In 5–40% of respiratory infections in children, the diagnostics
remain negative, suggesting that the patients might be infected with a yet
unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery
method based on recognition of restriction enzyme cleavage sites, ligation of
adaptors and subsequent amplification by PCR. However, direct discovery of
unknown pathogens in nasopharyngeal swabs is difficult due to the high
concentration of ribosomal RNA (rRNA) that acts as competitor. In the current
study we optimized VIDISCA by adjusting the reverse transcription enzymes and
decreasing rRNA amplification in the reverse transcription, using hexamer
oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA
templates was further reduced with oligonucleotides that anneal to rRNA but can
not be extended due to 3′-dideoxy-C6-modification. With these
modifications >90% reduction of rRNA amplification was established.
Further improvement of the VIDISCA sensitivity was obtained by high throughput
sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all
containing known respiratory viruses. We could identify the proper virus in the
majority of samples tested (11/18). The median load in the VIDISCA-454 positive
samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6).
Our results show that optimization of VIDISCA and subsequent
high-throughput-sequencing enhances sensitivity drastically and provides the
opportunity to perform virus discovery directly in patient material.
Abnormal nutrient metabolism is a hallmark of aging, and the underlying genetic and nutritional framework is rapidly being uncovered, particularly using C. elegans as a model. However, the direct metabolic consequences of perturbations in life history of C. elegans remain to be clarified. Based on recent advances in the metabolomics field, we optimized and validated a sensitive mass spectrometry (MS) platform for identification of major metabolite classes in worms and applied it to study age and diet related changes. Using this platform that allowed detection of over 600 metabolites in a sample of 2500 worms, we observed marked changes in fatty acids, amino acids and phospholipids during worm life history, which were independent from the germ-line. Worms underwent a striking shift in lipid metabolism after early adulthood that was at least partly controlled by the metabolic regulator AAK-2/AMPK. Most amino acids peaked during development, except aspartic acid and glycine, which accumulated in aged worms. Dietary intervention also influenced worm metabolite profiles and the regulation was highly specific depending on the metabolite class. Altogether, these MS-based methods are powerful tools to perform worm metabolomics for aging and metabolism-oriented studies.
Vaz, McDermott et al. identify variants in PCYT2, which encodes a key gene in phospholipid biosynthesis, in five individuals with a new complex hereditary spastic paraplegia. Functional studies in fibroblasts and a zebrafish model confirm the pathogenic nature of the variants, while lipidomic analysis reveals potential treatment strategies and plasma biomarkers.
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