THERMOSTABLE EXOSHELLS AS TOOLS FOR RECOMBINANT PROTEIN EXPRESSION AND IN VIVO DELIVERY

Ph.DDOCTOR OF PHILOSOPH

miRNA-Based Therapeutics for Heart Failure: Why Not?

International audienceNo abstract availabl

Thermostable exoshells fold and stabilize recombinant proteins

The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.NMRC (Natl Medical Research Council, S’pore)Published versio

Surface protein engineering increases the circulation time of a cell membrane-based nanotherapeutic

Mammalian cell membranes are often incompatible with chemical modifications typically used to increase circulation half-life. Using cellular nanoghosts as a model, we show that proline-alanine-serine (PAS) peptide sequences expressed on the membrane surface can extend the circulation time of a cell membrane derived nanotherapeutic. Membrane expression of a PAS 40 repeat sequence decreased protein binding and resulted in a 90% decrease in macrophage uptake when compared with non-PASylated controls (P ≤ 0.05). PASylation also extended circulation half-life (t1/2 = 37 h) compared with non-PASylated controls (t1/2 = 10.5 h) (P ≤ 0.005), resulting in ~7-fold higher in vivo serum concentrations at 24 h and 48 h (P ≤ 0.005). Genetically engineered membrane expression of PAS repeats may offer an alternative to PEGylation and provide extended circulation times for cellular membrane-derived nanotherapeutics.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)National Medical Research Council (NMRC)CLD acknowledges support from the National Medical Research Council, NMRC/CSAINV17nov-0008, XCJ thanks support by A*STAR Biomedical Research Council (IAF-PP grant), and Singapore Ministry of Education Tier-1 Academic Research Funds (RG 131/15)

Surface protein engineering increases the circulation time of a cell membrane-based nanotherapeutic

10.1016/j.nano.2019.02.024NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE18169-17

Bioorthogonal Catalysis for Treatment of Solid Tumors Using Thermostable, Self-Assembling, Single Enzyme Nanoparticles and Natural Product Conversion with Indole-3-acetic Acid

10.1021/acsnano.1c11560ACS NANO16

data_sheet_1_The Importance of Sex Stratification in Autoimmune Disease Biomarker Research: A Systematic Review.DOCX

<p>The immune system is highly dynamic and regulated by many baseline characteristic factors. As such, significant variability may exist among different patient groups suffering from the same autoimmune disease (AD). However, contemporary research practices tend to take the reductionist aggregate approach: they do not segment AD patients before embarking on biomarker discovery. This approach has been productive: many novel AD biomarkers have recently been discovered. Yet, subsequent validation studies of these biomarkers tend to suffer from a lack of specificity, sensitivity, and reproducibility which hamper their translation for clinical use. To enhance reproducibility in validation studies, an optimal discovery-phase study design is paramount: one which takes into account different parameters affecting the immune system biology. In this systematic review, we highlight need for stratification in one such parameter, i.e., sex stratification. We will first explore sex differences in immune system biology and AD prevalence, followed by reported sex-bias in the clinical phenotypes of two ADs—one which more commonly affects females: systemic lupus erythematosus, and one which more commonly affects males: ankylosing spondylitis. The practice of sex stratification in biomarker research may not only advance the discovery of sex-specific AD biomarkers but more importantly, promote reproducibility in subsequent validation studies, thus easing the translation of these novel biomarkers from bench to bedside to improve AD diagnosis. In addition, such practice will also promote deeper understanding for differential AD pathophysiology in males and females, which will be useful for the development of more effective interventions for each sex type.</p

CAVIN1-Mediated hERG Dynamics: A Novel Mechanism Underlying the Interindividual Variability in Drug-Induced Long QT

International audienceBACKGROUND: Drug-induced QT prolongation (diLQT) is a feared side effect that could expose susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG (human ether-a-go-go-related gene) channels that generate the delayed-rectifier potassium current (I Kr ) and thereby regulate the late repolarization phase. There is an important interindividual susceptibility to develop diLQT, which is of unknown origin but can be reproduced in patient-specific induced pluripotent stem cell–derived cardiomyocytes (iPS-CMs). We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT. METHODS: We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest sensitivity (HS) or lowest sensitivity (LS) to sotalol administration in vivo (ie, on the basis of the measure of the maximal change in QT interval 3 hours after administration). Specific small interfering RNAs and CAVIN1-T2A-GFP adenovirus were used to manipulate CAVIN1 expression. RESULTS: Whereas HS and LS iPS-CMs showed similar electrophysiological characteristics at baseline, the late repolarization phase was prolonged and I Kr significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. I Kr reduction was caused by a rapid translocation of hERG channel from the membrane to the cytoskeleton-associated fractions upon sotalol application. CAVIN1 , essential for caveolae biogenesis, was 2× more highly expressed in HS iPS-CMs, and its knockdown by small interfering RNA reduced their sensitivity to sotalol. CAVIN1 overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. We found that treatment with sotalol promoted translocation of the hERG channel from the plasma membrane to the cytoskeleton fractions in a process dependent on CAVIN1 (caveolae associated protein 1) expression. CAVIN1 silencing reduced the number of caveolae at the membrane and abrogated the translocation of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers, such as E4031, vandetanib, and clarithromycin. CONCLUSIONS: Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the interindividual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1

The Importance of Sex Stratification in Autoimmune Disease Biomarker Research: A Systematic Review

The immune system is highly dynamic and regulated by many baseline characteristic factors. As such, significant variability may exist among different patient groups suffering from the same autoimmune disease (AD). However, contemporary research practices tend to take the reductionist aggregate approach: they do not segment AD patients before embarking on biomarker discovery. This approach has been productive: many novel AD biomarkers have recently been discovered. Yet, subsequent validation studies of these biomarkers tend to suffer from a lack of specificity, sensitivity, and reproducibility which hamper their translation for clinical use. To enhance reproducibility in validation studies, an optimal discovery-phase study design is paramount: one which takes into account different parameters affecting the immune system biology. In this systematic review, we highlight need for stratification in one such parameter, i.e., sex stratification. We will first explore sex differences in immune system biology and AD prevalence, followed by reported sex-bias in the clinical phenotypes of two ADs—one which more commonly affects females: systemic lupus erythematosus, and one which more commonly affects males: ankylosing spondylitis. The practice of sex stratification in biomarker research may not only advance the discovery of sex-specific AD biomarkers but more importantly, promote reproducibility in subsequent validation studies, thus easing the translation of these novel biomarkers from bench to bedside to improve AD diagnosis. In addition, such practice will also promote deeper understanding for differential AD pathophysiology in males and females, which will be useful for the development of more effective interventions for each sex type