Versatile enzymatic system for the production of guanosine polyphosphates

Posters - ED02 Signalling and systems biology: abstract no. ED02/20During periods of environmental stress, bacteria synthesize guanosine tetraphosphate (ppGpp, magic spot I) and guanosine pentaphosphate (pppGpp, magic spot II) in a process known as the stringent response. These intracellular allarmone molecules ‘reprogramme’ the transcriptional and translational machinery to help the cell conserve scarce resources. Existing methods for the production of guanosine polyphosphates are either technically difficult or inefficient, hindering investigations into their biological roles. We have developed a simple and efficient one-step enzymatic method for the production of guanosine polyphosphates using a recombinant protein cloned from Staphylococcus aureus. The purified enzyme efficiently catalyses the formation of pppGpp (and AMP) from GTP + ATP; and ppGpp (and AMP) from GDP + ATP. Notably, it also catalyses the synthesis of pGpp (guanosine 5’-monophosphate 3’-diphosphate, and AMP) from GMP + ATP; albeit with reduced efficiency. The reverse reactions are not catalysed, leading to high conversion rates. Guanosine polyphosphate products can be obtained in a homogeneous form using a combination of anion exchange chromatography followed by desalting. Our approach can be used to produce guanosine polyphosphates on a multi-milligram scale. Furthermore, our results also suggest that a third ‘magic spot’ allarmone may be formed within certain bacterial species.postprintThe Apring 2010 Meeting of the Society for General Microbiology (SGM), Edinburgh, U.K., 29 March-1 April 2010. In Abstract Book of the SGM Spring 2010 Meeting, 2010, p. 9

Applying Web 2.0 in medical-related organizations

This study investigated the application of Web 2.0 to medical-related organizations. Thirty organizations participated in an online survey asking their perceived purposes, benefits and difficulties in using Web 2.0. The selected organizations fell into three categories: university medical libraries, hospitals, and non-profit organizations. Fourteen (46.7%) organizations were currently using Web 2.0, ten (33.3%) planned to use it in the future and six (20%) would not consider using it. A phone interview was further conducted with eight organizations (26.7%) about their opinion on Web 2.0. Results showed that most participants found the application of Web 2.0 beneficial to their organizations. Implications of this study for helping medical-related organizations make decisions regarding the use of Web 2.0 technologies in their organizations are discussed.postprintThe 6th International Conference on Knowledge Management (ICKM 2009), Hong Kong, 3-4 December 2009. In Proceedings of ICKM, 2009, p. 1-1

Sedlin and prostaglandin E2 dehydrogenase - interactions and implications for spondyloepiphyseal dysplasia tarda

Session D - Genomic Disorders: abstract no. D020Spondyloepiphyseal dysplasia tarda (SEDT) is a rare X-linked, late-onset skeletal disease. Affected individuals develop phenotypes in their early childhood, displaying barrel-shaped chests, vertebral bodies malformation, flattened disc spaces and premature osteoarthritis in weight-bearing joints. The disease was found linked to the gene SEDL coding for the protein sedlin. Sedlin is one of the subunits of the TRAPP (Transport Protein Particle) complex, which is responsible for vesicle tethering during endoplasmic reticulum-to-golgi transport. Although sedlin is known to function in intracellular trafficking, the reason why mutations in a trafficking protein lead to a skeletal disease remains unknown. To address this, four missense mutations (D47Y, S73L, F83S and V130D) of sedlin observed in SEDT patients were studied. Except D47Y, the other three mutations cause proteosomal degradation of sedlin in cultured cells, whereas the D47Y mutation had a minor effect on Bet3 binding to sedlin. Pull-down assay was performed to identify novel sedlin interacting partners. 15-hydroxyprostaglandin dehydrogenase (PGDH) was pulled down and the interaction was confirmed in cell culture system. Sedlin activates PGDH activity in vitro. By confocal microscopy, sedlin was also found to colocalize with PGDH in the cytosol. PGDH catalyzes the degradation of prostaglandin E2, which affects cartilage and bone growth. Further investigation is ongoing to understand the function of sedlin and the mechanism of disease for SEDT.postprintThe 1st International Congress on Research of Rare and Orphan Diseases (RE(ACT) 2012), Basel, Switzerland, 29 February-2 March 2012. In Brochure of RE(ACT)® 2012, 2012, p. 11

The effect of simultaneous dual-focus integration on the global flash multifocal electroretinogram in the human eye

202205 bcfcAccepted ManuscriptRGCPublishe

Embryonic Disruption Of The Candidate Dyslexia Susceptibility Gene Homolog Kiaa0319-like Results In Neuronal Migration Disorders.

Developmental dyslexia, the most common childhood learning disorder, is highly heritable, and recent studies have identified KIAA0319-Like (KIAA0319L) as a candidate dyslexia susceptibility gene at the 1p36-34 (DYX8) locus. In this experiment, we investigated the anatomical effects of knocking down this gene during rat corticogenesis. Cortical progenitor cells were transfected using in utero electroporation on embryonic day (E) 15.5 with plasmids encoding either: (1) Kiaa0319l shRNA, (2) an expression construct for human KIAA0319L, (3) Kiaa0319l shRNA + KIAA0319L expression construct (rescue), or (4) controls (scrambled Kiaa0319l shRNA or empty expression vector). Mothers were injected with BrdU at either E13.5, E15.5, or E17.5. Disruption of Kiaa0319l function (by knockdown, overexpression, or rescue) resulted in the formation of large nodular periventricular heterotopia in approximately 25% of the rats, and these heterotopia can be seen as early as postnatal day 1. Only a small subset of heterotopic neurons had been transfected, indicating non-cell autonomous effects of the transfection. Most heterotopic neurons were generated in mid- to late-gestation, and laminar markers suggest that they were destined for upper cortical laminæ. Finally, we found that transfected neurons in the cerebral cortex were located in their expected laminæ. These results indicate that KIAA0319L is the fourth of four candidate dyslexia susceptibility genes that is involved in neuronal migration, which supports the association of abnormal neuronal migration with developmental dyslexia