The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications

BACKGROUND: Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme. RESULTS: Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophila's DHFR-TS is presented. A first aspect of the presented work relates to destruction of DHFR-TS enzyme function in an alveolate thereby causing an auxotrophy for thymidine. A second aspect is to knock in an expression cassette encoding for a foreign gene with subsequent expression of the target protein. CONCLUSION: This system avoids the use of antibiotics or other drugs and therefore is of high interest for biotechnological applications

A recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila

BACKGROUND: Tetrahymena thermophila is one of the best characterized unicellular eukaryotes and its genome is sequenced in its entirety. However, the AT-richness of the genome and an unusual codon usage cause problems in cloning and expression of the ciliate DNA. To overcome these technical hiatuses we developed a Cre-dependent recombinase system. RESULTS: We created novel donor and acceptor vectors that facilitate the transfer of expression cassettes from the donor into novel acceptor plasmid. Expression vectors were used that encode the 19 kDa C-terminus of the MSP1 protein of Plasmodium falciparum and a blasticidin S (bsdR) resistance gene, respectively. The functional expression of these genes was demonstrated by western blot analysis with MSP1 specific antibodies and by a blasticidin growing assay. CONCLUSION: The Cre dependent recombinase system in combination with the modular structure of the donor vectors ease cloning and expression of foreign genes in the ciliate system, providing a powerful tool for protistology research in future

Subject-Specific Ablation of Pathologic Conduction Patterns Beyond the Pulmonary Veins: A Personalised Modelling Approach

Improving patient outcomes with ablation of non-paroxysmal AF (PsAF) has proved challenging using a population-based treatment approach due to large interindividual variability in the underlying electroanatomical substrate. Ablation of pathologic conduction patterns outside of pulmonary vein isolation (PVI) has recently shown encouraging results in PsAF patients returning for their first or second retreatment (76% freedom from AF recorded in the RECOVER AF trial). However, the optimal targets and best sequence of ablation lesions are still unknown, and testing different sequences, types, and methods of ablation cannot be performed clinically on a single patient or patient cohort. Considering the predictive potential of computational modelling, a small exploratory subset of patients (N=4) enrolled in the ongoing DISCOVER trial was used to create patient-specific models of left atrial electrophysiology. The subject-specific models displayed a high correlation between simulated targets and clinical targets. AF complexity was highest in all patients prior to therapy. PVI caused a marginal decrease in complexity across the cohort whereas PVI+PCP showed an extensive decrease in the AF complexity across the patients and resulted in AF termination in all patients

Expression, secretion and surface display of a human alkaline phosphatase by the ciliate Tetrahymena thermophila

<p>Abstract</p> <p>Background</p> <p><it>Tetrahymena thermophila </it>possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites <it>Ichthyophthirius multifiliis </it>and <it>Plasmodium falciparum </it>and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that <it>T. thermophila </it>is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using <it>T. thermophila </it>and thereby presents a powerful tool for the optimization of the ciliate-based expression system.</p> <p>Results</p> <p>Functional and full length human intestinal alkaline phosphatase was expressed by <it>T. thermophila </it>using a codon-adapted gene containing the native signal-peptide and GPI (Glycosylphosphatidylinositol) anchor attachment signal. HiAP activity in the cell extract of transformants suggested that the hiAP gene was successfully expressed. Furthermore, it was demonstrated that the enzyme was modified with N-glycosylation and localized on the surface membrane by the C-terminal GPI anchor. A C-terminally truncated version of hiAP lacking the GPI anchor signal peptide was secreted into the medium as an active enzyme. In a first approach to establish a high level expression system up to 14,000 U/liter were produced in a time frame of two days, which exceeds the production rate of other published expression systems for this enzyme.</p> <p>Conclusions</p> <p>With the expression of hiAP, not only a protein of commercial interest could be produced, but also a reporter enzyme that offers the possibility to analyze <it>T. thermophila </it>genes that play a role in the regulation of protein secretion. Additionally, the fact that ciliates do not secrete an endogenous alkaline phosphatase provides the possibility to use the truncated hiAP as a reporter enzyme, allowing the quantification of measures that will be necessary for further optimization of the host strains and the fermentation processes.</p

