Inflammatory gene expression by human colonic smooth muscle cells

Intestinal mucosal cells and invading leukocytes produce inappropriate levels of cytokines and chemokines in human colitis. However, smooth muscle cells of the airway and vasculature also synthesize cytokines and chemokines. To determine whether human colonic myocytes can synthesize proinflammatory mediators, strips of circular smooth muscle and smooth muscle cells were isolated from human colon. Myocytes and muscle strips were stimulated with 10 ng/ml of IL-1beta, TNF-alpha, and IFN-gamma, respectively. Expression of mRNA for IL-1beta, IL-6, IL-8, and cyclooxygenase-2 (COX-2) was induced within 2 h and continued to increase for 8-12 h. Regulated on activation, normal T cell-expressed and -secreted (RANTES) mRNA expression was slower, appearing at 8 h and increasing linearly through 20 h. Expression of all five mRNAs was inhibited by 0.1 microM MG-132, a proteosome inhibitor that blocks NF-kappaB activation. Expression of IL-1beta, IL-6, IL-8, and COX-2 mRNA was reduced by 30 microM PP1, an Src family tyrosine kinase inhibitor, and by 25 microM SB-203580, a p38 MAPK inhibitor. MAPK/extracellular regulated kinase-1 inhibitor PD-98059 (25 microM) was much less effective. In conclusion, human colonic smooth muscle cells can synthesize and secrete interleukins (IL-1beta and IL-6) and chemokines (IL-8 and RANTES) and upregulate expression of COX-2. Regulation of cytokine, chemokine, and COX-2 mRNA depends on multiple signaling pathways, including Src-family kinases, extracellular regulated kinase, p38 MAPKs, and NF-kappaB. SB-203580 was a consistent, efficacious inhibitor of inflammatory gene expression, suggesting an important role of p38 MAPK in synthetic functions of human colonic smooth muscle.     

Asaia sp., an unusual spoilage organism of fruit-flavored bottled water

A gram-negative bacillus was isolated from a batch of fruit-flavored bottled water, which had spoiled as a result of bacterial overgrowth (>10(6) CFU/ml). The spoilage organism was extremely difficult to identify phenotypically and was poorly identified as Pasturella sp. (78.7% identification profile) employing the API 20NE identification scheme, which gave the profile 5040000. Molecular identification through PCR amplification of a partial region of the 16S rRNA gene followed by direct automated sequencing of the PCR amplicon allowed identification of the organism. Due to the sequence identity (100%) between the spoilage organism and a reference strain in GenBank, the spoilage isolate was considered to be an Asaia sp., a recently described genus and member of the acetic acid bacteria. This is the first report of Asaia sp. causing spoilage of a foodstuff and highlights the benefits of molecular identification techniques based on 16S rRNA gene sequences in the identification of unusual spoilage organisms.     

Clinical development of therapeutic recombinant proteins

Only a small subset of the therapeutics that enter clinical studies will prove to be safe and effective in humans and gain approval for marketing. The success of the products and, by inference, the sponsoring companies can be measured by tracking advancement through the clinical phase and review transitions to marketing approval. To determine phase transition probabilities and approval success rates for recombinant protein (rDNA) therapeutics, the Tufts Center for the Study of Drug Development collected data for 271 rDNA therapeutics that entered clinical study between 1980 and 2002. The data were stratified into eight therapeutic categories. Approval success rates were calculated for rDNA therapeutics with two possible fates: (i) approval in any country and (ii) U.S. approval only. Global approval success rates ranged from 23% to 63%, and U.S. approval success rates ranged from 17% to 58%. Trends in clinical phase lengths over five time periods and an overview of the rDNA therapeutics currently under Food and Drug Administration review are discussed.     

A defect in a novel ADAMTS family member is the cause of the belted white-spotting mutation

Several features of the pigment defect in belted (bt) mutant mice suggest that it occurs as a result of a defect in melanocyte development that is unique from those described for other classical white-spotting mutations. We report here that bt mice carry mutations in Adamts20, a novel member of the ADAMTS family of secreted metalloproteases. Adamts20 shows a highly dynamic pattern of expression in the developing embryo that generally precedes the appearance of melanoblasts in the same region, and is not expressed in the migrating cells themselves. Adamts20 shows remarkable homology with GON-1, an ADAMTS family protease required for distal tip cell migration in C. elegans. Our results suggest that the role of ADAMTS proteases in the regulation of cell migration has been conserved in mammalian development.     

Characterisation of the Small RNAs in the Biomedically Important Green-Bottle Blowfly Lucilia sericata

Background

The green bottle fly maggot, Lucilia sericata, is a species with importance in medicine, agriculture and forensics. Improved understanding of this species’ biology is of great potential benefit to many research communities. MicroRNAs (miRNA) are a short non-protein coding regulatory RNA, which directly regulate a host of protein coding genes at the translational level. They have been shown to have developmental and tissue specific distributions where they impact directly on gene regulation. In order to improve understanding of the biology of L. sericata maggots we have performed small RNA-sequencing of their secretions and tissue at different developmental stages.

Results

We have successfully isolated RNA from the secretions of L. sericata maggots. Illumina small RNA-sequencing of these secretions and the three tissues (crop, salivary gland, gut) revealed that the most common small RNA fragments were derived from ribosomal RNA and transfer RNAs of both insect and bacterial origins. These RNA fragments were highly specific, with the most common tRNAs, such as GlyGCC, predominantly represented by reads derived from the 5’ end of the mature maggot tRNA. Each library also had a unique profile of miRNAs with a high abundance of miR-10-5p in the maggot secretions and gut and miR-8 in the food storage organ the crop and salivary glands. The pattern of small RNAs in the bioactive maggot secretions suggests they originate from a combination of saliva, foregut and hindgut tissues. Droplet digital RT-PCR validation of the RNA-sequencing data shows that not only are there differences in the tissue profiles for miRNAs and small RNA fragments but that these are also modulated through developmental stages of the insect.

Conclusions

We have identified the small-RNAome of the medicinal maggots L. sericata and shown that there are distinct subsets of miRNAs expressed in specific tissues that also alter during the development of the insect. Furthermore there are very specific RNA fragments derived from other non-coding RNAs present in tissues and in the secretions. This new knowledge has applicability in diverse research fields including wound healing, agriculture and forensics.