2 measured by single-molecule array associates with systemic disease manifestations in Sjögren's syndrome.

OBJECTIVES: Type I IFN (IFN-I) activation is a prominent feature of primary SS (pSS), SLE and SSc. Ultrasensitive single-molecule array (Simoa) technology has facilitated the measurement of subfemtomolar concentrations of IFNs. Here we aimed to measure IFN-α2 in serum from pSS, SLE and SSc using a Simoa immunoassay and correlate these levels to blood IFN-stimulated gene (ISG) expression and disease activity. METHODS: Serum IFN-α2 was measured in patients with pSS (n = 85 and n = 110), SLE (n = 24) and SSc (n = 23) and healthy controls (HCs; n = 68) using an IFN-α Simoa assay on an HD-X analyser. IFN-I pathway activation was additionally determined from serum by an IFN-I reporter assay and paired samples of whole blood ISG expression of IFI44, IFI44L, IFIT1, IFIT3 and MxA by RT-PCR or myxovirus resistance protein 1 (MxA) protein ELISA. RESULTS: Serum IFN-α2 levels were elevated in pSS (median 61.3 fg/ml) compared with HCs (median ≤5 fg/ml, P < 0.001) and SSc (median 11.6 fg/ml, P = 0.043), lower compared with SLE (median 313.5 fg/ml, P = 0.068) and positively correlated with blood ISG expression (r = 0.66-0.94, P < 0.001). Comparable to MxA ELISA [area under the curve (AUC) 0.93], IFN-α2 measurement using Simoa identified pSS with high ISG expression (AUC 0.90) with 80-93% specificity and 71-84% sensitivity. Blinded validation in an independent pSS cohort yielded a comparable accuracy. Multiple regression indicated independent associations of autoantibodies, IgG, HCQ treatment, cutaneous disease and a history of extraglandular manifestations with serum IFN-α2 concentrations in pSS. CONCLUSION: Simoa serum IFN-α2 reflects blood ISG expression in pSS, SLE and SSc. In light of IFN-targeting treatments, Simoa could potentially be applied for patient stratification or retrospective analysis of historical cohorts.

Neuron-specific biomarkers predict hypo- and hyperalgesia in individuals with diabetic peripheral neuropathy.

AIMS/HYPOTHESIS: The individual risk of progression of diabetic peripheral neuropathy is difficult to predict for each individual. Mutations in proteins that are responsible for the process of myelination are known to cause neurodegeneration and display alteration in experimental models of diabetic neuropathy. In a prospective observational human pilot study, we investigated myelin-specific circulating mRNA targets, which have been identified in vitro, for their capacity in the diagnosis and prediction of diabetic neuropathy. The most promising candidate was tested against the recently established biomarker of neural damage, neurofilament light chain protein. METHODS: Schwann cells were cultured under high-glucose conditions and mRNAs of various myelin-specific genes were screened intra- and extracellularly. Ninety-two participants with type 2 diabetes and 30 control participants were enrolled and evaluated for peripheral neuropathy using neuropathy deficit scores, neuropathy symptom scores and nerve conduction studies as well as quantitative sensory testing at baseline and after 12/24 months of a follow-up period. Magnetic resonance neurography of the sciatic nerve was performed in 37 individuals. Neurofilament light chain protein and four myelin-specific mRNA transcripts derived from in vitro screenings were measured in the serum of all participants. The results were tested for associations with specific neuropathic deficits, fractional anisotropy and the progression of neuropathic deficits at baseline and after 12 and 24 months. RESULTS: In neuronal Schwann cells and human nerve sections, myelin protein zero was identified as the strongest candidate for a biomarker study. Circulating mRNA of myelin protein zero was decreased significantly in participants with diabetic neuropathy (p < 0.001), whereas neurofilament light chain protein showed increased levels in participants with diabetic neuropathy (p < 0.05). Both variables were linked to altered electrophysiology, fractional anisotropy and quantitative sensory testing. In a receiver-operating characteristic curve analysis myelin protein zero improved the diagnostic performance significantly in combination with a standard model (diabetes duration, age, BMI, HbA1c) from an AUC of 0.681 to 0.836 for the detection of diabetic peripheral neuropathy. A follow-up study revealed that increased neurofilament light chain was associated with the development of a hyperalgesic phenotype (p < 0.05), whereas decreased myelin protein zero predicted hypoalgesia (p < 0.001) and progressive loss of nerve function 24 months in advance (HR of 6.519). CONCLUSIONS/INTERPRETATION: This study introduces a dynamic and non-invasive assessment strategy for the underlying pathogenesis of diabetic peripheral neuropathy. The diagnosis of axonal degeneration, associated with hyperalgesia, and demyelination, linked to hypoalgesia, could benefit from the usage of neurofilament light chain protein and circulating mRNA of myelin protein zero as potential biomarkers.