Secretion of functional human enzymes by Tetrahymena thermophila

BACKGROUND: The non-pathogenic ciliate Tetrahymena thermophila is one of the best-characterized unicellular eucaryotes used in various research fields. Previous work has shown that this unicellular organism provides many biological features to become a high-quality expression system, like multiplying to high cell densities with short generation times in bioreactors. In addition, the expression of surface antigens from the malaria parasite Plasmodium falciparum and the ciliate Ichthyophthirius multifiliis suggests that T. thermophila might play an important role in vaccine development. However, the expression of functional mammalian or human enzymes remains so far to be seen. RESULTS: We have been able to express a human enzyme in T. thermophila using expression modules that encode a fusion protein consisting of the endogenous phospholipase A(1 )precursor and mature human DNaseI. The recombinant human enzyme is active, indicating that also disulfide bridges are correctly formed. Furthermore, a detailed N-glycan structure of the recombinant enzyme is presented, illustrating a very consistent glycosylation pattern. CONCLUSION: The ciliate expression system has the potential to become an excellent expression system. However, additional optimisation steps including host strain improvement as wells as measures to increase the yield of expression are necessary to be able to provide an alternative to the common E. coli and yeast-based systems as well as to transformed mammalian cell lines

Calculation of Left Ventricular Relaxation Time Constant-Tau in Humans by Continuous-Wave Doppler

Left ventricular relaxation time constant, Tau, is the best index to evaluate left ventricular diastolic function, but the measurement is only available traditionally in catheter lab. In Echo lab, several methods of non-invasive measurement of Tau have been tried since 1992, however almost all the methods are still utilizing the same formula to calculate Tau as in catheter lab, which makes them inconvenient, time-consuming and sometimes not very accurate. Based on Weiss’ formula and simplified Bernoulli’s equation, a simple method is developed by pure mathematical derivative to calculate Tau by continuous-wave Doppler in patients with mitral regurgitation

Influence of Anaesthesia on Mobilisation Following Hip Fracture Surgery : An Observational Study: 麻醉技術對髖部骨折病人術後活動能力的影響：一項觀察性研究

Background Anaesthetic technique can influence mortality and morbidity following hip fracture surgery. However, its influence on postoperative mobilisation is not clear. In this study, we evaluated the influence of anaesthetic technique on postoperative mobilisation. Methods In this prospective observational study, we included all consecutive patients who underwent surgery for hip fracture between 1 January 2012 and 31 December 2013 at our institution. Any patients who died prior to mobilisation or who could not be followed up after surgery were excluded. Data was collected on demographics, clinical characteristics, anaesthesia technique and surgical factors, and date and time of admission, operation, first mobilisation and discharge. Results Of the 1040 patients included in the analysis, 264 received general anaesthesia only (Group GA), 322 received general anaesthesia with regional anaesthesia (Group GARA), and 454 received central neuraxial blockade anaesthesia with or without sedation (Group CNB). There was no significant difference in age (p = 0.56), sex (p = 0.23), number of comorbidities (p = 0.06), residential status (p = 0.18), time to surgery (p = 0.10) and length of hospital stay (p = 0.30) between the three groups. There was a statistically significant difference in ASA grade (p = 0.01), implant type used (p = 0.04), grade of operating surgeon (p = 0.02) and grade of anaesthetist during surgery (p = 0.004) among the three groups. Patients in Group GARA had a median time-to-first mobilisation of 23.8 hours after surgery, compared to 24.1 hours in Group GA and 24.3 hours in Group CNB. This difference was not statistically significant after controlling for confounding factors (p = 0.45). Conclusion Our results show that anaesthetic technique does not influence time-to-first mobilisation after hip fracture surgery