Analytical and Clinical Performance of Amyloid-Beta Peptides Measurements in CSF of ADNIGO/2 Participants by an LC–MS/MS Reference Method

BACKGROUND: Cerebrospinal fluid (CSF) amyloid-β1-42 (Aβ42) reliably detects brain amyloidosis based on its high concordance with plaque burden at autopsy and with amyloid positron emission tomography (PET) ligand retention observed in several studies. Low CSF Aβ42 concentrations in normal aging and dementia are associated with the presence of fibrillary Aβ across brain regions detected by amyloid PET imaging. METHODS: An LC-MS/MS reference method for Aβ42, modified by adding Aβ40 and Aβ38 peptides to calibrators, was used to analyze 1445 CSF samples from ADNIGO/2 participants. Seventy runs were completed using 2 different lots of calibrators. For preparation of Aβ42 calibrators and controls spiking solution, reference Aβ42 standard with certified concentration was obtained from EC-JRC-IRMM (Belgium). Aβ40 and Aβ38 standards were purchased from rPeptide. Aβ42 calibrators' accuracy was established using CSF-based Aβ42 Certified Reference Materials (CRM). RESULTS: CRM-adjusted Aβ42 calibrator concentrations were calculated using the regression equation Y (CRM-adjusted) = 0.89X (calibrators) + 32.6. Control samples and CSF pools yielded imprecision ranging from 6.5 to 10.2% (Aβ42) and 2.2 to 7.0% (Aβ40). None of the CSF pools showed statistically significant differences in Aβ42 concentrations across 2 different calibrator lots. Comparison of Aβ42 with Aβ42/Aβ40 showed that the ratio improved concordance with concurrent [18F]-florbetapir PET as a measure of fibrillar Aβ (n = 766) from 81 to 88%. CONCLUSIONS: Long-term performance assessment substantiates our modified LC-MS/MS reference method for 3 Aβ peptides. The improved diagnostic performance of the CSF ratio Aβ42/Aβ40 suggests that Aβ42 and Aβ40 should be measured together and supports the need for an Aβ40 CRM

Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrioanguillarum strain 775

Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.

The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1), which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR) leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1), revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS) of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC) was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state

A novel pathway producing dimethylsulphide in bacteria is widespread in soil environments

The volatile compound dimethylsulphide (DMS) is important in climate regulation, the sulphur cycle and signalling to higher organisms. Microbial catabolism of the marine osmolyte dimethylsulphoniopropionate (DMSP) is thought to be the major biological process generating DMS. Here we report the discovery and characterisation of the first gene for DMSP-independent DMS production in any bacterium. This gene, mddA, encodes a methyltransferase that methylates methanethiol (MeSH) and generates DMS. MddA functions in many taxonomically diverse bacteria including sediment-dwelling pseudomonads, nitrogen-fixing bradyrhizobia and cyanobacteria, and mycobacteria, including the pathogen Mycobacterium tuberculosis. The mddA gene is present in metagenomes from varied environments, being particularly abundant in soil environments, where it is predicted to occur in up to 76% of bacteria. This novel pathway may significantly contribute to global DMS emissions, especially in terrestrial environments, and could represent a shift from the notion that DMSP is the only significant precursor of DMS

An Age-Dependent Pharmacokinetic Study of Intravenous and Oral Mycophenolate Mofetil in Combination with Tacrolimus for GVHD Prophylaxis in Pediatric Allogeneic Stem Cell Transplantation Recipients

Acute graft-versus-host disease (aGVHD) still remains a major limiting factor following allogeneic stem cell transplantation (AlloSCT) in pediatric recipients. Mycophenolate mofetil (MMF), an uncompetitive selective inhibitor of inosine monophosphate dehydrogenase, is a new immunosuppressant agent without major mucosal, hepatic, or renal toxicity compared to other prophylactic aGVHD immunosuppressant drugs. Although there has been an extensive pharmacokinetic (PK) experience with MMF administration following solid organ transplantation in children, there is a paucity of PK data following its use in pediatric AlloSCT recipients. We investigated the safety and PK of MMF as GVHD prophylaxis following intravenous (i.v.) and oral (p.o.) administration (900 mg/m2 every 6 hours) in conjunction with tacrolimus, after myeloablative (MA) and nonmyeloablative (NMA) conditioning and AlloSCT in 3 distinct age groups of pediatric AlloSCT recipients (0-6 years, 6-12 years, and 12-16 years). Mycophenolic acid (MPA) in plasma samples was measured either by high-performance liquid chromatography (HPLC) or liquid chromatography/mass spectrometry (LC/MS/MS) as we have previously described. Plasma samples were obtained at baseline and at 0.5, 1, 2, 3, 4, and 6 hours after i.v. dosing on days +1, +7, +14, and at 2 time points between day +45 and +100 after p.o. administration post AlloSCT. MPA PK analysis included AUC (0-6 hours), Cmax, Tmax, Css, Vss, C trough (C0), CL, and T½. Thirty-eight patients, with a median age of 8 years (0.33-16 years), 20/18 M:F ratio, 21/17 malignant/nonmalignant disease, 17/21 MA: NMA conditioning, 16 of 22 related/unrelated allografts. Median time to myeloid and platelet engraftment was 18 and 31 days, respectively. Mean donor chimerism on day +60 and +100 was 83% and 90%, respectively. Probability of developing aGVHD grade II-IV and extensive chronic GVHD (cGVHD) was 54% and 34%, respectively. There was significant intra- and interpatient MMF PK variability. There was a significant increase in i.v. MPA area under the curve (AUC)0-6hour and Cmax (P < .0003) and a significant decrease in CLss (P < .002) and Vss (P < .001) on day +14 versus day +7. Children <12 years of age had a significant increase in i.v. MPA Tmax (P = .01), Vss (P = .028), and CLss (P < .001) compared to the older age group. There was a trend in increased i.v. MPA CLss following MA versus NMA conditioning (P < .054); i.v. and p.o. MMF administration (900 mg/m2 every 6 hours) in combination with tacrolimus was well tolerated in pediatric AlloSCT recipients. There was a significant increase in MPA exposure on day +14 versus day +7, suggesting improved enterohepatic recirculation at day +14 post-AlloSCT. Children <12 years of age appear to have a significantly different MPA PK profile compared to older children and adolescents and may require more frequent dosing

