Search

The haploid Saccharomyces cerevisiae strain CEN.PK113-7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C. However, these assemblies contain large sequence gaps, particularly in subtelomeric regions, and the assumption of perfect homology to S288C for scaffolding introduces bias. In this study, we obtained a near-complete genome assembly of CEN.PK113-7D using only Oxford Nanopore Technology's MinION sequencing platform. Fifteen of the 16 chromosomes, the mitochondrial genome and the 2-μm plasmid are assembled in single contigs and all but one chromosome starts or ends in a telomere repeat. This improved genome assembly contains 770 Kbp of added sequence containing 248 gene annotations in comparison to the previous assembly of CEN.PK113-7D. Many of these genes encode functions determining fitness in specific growth conditions and are therefore highly relevant for various industrial applications. Furthermore, we discovered a translocation between chromosomes III and VIII that caused misidentification of a MAL locus in the previous CEN.PK113-7D assembly. This study demonstrates the power of long-read sequencing by providing a high-quality reference assembly and annotation of CEN.PK113-7D and places a caveat on assumed genome stability of microorganisms., (© FEMS 2017.)

The CSF biomarkers beta-amyloid peptide (Abeta(1-42)), total tau protein (T-tau) and tau phosphorylated at threonine 181 (P-tau(181P)) were determined in autopsy-confirmed Alzheimer's disease patients in order to study possible associations with the epsilon4 allele of APOE and density and spread of plaques (SP) and tangles (NFT). CSF levels of Abeta(1-42), T-tau and P-tau(181P) were determined in 50 Alzheimer's disease patients using commercially available single parameter ELISA kits (INNOTEST(R)). Genomic DNA was extracted from whole blood and the APOE genotype was determined using standard methods. Tangle burden was assessed by means of Braak's NFT stages (I-VI), whereas the plaque burden was assessed by means of Braak's SP stages (A-C). CSF biomarker levels were not different when comparing epsilon4 carriers (n = 21) and non-carriers (n = 29) (P > 0.05 for all comparisons). No significant correlations between the number of epsilon4 alleles (0, 1 or 2) and CSF levels of Abeta(1-42) (Spearman Rank Order: r = -0.057, P = 0.695), T-tau (r = 0.104, P = 0.472) and P-tau(181P) (r = 0.062, P = 0.668) were found. Braak's SP (Abeta(1-42): r = -0.155, P = 0.280; T-tau: r = -0.044, P = 0.763; P-tau(181P): r = -0.010, P = 0.947) and NFT (Abeta(1-42): r = -0.145, P = 0.315; T-tau: r = 0.117, P = 0.415; P-tau(181P): r = 0.150, P = 0.296) stages were not significantly correlated with CSF biomarker levels. In conclusion, CSF levels of Abeta(1-42), T-tau and P-tau(181P) were not associated with epsilon4, tangle or plaque burden in 50 autopsy-confirmed Alzheimer's disease patients. In the light of future biomarker applications like monitoring of disease progression and as allocortical neuropathological changes significantly contribute to clinical symptoms, the concept of in vivo surrogate biomarkers should be further explored.

It is well established that Alzheimer's disease causing mutations in APP, PSEN1 and PSEN2 lead to a relative increased production of Abeta42, thereby fostering its deposition in plaques. Recently others and we showed that amyloid precursor protein (APP) overproduction, either as a result of genomic locus duplication or altered regulatory sequences in the APP promoter region, leads to early-onset disease. Here, we have expanded our study of genetic variability in the APP promoter to a large group of well-documented Belgian patients (n = 750, mean onset age = 75.0 +/- 8.6, range = 37-96). We identified three different APP promoter mutations (-369C-->G, -534G-->A and -479C-->T) in seven patients. In patients with onset < or =70 years (n = 204), we identified one patient carrying the London APP V717I mutation while no patients carried an APP locus duplication, indicating that APP promoter mutations (n = 2) were more frequently associated with increased risk for early-onset Alzheimer's disease. The two mutations (-369C-->G and -534G-->A) increasing APP promoter activity by nearly 2-fold and mimicking an APP duplication, appeared in probands of families with multiple patients with dementia. The -479C-->T mutation that increased APP expression only mildly (1.2-fold), was observed in four patients with onset ages ranging from 62 to 79 years (mean 71.5 years), suggesting that its contribution to disease risk is more pronounced at later age due to modulating factors. In conclusion, we provided evidence that mutations in APP regulatory sequences are more frequent than APP coding mutations, and that increased APP transcriptional activity constitutes a risk factor for Alzheimer's disease with onset ages inversely correlated with levels of APP expression.

We assessed the impact of amyloid precursor protein (APP) gene locus duplications in early onset Alzheimer's disease in a Dutch population-based sample. Using real-time PCR and an in-house-developed multiplex amplicon quantification assay, we identified a genomic APP duplication in 1 out of 10 multigenerational families segregating early onset Alzheimer's disease. In this family, cerebral amyloid angiopathy (CAA) coincided with this disease. The duplicated genomic region included no other genes than APP and extended maximally over 0.7 Mb. In a sample of 65 familial early onset patients, we observed the same APP genomic duplication in one patient (1.7%), while in 36 isolated patients duplications in the APP locus were absent. This indicated that APP locus duplications explained <2% of familial, non-autosomal dominant Alzheimer's disease and are an infrequent cause of de novo mutation. Our findings corroborated a recent French study, and indicated that investigating genomic duplications in the APP locus in families segregating Alzheimer's disease and CAA should be considered.

We present a modification of the HemoQuant assay, a good but lengthy and tedious method for determining heme in feces by means of its transformation to porphyrins. The laborious extraction procedure was replaced by a simple centrifugation procedure. The nonhomogeneous hot oxalic acid suspension was replaced by acetic acid. We observed no significant difference in results between samples analyzed by the older method vs the present modification (r = 0.996, n = 52). Mean (and SD) analytical recoveries of added hemoglobin and protoporphyrin were 99% (7%) and 93% (6%), respectively. The analytical procedure can now be automated by using discrete samplers and a flow-through fluorometer. Initial sampling and dilution of feces are still done manually, however. The excellent specificity, sensitivity, and overall analytical performance of the original method are retained, while circumventing the practical inconveniences of this reliable screening test for occult blood in feces.