Comparison of Bead-Based Fluorescence Versus Planar Electrochemiluminescence Multiplex Immunoassays for Measuring Cytokines in Human Plasma.

This study compared two 96-well multiplex immunoassay platforms for analytical performance in assessing cytokine concentrations in standards, quality controls and human plasma samples (n = 62), and evaluated assay time requirements. Assays included a bead-based fluorescence MILLIPLEX® assay/Luminex fluorescence platform (LMX) and three kits from Meso Scale Discovery (MSD) in planar electrochemiluminescence format. The LMX kit evaluated 21 cytokines and the MSD kits evaluated 10 cytokines each, with 16 overlapping cytokines between platforms. Both assays provided good reproducibility in standard curves for all analytes. Interassay CVs of shared analytes showed average kit quality control CVs ranging 1.9-18.2% for LMX and 2.4-13.9% for MSD. The MSD platform had lower LLoQs than LMX for 14/16 shared cytokines. For IL-17, the LLoQ was lower with LMX than MSD, and the LLoQs for IL-6 were similar. Although MSD calibration curves indicated lower LLoQs for most of those analytes, many more cytokines in human plasma samples were not detected by MSD than by LMX. The ULoQs were higher in LMX versus MSD assays for 13/16 shared analytes, lower than MSD for IL-17, and equivalent between assays for IL-6 and MIP-1α. Bland-Altman plots indicated that MSD classified 13/16 shared analytes as concentrations lower than by LMX. Time and motion analysis indicated that total mean assay times were 20 h 28 m and 21 h 33 m for LMX and MSD, respectively, including an overnight (17 h) incubation. The MSD assays employed a manufacturer-approved overnight incubation instead of the standard 2-h incubation, which kit instructions suggest might increase detection sensitivity. Hands-on labor time averaged 1 h 37 m for LMX and 2 h 33 m for MSD. In summary, assay selection factors should include selection of specific markers of interest, time and cost considerations, and anticipated cytokine concentrations in prospective samples.

Development and evaluation of an ultrasensitive free VEGF-A immunoassay for analysis of human aqueous humor.

Aim: Novel bifunctional VEGF-A neutralizing therapies are being developed for the treatment of retinal vascular diseases such as age-related macular degeneration and diabetic retinopathy. In developing new therapeutic drugs, only small aqueous humor sample volumes are available for analyzing several parameters. Highly sensitive detection methods must be applied in analyzing VEGF-A levels in ocular fluids in order to demonstrate VEGF-A suppression following drug administration. Experimental: A highly sensitive immunoassay for VEGF-A was developed on the single molecule array (Simoa) platform, and validated before being used for the analysis of clinical aqueous humor samples from patients treated with anti-VEGF-A therapeutics. Results: This highly sensitive immunoassay allows the detection of baseline VEGF-A levels and suppression effects after drug administration, even in sample volumes as low as 12 µl. Conclusion: The Simoa VEGF-A assay is a valuable tool for the reliable monitoring of VEGF-A suppression after intravitreal administration of anti-VEGF-A drugs.

