No abstract

High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification.

No abstract

Most sporadic gastrointestinal stromal tumours (GISTs) have somatic c-kit gene mutations that are considered to be causal. Neurofibromatosis type 1 (NF1) is caused by mutations of the NF1 gene and NF1 patients have an increased risk of developing GISTs. Since most neoplasms are considered to develop as a result of the combination of several gene mutations, these findings suggest that GISTs from NF1 patients might have somatic c-kit gene mutations and that sporadic GISTs from non-NF1 patients might have somatic NF1 gene mutations. The present study analysed 29 GISTs from seven NF1 patients for c-kit gene mutations and ten sporadic GISTs from ten non-NF1 patients for NF1 mutations. Exons 9, 11, 13, and 17 of the c-kit gene were amplified and directly sequenced after the extraction of genomic DNA from wax-embedded tissues from 26 GISTs from five NF1 patients. The whole coding region of the c-kit cDNA and the whole coding region of the NF1 cDNA were amplified and directly sequenced after RNA extraction and cDNA synthesis in three fresh GIST tissues from two NF1 patients and ten fresh GIST tissues from ten non-NF1 patients. Of the ten sporadic GISTs, eight had heterozygous mutations at exon 11, and one at exon 9, of c-kit. Heterozygous NF1 gene mutations were detected in GISTs from the two NF1 patients from whom fresh tissues were available. None of the 29 GISTs derived from NF1 patients had detectable c-kit gene mutations and none of the ten GISTs derived from non-NF1 patients had detectable NF1 mutations. These results suggest that the pathogenesis of GISTs in NF1 patients is different from that in non-NF1 patients.

Serotonin [5-hydroxytryptamine (5-HT)] is a major therapeutic target of psychiatric disorders. Tryptophan hydroxylase (TPH) catalyzesthe rate-limiting reaction in the biosynthesis of 5-HT. Two isoforms (TPH1 and TPH2) having tryptophan hydroxylating activity were identified. Association studies have revealed possible TPH1 involvement in psychiatric conditions and behavioral traits. However, TPH1 mRNA was reported to be mainly expressed in the pineal gland and the periphery and to be barely detected in the brain. Therefore, contribution of TPH1 to brain 5-HT levels is not known, and the mechanisms how TPH1 possibly contributes to the pathogenesis of psychiatric disorders are not understood. Here, we show an unexpected role of TPH1 in the developing brain. We found that TPH1 is expressed preferentially during the late developmental stage in the mouse brain. TPH1 showed higher affinity to tryptophan and stronger enzyme activity than TPH2 in a condition reflecting that of the developing brainstem. Low 5-HT contents in the raphe nucleus were seen during development in New Zealand white (NZW) and SWR mice having common functional polymorphisms in the TPH1 gene. However, the 5-HT contents in these mice were not reduced in adulthood. In adult NZW and SWR mice, depression-related behavior was observed. Considering an involvement of developmental brain disturbance in psychiatric disorders, TPH1 may act specifically on development of 5-HT neurons, and thereby influence behavior later in life.