Directed evolution of cyclic peptides for inhibition of autophagy

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B—two essential components of the autophagosome maturation machinery—with mid-nanomolar affinities and disrupted protein–protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

SUPR peptide mRNA display was used to evolve a cell-permeable, macrocyclic peptide for autophagy inhibition.     

The Application of 2D NMR Techniques in the Structural Assignment of the Diterpenoid Alkaloid,Delphinine

The two dimensional inverse detected heteronuclear correlation experiment HMQC and the homonuclear correlation experiments COSY and ROESY were performed on the natural alkaloid, delphinine enabling complete assignments of the ¹H and ¹³C spectra. The stereochemistry of ring A and B have been determined in solution. The results suggest that the application of direct correlation multipulse NMR techniques allows for unambiguous structural assignment of delphinine.     

Hard X‐ray phase‐contrast tomography of non‐homogeneous specimens: grating interferometry versus propagation‐based imaging

X‐ray phase‐contrast imaging is an effective approach to drastically increase the contrast and sensitivity of microtomographic techniques. Numerous approaches to depict the real part of the complex‐valued refractive index of a specimen are nowadays available. A comparative study using experimental data from grating‐based interferometry and propagation‐based phase contrast combined with single‐distance phase retrieval applied to a non‐homogeneous sample is presented (acquired at beamline ID19‐ESRF). It is shown that grating‐based interferometry can handle density gradients in a superior manner. The study underlines the complementarity of the two techniques for practical applications.     

Conformational study of insect adipokinetic hormones using NMR constrained molecular dynamics

Mem-CC (pGlu-Leu-Asn-Tyr-Ser-Pro-Asp-Trp-NH₂), Tem-HrTH (pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-NH₂) and Del-CC (pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn-NH₂) are adipokinetic hormones, isolated from the corpora cardiaca of different insect species. These hormones regulate energy metabolism during flight and so are intimately involved in an insect's mobility. Secondary structural elements of these peptides and the N7 analogue, [N7]-Mem-CC (pGlu-Leu-Asn-Tyr-Ser-Pro-Asn-Trp-NH₂), have been determined in dimethylsulfoxide solution using NMR restrained molecular mechanic simulations. The neuropeptides were all found to have an extended structure for the first 4 residues and a -turn between residues 4–8. For Tem-HrTH and Del-CC, asparagine (N7) which is postulated to be involved in receptor binding and/or activation, projects outward form the -turn. Mem-CC does not have an asparagine at position 7 while, for [N7]-Mem-CC, the N7 sidechain folds inside the -turn preventing its interaction with the receptor.     

NMR Study of Insect Adipokinetic Hormones

Mem-CC (pGlu-Leu-Asn-Tyr-Ser-Pro-Asp-Trp-NH₂), Tem-HrTH (pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-NH₂) and Del-CC (pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-GIy-Asn-NH₂) are adipokinetic hormones, isolated from the corpora cardiaca of different insect species. These hormones regulate energy metabolism during flight and so are intimately involved in an insect's mobility. In an attempt to measure the solution conformations of these peptides, their ₁H nmr spectra have been recorded in dimethylsulfoxide solution. the N7 analogue, [N7]-Mem-CC (pGlu-Leu-Asn-Tyr-Ser-Pro-Asn-Trp-NH₂) has also been studied. the solution dynamics of Tem-HrTH has been simulated in dmso and in a water droplet. These results have been correlated with measured coupling constants. the molecule was found to have a (β-turn at proline with asparagine (N7), which is postulated to be involved in receptor binding and/or activation, projecting outward from the turn.     

Applying an alternative mathematics pedagogy for students with weak mathematics: meta-analysis of alternative pedagogies

Student mathematics performance and the need for work-ready graduates to be mathematics-competent is a core issue for many universities. While both student and teacher are responsible for learning outcomes, there is a need to explicitly acknowledge the weak mathematics foundation of many university students. A systematic literature review was undertaken of identified innovations and/or interventions that may lead to improvement in student outcomes for university mathematics-based units of study. The review revealed the importance of understanding the foundations of student performance in higher education mathematics learning, especially in first year. Pre-university mathematics skills were identified as significant in student retention and mathematics success at university, and a specific focus on student pre-university mathematics skill level was found to be more effective in providing help, rather than simply focusing on a particular at-risk group. Diagnostics tools were found to be important in identifying (1) student background and (2) appropriate intervention. The studies highlighted the importance of appropriate and validated interventions in mathematics teaching and learning, and the need to improve the learning model for mathematics-based subjects, communication and technology innovations.     

Adsorption reactions of alkali-halide nanocrystals: Identification of an important surface defect

The adsorption reactions of alkali-halide clusters have been investigated on a size-selected basis using flow-reactor methods. The reactivity of larger sodium-fluoride clusters [Na_nF_n?1]⁺ toward polar molecules NH₃ and H₂O shows a distinctive pattern as a function ofn at ambient temperature. Comparison with computed structures shows that aparticular kind of defect greatly facilitates the initial adsorption process. This defect can be formed by removal of an ion-pair from the face of an otherwise perfect nanocrystal, to create a basket-like opening for the adsorbed molecule. It is shown that this kind of defect occurs as a most stable low-temperature structure only to a certain size, after which a less reactive defect takes its place. The implications of these findings for the adsorption reactivity of the surfaces of ionic solids are briefly discussed.     

Phase and mixing behavior in two-component lipid bilayers: a molecular dynamics study in DLPC/DSPC mixtures

Phase and mixing behavior of dilauroylphosphatidylcholine (DLPC)/distearoylphosphatidylcholine (DSPC) lipid mixtures are studied by molecular dynamics simulations with use of a coarse-grained model over a wide range of concentrations. The results reveal that phase transformations from the fluid to the gel state can be followed over a microsecond time scale. The changes in structure suggest regions of phase coexistence allowing us to outline the entire phase diagram for this lipid mixture using a molecular based model. We show that simulations yield good agreement with the experimental phase diagram. We also address the effect of macroscopic phase separation on the determination of the transition temperature, different leaflet composition, and finite size effects. This study may have implications on lateral membrane organization and the associated processes dependent on these membrane regions on different time and length scales.