Tau plasma levels in subjective cognitive decline: Results from the DELCODE study.

Previous studies have demonstrated increased tau plasma levels in patients with Alzheimer's disease (AD) and mild cognitive impairment (MCI) due to AD. Much less is known whether increased tau plasma levels can already be detected in the pre-MCI stage of subjective cognitive decline (SCD). In the present study we measured tau plasma levels in 111 SCD patients and 134 age- and gender-matched cognitively healthy controls participating in the DZNE (German Center for Neurodegenerative Diseases) longitudinal study on cognition and dementia (DELCODE). Tau plasma levels were measured using ultra-sensitive, single-molecule array (Simoa) technology. We found no significant different tau plasma levels in SCD (3.4 pg/ml) compared with healthy controls (3.6 pg/ml) after controlling for age, gender, and education (p = 0.137). In addition, tau plasma levels did not correlate with Aß42 (r = 0.073; p = 0.634), tau (r = -0.179; p = 0.240), and p-tau181 (r = -0.208; p = 0.171) cerebrospinal fluid (CSF) levels in a subgroup of 45 SCD patients with available CSF. In conclusion, plasma tau is not increased in SCD patients. In addition, the lack of correlation between tau in plasma and CSF in the examined cohort suggests that tau levels are affected by different factors in both biofluids.

Elevated levels of circulating CDH5 and FABP1 in association with human drug-induced liver injury.

BACKGROUND & AIMS: The occurrence of drug-induced liver injury (DILI) is a major issue in all phases of drug development. To identify novel biomarker candidates associated with DILI, we utilised an affinity proteomics strategy, where antibody suspension bead arrays were applied to profile plasma and serum samples from human DILI cases and controls. METHODS: An initial screening was performed using 4594 randomly selected antibodies, representing 3450 human proteins. Resulting candidate proteins together with proposed DILI biomarker candidates generated a DILI array of 251 proteins for subsequent target analysis and verifications. In total, 1196 samples from 241 individuals across four independent cohorts were profiled: healthy volunteers receiving acetaminophen, patients with human immunodeficiency virus and/or tuberculosis receiving treatment, DILI cases originating from a wide spectrum of drugs, and healthy volunteers receiving heparins. RESULTS: We observed elevated levels of cadherin 5, type 2 (CDH5) and fatty acid-binding protein 1 (FABP1) in DILI cases. In the two longitudinal cohorts, CDH5 was elevated already at baseline. FABP1 was elevated after treatment initiation and seemed to respond more rapidly than alanine aminotransferase (ALT). The elevations were verified in the DILI cases treated with various drugs. In the heparin cohort, CDH5 was stable over time whereas FABP1 was elevated. CONCLUSIONS: These results suggest that CDH5 may have value as a susceptibility marker for DILI. FABP1 was identified as a biomarker candidate with superior characteristics regarding tissue distribution and kinetics compared to ALT but likely with limited predictive value for the development of severe DILI. Further studies are needed to determine the clinical utility of the proposed markers.

Prevention of tau increase in cerebrospinal fluid of APP transgenic mice suggests downstream effect of BACE1 inhibition.

INTRODUCTION: The inhibition of the ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a main therapeutic approach for the treatment of Alzheimer's disease (AD). We previously reported an age-related increase of tau protein in the cerebrospinal fluid (CSF) of amyloid ß (Aß) precursor protein (APP) transgenic mice. METHODS: APP transgenic mice were treated with a potent BACE1 inhibitor. CSF tau and CSF Aß levels were assessed. A novel high-sensitivity tau sandwich immunoassay was developed. RESULTS: We demonstrate that long-term BACE1 inhibition prevents CSF tau increase both in early-depositing APP transgenic mice and APP transgenic mice with moderate Aß pathology. DISCUSSION: Our results demonstrate that BACE1 inhibition not only reduces Aß generation but also downstream AD pathophysiology. The tight correlation between Aß aggregation in brain and CSF tau levels renders CSF tau a valuable marker to predict the effectiveness of BACE1 inhibitors in current clinical trials.

