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.     

Crystallization and preliminary X-ray investigation of recombinant human interleukin 10

Crystals of recombinant human interleukin 10 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space group P4₁2₁2 or P4₃2₁2; the unit cell axes are a = 36.5 Å and c = 221.9 Å. There is the equivalent of one polypeptide chain in the asymmetric unit. The crystals are stable to X-rays and diffract to at least 2.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Automated colorimetric screen for apyrase inhibitors

Apyrases are enzymes that efficiently hydrolyze ATP and ADP and may operate both inside and outside the cell. Although apyrases are important to a variety of cellular mechanisms and uses in industry, there are no available apyrase-specific inhibitors. Colorimetric assays based on the Fiske-Subbarow method for measuring inorganic phosphate are able to detect the release of inorganic phosphate from ATP and other nucleotides. We found that this type of assay could be automated and used to screen for apyrase-inhibiting compounds by assaying for a reduction in released phosphate in the presence of potential inhibitors. The automation of this assay allowed for the successful screening of a commercially available compound library. Several low molecular weight compounds were identified that, when used at micromolar concentrations, effectively inhibited apyrase activity.     

Spondylitis in a humpback whale (Megaptera novaeangliae) from the southeast Pacific

A 7.25 m long male humpback whale (Megaptera novaeangliae) with spondylitis was found beached on August 13, 1994 at Ancon, Ecuador (2 degrees 23' S, 80 degrees 47' W). The condition involved at least 11 vertebrae, 7 lumbar (L4 to L11) and 4 caudal (Ca1 to Ca4). Partial fusion of vertebrae was observed as a result of intervertebral bony proliferation, likely impeding full motion. The relatively young age of this specimen and the severity of the deformities suggest an infectious, rather than degenerative, process. The gross findings are most consistent with some type of spondyloarthritis. Although this condition has previously been identified in a number of cetacean species, the pathogenesis, population impact and ecologic implications have not been fully assessed. This is the third case described for humpback whales and the first for a humpback whale from the SE Pacific.     

The reproductive flight phenology of a neotropical ant assemblage

1. Alate flights reflect an ant colony's investment in sexual reproduction and dispersal yet little is known about community‐wide patterns of alate phenology. Two Malaise traps (for 2 years) and two light traps (for 1 year) were used to explore the flight phenologies of 22 common neotropical species from Barro Colorado Island, Panama. 2. The traps caught 23 182 individuals and 286 species/morphospecies. The two trap methods shared only 18 species. Samples also differed in sexual composition: light trap samples were 80% female, Malaise trap samples were 2.6% female. 3. Of 22 common species, all but one flew over half the year, with about half flying every month of the year. These data, combined with a literature review, suggest a latitudinal gradient in alate flight season: one north temperate assemblage (42°N) averaged 1.6 lunar months per species. The ever‐warm tropical year provides a larger flight window that allows a diversity of phenologies, from continuous to strongly pulsed. 4. Rainfall was correlated with alate flights in one‐third of the species. Quantile regression suggested that high weekly rainfall was necessary but not sufficient to produce alate flights in about a quarter of the species. 5. By decreasing the number of nests releasing alates on a given day, long flight seasons may lower the probability of finding a mate. At the same time, long flight seasons may increase the opportunity of finding vacant nest sites. High population densities and high incidence of nest disturbance in this community may ameliorate the first cost while enhancing the second benefit.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.