Use of genetically modified bacteria for drug delivery in humans: Revisiting the safety aspect

The use of live, genetically modified bacteria as delivery vehicles for biologics is of considerable interest scientifically and has attracted significant commercial investment. We have pioneered the use of the commensal gut bacterium Bacteroides ovatus for the oral delivery of therapeutics to the gastrointestinal tract. Here we report on our investigations of the biological safety of engineered B. ovatus bacteria that includes the use of thymineless death as a containment strategy and the potential for the spread of transgenes in vivo in the mammalian gastrointestinal tract. We demonstrate the ability of GM-strains of Bacteroides to survive thymine starvation and overcome it through the exchange of genetic material. We also provide evidence for horizontal gene transfer in the mammalian gastrointestinal tract resulting in transgene-carrying wild type bacteria. These findings sound a strong note of caution on the employment of live genetically modified bacteria for the delivery of biologics

The Alzheimer's Disease Neuroimaging Initiative 2 Biomarker Core: A review of progress and plans

INTRODUCTION: We describe Alzheimer's Disease Neuroimaging Initiative (ADNI) Biomarker Core progress including: the Biobank; cerebrospinal fluid (CSF) amyloid beta (Aβ1-42), t-tau, and p-tau181 analytical performance, definition of Alzheimer's disease (AD) profile for plaque, and tangle burden detection and increased risk for progression to AD; AD disease heterogeneity; progress in standardization; and new studies using ADNI biofluids. METHODS: Review publications authored or coauthored by ADNI Biomarker core faculty and selected non-ADNI studies to deepen the understanding and interpretation of CSF Aβ1-42, t-tau, and p-tau181 data. RESULTS: CSF AD biomarker measurements with the qualified AlzBio3 immunoassay detects neuropathologic AD hallmarks in preclinical and prodromal disease stages, based on CSF studies in non-ADNI living subjects followed by the autopsy confirmation of AD. Collaboration across ADNI cores generated the temporal ordering model of AD biomarkers varying across individuals because of genetic/environmental factors that increase/decrease resilience to AD pathologies. DISCUSSION: Further studies will refine this model and enable the use of biomarkers studied in ADNI clinically and in disease-modifying therapeutic trials

Near real-time input to a propagation model for nowcasting of HF communications with aircraft on polar routes

There is a need for improved techniques for nowcasting and forecasting (over several hours) HF propagation at northerly latitudes to support airlines operating over the increasingly popular trans-polar routes. In this paper the assimilation of real-time measurements into a propagation model developed by the authors is described, including ionosonde measurements and Total Electron Content (TEC) measurements to define the main parameters of the ionosphere. The effects of D-region absorption in the polar cap and auroral regions are integrated with the model through satellite measurements of the flux of energetic solar protons (>1 MeV) and the X-ray flux in the 0.1-0.8 nm band, and ground-based magnetometer measurements which form the Kp and Dst indices of geomagnetic activity. The model incorporates various features (e.g. convecting patches of enhanced plasma density) of the polar ionosphere that are, in particular, responsible for off-great circle propagation and lead to propagation at times and frequencies not expected from on-great circle propagation alone. The model development is supported by the collection of HF propagation measurements over several paths within the polar cap, crossing the auroral oval, and along the mid-latitude trough

The luxS mutation causes loosely-bound biofilms in Shewanella oneidensis

<p>Abstract</p> <p>Background</p> <p>The <it>luxS </it>gene in <it>Shewanella oneidensis </it>was shown to encode an autoinducer-2 (AI-2)-like molecule, the postulated universal bacterial signal, but the impaired biofilm growth of a <it>luxS </it>deficient mutant could not be restored by AI-2, indicating it might not have a signalling role in this organism.</p> <p>Findings</p> <p>Here, we provide further evidence regarding the metabolic role of a <it>luxS </it>mutation in <it>S. oneidensis</it>. We constructed a <it>luxS </it>mutant and compared its phenotype to a wild type control with respect to its ability to remove AI-2 from the medium, expression of secreted proteins and biofilm formation. We show that <it>S. oneidensis </it>has a cell-dependent mechanism by which AI-2 is depleted from the medium by uptake or degradation at the end of the exponential growth phase. As AI-2 depletion is equally active in the <it>luxS </it>mutant and thus does not require AI-2 as an inducer, it appears to be an unspecific mechanism suggesting that AI-2 for <it>S. oneidensis </it>is a metabolite which is imported under nutrient limitation. Secreted proteins were studied by iTraq labelling and liquid chromatography mass spectrometry (LC-MS) detection. Differences between wild type and mutant were small. Proteins related to flagellar and twitching motility were slightly up-regulated in the <it>luxS </it>mutant, in accordance with its loose biofilm structure. An enzyme related to cysteine metabolism was also up-regulated, probably compensating for the lack of the LuxS enzyme. The <it>luxS </it>mutant developed an undifferentiated, loosely-connected biofilm which covered the glass surface more homogenously than the wild type control, which formed compact aggregates with large voids in between.</p> <p>Conclusions</p> <p>The data confirm the role of the LuxS enzyme for biofilm growth in <it>S. oneidensis </it>and make it unlikely that AI-2 has a signalling role in this organism.</p