Development and Validation of an Ultrasensitive Procalcitonin Sandwich Immunoassay.

Procalcitonin (PCT) is well established as a highly specific biomarker for the detection of bacterial infections and sepsis. However, the currently available diagnostic tests are not able to detect very low or very early increases of PCT or even baseline levels in healthy individuals or patients with non-bacterial infections. In order to be able to detect these very low concentrations of PCT, a sandwich immunoassay was developed using high sensitivity Single Molecule Array technology (Simoa). The assay was thoroughly validated and applied to analyze human cerebrospinal fluid (CSF) and serum samples from patients with bacterial or viral meningitis as well as CSF, serum, and K2 EDTA plasma from healthy control subjects. A 50-fold increase in sensitivity compared to the current gold standard assays was achieved, which was sensitive enough for the detection of baseline PCT levels. Both serum and CSF showed significantly elevated PCT levels in patients with bacterial meningitis compared to patients with viral meningitis and the healthy control group. Procalcitonin concentration levels for patients with viral meningitis and the control group could be measured, but were not significantly different. The determination of PCT in the low pg·mL-1 range could help to improve the monitoring of bacterial infectious diseases, as PCT level changes could be detected earlier.

Study of the Humoral Immune Response towards HCV Genotype 4 Using a Bead-Based Multiplex Serological Assay.

Hepatitis C is one of the leading causes of hepatocellular carcinoma and remains at a high prevalence in Egypt and other resource-limited countries. Several hepatitis C virus (HCV) genotypes are distributed throughout the world, with genotype 4 being most common in North and Central Africa. We developed a multiplex serological assay for the detection of the HCV specific humoral immune response, with a focus on genotype 4. For the multiplex HCV assay we used twelve antigenic regions of different HCV proteins (core, and non-structural (NS) proteins NS3, NS4, NS5A, NS5B) and validated the assay technically and clinically. In comparison to a commercially available test, our assay revealed a higher sensitivity for genotype 4, and is therefore more suited for studying immune seroconversion in samples from acutely infected Egyptian HCV patients. Furthermore, our assay discriminates acutely and chronically infected HCV patients. Of 296 well characterized HCV patient samples, 83.9% of the acute samples and 86.5% of the chronic samples could be correctly classified. In sum, this newly developed serological HCV assay has a higher sensitivity for HCV genotype 4, and can thus improve diagnostic accuracy. Through the discrimination of acutely and chronically infected HCV patients the assay may be useful in supporting clinical management of HCV patients.

Lettuce costunolide synthase (CYP71BL2) and its homolog (CYP71BL1) from sunflower catalyze distinct regio- and stereoselective hydroxylations in sesquiterpene lactone metabolism.

Sesquiterpene lactones (STLs) are terpenoid natural products possessing the γ-lactone, well known for their diverse biological and medicinal activities. The occurrence of STLs is sporadic in nature, but most STLs have been isolated from plants in the Asteraceae family. Despite the implication of the γ-lactone group in many reported bioactivities of STLs, the biosynthetic origins of the γ-lactone ring remains elusive. Germacrene A acid (GAA) has been suggested as a central precursor of diverse STLs. The regioselective (C6 or C8) and stereoselective (α or ß) hydroxylation on a carbon of GAA adjacent to its carboxylic acid at C12 is responsible for the γ-lactone formation. Here, we report two cytochrome P450 monooxygenases (P450s) capable of catalyzing 6α- and 8ß-hydroxylation of GAA from lettuce and sunflower, respectively. To identify these P450s, sunflower trichomes were isolated to generate a trichome-specific transcript library, from which 10 P450 clones were retrieved. Expression of these clones in a yeast strain metabolically engineered to synthesize substrate GAA identified a P450 catalyzing 8ß-hydroxylation of GAA, but the STL was not formed by spontaneous lactonization. Subsequently, we identified the closest homolog of the GAA 8ß-hydroxylase from lettuce and discovered 6α-hydroxylation of GAA by the recombinant enzyme. The resulting 6α-hydroxy-GAA spontaneously undergoes a lactonization to yield the simplest form of STL, costunolide. Furthermore, we demonstrate the milligram per liter scale de novo synthesis of costunolide using the lettuce P450 in an engineered yeast strain, an important advance that will enable exploitation of STLs. Evolution and homology models of these two P450s are discussed.

