A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65)

<p>Abstract</p> <p>Background</p> <p>Human glutamic acid decarboxylase 65 (hGAD65) is a key autoantigen in type 1 diabetes, having much potential as an important marker for the prediction and diagnosis of type 1 diabetes, and for the development of novel antigen-specific therapies for the treatment of type 1 diabetes. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell culture-based expression systems or nuclear transformed plants is limited by low yield and low efficiency. Chloroplast transformation of the unicellular eukaryotic alga <it>Chlamydomonas reinhardtii </it>may offer a potential solution.</p> <p>Results</p> <p>A DNA cassette encoding full-length <it>hGAD65</it>, under the control of the <it>C. reinhardtii </it>chloroplast <it>rbc</it>L promoter and 5'- and 3'-UTRs, was constructed and introduced into the chloroplast genome of <it>C. reinhardtii </it>by particle bombardment. Integration of <it>hGAD65 </it>DNA into the algal chloroplast genome was confirmed by PCR. Transcriptional expression of <it>hGAD65 </it>was demonstrated by RT-PCR. Immunoblotting verified the expression and accumulation of the recombinant protein. The antigenicity of algal-derived hGAD65 was demonstrated with its immunoreactivity to diabetic sera by ELISA and by its ability to induce proliferation of spleen cells from NOD mice. Recombinant hGAD65 accumulated in transgenic algae, accounts for approximately 0.25–0.3% of its total soluble protein.</p> <p>Conclusion</p> <p>Our results demonstrate the potential value of <it>C. reinhardtii </it>chloroplasts as a novel platform for rapid mass production of immunologically active hGAD65. This demonstration opens the future possibility for using algal chloroplasts as novel bioreactors for the production of many other biologically active mammalian therapeutic proteins.</p

Regulatory capacity building and the governance of clinical stem cell research in China

While other works have explained difficulties in applying ‘international’ guidelines in the field of regenerative medicine in so-called low- and middle-income countries (LMICs) in terms of ‘international hegemony’, ‘political and ethical governance’ and ‘cosmopolitisation’, this article on stem cell regulation in China emphasises the particular complexities faced by large LMICs: the emergence of alternative regulatory arrangements made by stakeholders at a provincial level at home. On the basis of ethnographic and archival research of clinical stem cell research hubs, we have characterized six types of entrepreneurial ‘bionetworks’, each of which embodies a regulatory orientation that developed in interaction with China’s regulatory dilemmas. Rather than adopting guidelines from other countries, we argue that regulatory capacity building is more appropriately viewed as a relational concept, referring to the ability to develop regulatory requirements that can cater for different regulatory research needs on an international level and at home

Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4

The bromodomain and extra-terminal (BET) family of proteins, comprised of four members including BRD2, BRD3, BRD4 and the testis-specific isoform BRDT, largely function as transcriptional co-activators 1–3 and play critical roles in various cellular processes, including cell cycle, apoptosis, migration and invasion 4,5. As such, BET proteins enhance the oncogenic functions of major cancer drivers by either elevating their expression such as c-Myc in leukemia 6,7 or by promoting transcriptional activities of oncogenic factors such as AR and ERG in the prostate cancer setting 8. Pathologically, BET proteins are frequently overexpressed and clinically linked to various types of human cancers 5,9,10, therefore pursued as attractive therapeutic targets for selective inhibition in patients. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed 11,12 and shown promising outcomes in early clinical trials. Despite resistance to BET inhibitor has been documented in pre-clinical models 13–15 the molecular mechanisms underlying acquired resistance are largely unknown. Here, we report that Cullin 3SPOP earmarks BET proteins including BRD2, BRD3 and BRD4 for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of BET proteins, leading to their elevated abundance in SPOP-deficient prostate cancer. As a result, prostate cancer cells and prostate cancer patient-derived organoids harboring SPOP mutations are more resistant to BET inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor suppressor role of SPOP in prostate cancer by negatively controlling BET protein stability, and also provide a molecular mechanism for BET inhibitor resistance in prostate cancer patients bearing SPOP mutations

Light regulation of the RNA-binding activity of 46 kDa protein in chloroplast of Chlamydomonas reinhardtii

In this thesis project, I undertook a study of the light regulation of a 46 kDa RNA binding protein associated with chloroplast membranes in C. reinhardtii . The optimal conditions for binding of this protein were characterized. Initial purification steps using (NH 4 ) 2 SO 4 precipitation and affinity chromatography revealed that this protein is present at extremely low abundance. This activity is low in the dark and increases drastically within 1-10 minute following a shift to light. This activation does not require protein synthesis in either the chloroplast or the cytosol. Finally activation is abolished by a proton ionophore and an inhibitor of the cytochrome b 6 /f complex, and partially reduced by an inhibitor of photosystem II. With a previous finding that ADP inhibits the RNA-binding activity of the protein in vitro , these results support a role of ADP in repression of this binding activity

The Pricing of Vulnerable Options in a Fractional Brownian Motion Environment

Under the assumption of the stock price, interest rate, and default intensity obeying the stochastic differential equation driven by fractional Brownian motion, the jump-diffusion model is established for the financial market in fractional Brownian motion setting. With the changes of measures, the traditional pricing method is simplified and the general pricing formula is obtained for the European vulnerable option with stochastic interest rate. At the same time, the explicit expression for it comes into being

Optimisation of the operating-power of the circuit breaker interrupter

To reduce the operation power of the circuit breaker interrupter, here, the operation power is divided into two parts: the arc reaction force and kinetic energy of the moving parts, then the reaction force and the reactive force work calculation method are put forward. Based on the above method, the operating power of the original structure and the improved structure of the interrupter are obtained, and the comparison of the results shows that the improved structure can reduce the operating power

Investigation of CFRP-Countersunk Bolted Assembly Fatigue Damage under Three-Point Bending via Experimental and Numerical Analysis

In this research, the fatigue damage behavior under three-point bending of a composite joint incorporating a single countersunk fastener is investigated. Firstly, a self-developed fatigue test system was set up to test the fatigue characteristics of CFRP-countersunk bolted assembly under the displacement amplitude cycles of 103 to 106 to study the formation and expansion rule of damage and cracks. It found two typical damage processes, both of which involve some formal interface damage between fiber and matrix. Based on the experiment, a finite element fatigue damage analysis on this assembly was carried out according to the Hashin failure criterion. The simulation result shows an identical fatigue damage location and fatigue life with the experimental phenomenon. Moreover, it predicted the final fatigue life of the specimen under 10 hz cyclic loading with 1 mm displacement and 10 Nm bolt preloading. This research provides guidance for the engineering fatigue issues of single-bolted joint composite connection structures and provides a reference for the corresponding technical specifications formulation