Areca catechu-(Betel-nut)-induced whole transcriptome changes in a human monocyte cell line that may have relevance to diabetes and obesity; a pilot study.

BACKGROUND: Betel-nut consumption is the fourth most common addictive habit globally and there is good evidence linking the habit to obesity, type 2 diabetes (T2D) and the metabolic syndrome. The aim of our pilot study was to identify gene expression relevant to obesity, T2D and the metabolic syndrome using a genome-wide transcriptomic approach in a human monocyte cell line incubated with arecoline and its nitrosated products. RESULTS: The THP1 monocyte cell line was incubated separately with arecoline and 3-methylnitrosaminopropionaldehyde (MNPA) in triplicate for 24 h and pooled cDNA indexed paired-end libraries were sequenced (Illumina NextSeq 500). After incubation with arecoline and MNPA, 15 and 39 genes respectively had significant changes in their expression (q < 0.05, log fold change 1.5). Eighteen of those genes have reported associations with T2D and obesity in humans; of these genes there was most marked evidence for CLEC10A, MAPK8IP1, NEGR1, NQ01 and INHBE genes. CONCLUSIONS: Our preliminary studies have identified a large number of genes relevant to obesity, T2D and metabolic syndrome whose expression was changed significantly in human TPH1 cells following incubation with betel-nut derived arecoline or with MNPA. These findings require validation by further cell-based work and investigation amongst betel-chewing communities

Influence of pulsationless milking on teat canal keratin and mastitis

Twenty-four Holstein cows, producing at least 21 kg of milk/d, were used in two replicate experiments to determine the effect of presence or absence of pulsation on loss of teat canal keratin during machine milking. Left quarters were milked without pulsation and right quarters were milked with pulsation. On d 0 and 10, keratin was collected from one left and from one right teat canal of each cow prior to milking and from the remaining two teat canals after milking. Milk was collected for assessment of SCC and bacteriological status on d 0 and approximately every 3 d until d 18. Quantity of keratin recovered before milking on d 10 did not differ between teats milked with or without pulsation, but loss of keratin because of milking was greater from teats milked with pulsation. By d 7, 30% (12 of 43) of quarters milked without pulsation had become infected, but no (0 of 47) quarters milked with pulsation were infected. By d 14 to 16, new infections had increased to 68% (28 of 41) of quarters milked without pulsation and 2% (1 of 43) in quarters milked with pulsation; mean SCC in pulsationless quarters increased sevenfold relative to pulsation quarters. Protein and water content of keratin did not differ because of treatment, and changes in lipid composition were minor. Histological analysis of the teats of 4 cows indicated that the mean diameter of the teat canal, within 2 h after milking, was greater without pulsation than with pulsation (680 vs. 483 mum)

Genome-wide analysis of allelic expression imbalance in human primary cells by high-throughput transcriptome resequencing

Many disease-associated variants identified by genome-wide association (GWA) studies are expected to regulate gene expression. Allele-specific expression (ASE) quantifies transcription from both haplotypes using individuals heterozygous at tested SNPs. We performed deep human transcriptome-wide resequencing (RNA-seq) for ASE analysis and expression quantitative trait locus discovery. We resequenced double poly(A)-selected RNA from primary CD4(+) T cells (n = 4 individuals, both activated and untreated conditions) and developed tools for paired-end RNA-seq alignment and ASE analysis. We generated an average of 20 million uniquely mapping 45 base reads per sample. We obtained sufficient read depth to test 1371 unique transcripts for ASE. Multiple biases inflate the false discovery rate which we estimate to be approximately 50% for random SNPs. However, after controlling for these biases and considering the subset of SNPs that pass HapMap QC, 4.6% of heterozygous SNP-sample pairs show evidence of imbalance (P &lt;0.001). We validated four findings by both bacterial cloning and Sanger sequencing assays. We also found convincing evidence for allelic imbalance at multiple reporter exonic SNPs in CD6 for two samples heterozygous at the multiple sclerosis-associated variant rs17824933, linking GWA findings with variation in gene expression. Finally, we show in CD4(+) T cells from a further individual that high-throughput sequencing of genomic DNA and RNA-seq following enrichment for targeted gene sequences by sequence capture methods offers an unbiased means to increase the read depth for transcripts of interest, and therefore a method to investigate the regulatory role of many disease-associated genetic variants