The nucleotide sequence and genome organization of Plasmopara halstedii virus.

BACKGROUND: Only very few viruses of Oomycetes have been studied in detail. Isometric virions were found in different isolates of the oomycete Plasmopara halstedii, the downy mildew pathogen of sunflower. However, complete nucleotide sequences and data on the genome organization were lacking. METHODS: Viral RNA of different P. halstedii isolates was subjected to nucleotide sequencing and analysis of the viral genome. The N-terminal sequence of the viral coat protein was determined using Top-Down MALDI-TOF analysis. RESULTS: The complete nucleotide sequences of both single-stranded RNA segments (RNA1 and RNA2) were established. RNA1 consisted of 2793 nucleotides (nt) exclusive its 3' poly(A) tract and a single open-reading frame (ORF1) of 2745 nt. ORF1 was framed by a 5' untranslated region (5' UTR) of 18 nt and a 3' untranslated region (3' UTR) of 30 nt. ORF1 contained motifs of RNA-dependent RNA polymerases (RdRp) and showed similarities to RdRp of Scleropthora macrospora virus A (SmV A) and viruses within the Nodaviridae family. RNA2 consisted of 1526 nt exclusive its 3' poly(A) tract and a second ORF (ORF2) of 1128 nt. ORF2 coded for the single viral coat protein (CP) and was framed by a 5' UTR of 164 nt and a 3' UTR of 234 nt. The deduced amino acid sequence of ORF2 was verified by nano-LC-ESI-MS/MS experiments. Top-Down MALDI-TOF analysis revealed the N-terminal sequence of the CP. The N-terminal sequence represented a region within ORF2 suggesting a proteolytic processing of the CP in vivo. The CP showed similarities to CP of SmV A and viruses within the Tombusviridae family. Fragments of RNA1 (ca. 1.9 kb) and RNA2 (ca. 1.4 kb) were used to analyze the nucleotide sequence variation of virions in different P. halstedii isolates. Viral sequence variation was 0.3% or less regardless of their host's pathotypes, the geographical origin and the sensitivity towards the fungicide metalaxyl. CONCLUSIONS: The results showed the presence of a single and new virus type in different P. halstedii isolates. Insignificant viral sequence variation indicated that the virus did not account for differences in pathogenicity of the oomycete P. halstedii.

Identification and functional characterization of a new sunflower germacrene A synthase (HaGAS3).

Sesquiterpenes and sesquiterpene lactones are major natural compounds found in linear and capitate glandular trichomes of sunflower, Helianthus annuus L. In addition to two recently identified germacrene A synthases HaGAS1 and HaGAS2, found in capitate trichome gland cells, reverse transcription-PCR experiments have now allowed identification of a third enzyme of this type, HaGAS3. Its cDNA sequence was established and its functional characterization as a germacrene A synthase was achieved through in vitro expression in engineered yeast, and by GC-MS experiments. PCR and RT-PCR experiments with cDNA from different plant organs revealed that the new enzyme is expressed independently from the other two. While these latter two were expressed in plant organs bearing capitate glandular trichomes and in roots, the new enzyme occurred in plant tissues not linked to the presence of specific trichomes (for example, cotyledons), and was absent in roots. The experiments show that independently regulated pathways for the first cyclic sesquiterpene, germacrene A, are present in sunflower.

Biochemical conservation and evolution of germacrene A oxidase in asteraceae.

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes approximately 8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.