Sweeter and stronger: Enhancing sweetness and stability of the single chain monellin MNEI through molecular design

Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interactio

Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding.

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties

Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers

Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed

Physiological and ultrastructural effects of acute ozone fumigation in the lichen Xanthoria parietina: the role of parietin and hydration state

The physiological and ultrastructural effects induced by acute exposure to ozone (O3) were investigated in the lichen Xanthoria parietina. Our working hypothesis was that parietin content and hydration of the thalli may play a role in the modulation of the effects of O3 exposure. Four batches of X. parietina samples, dry and wet, with (P+) and without (Pâ\u88\u92) parietin, were fumigated for 1 h with 3 ppm O3. The effects of O3 were assessed immediately after the fumigation and after one week of recovery under controlled conditions. O3 fumigation caused physiological and ultrastructural impairment both to the photobiont and the mycobiont, irrespective if samples were fumigated wet or dry, and P+ or Pâ\u88\u92. However, one week after fumigation, a recovery was observed in P+ samples for the photobiont and in dry samples for the mycobiont. We suggest that the hydration state may play a major role in determining the severity of the damage, while the presence of parietin may promote the recovery. Our results provide physiological and ultrastructural basis to explain the ecological insensitivity of lichens to high environmental levels of ozone occurring during dry Mediterranean summers

3D tortuosity and diffusion characterization in the human mineralized collagen fibril using a random walk model

Bone tissue is mainly composed at the nanoscale of apatite minerals, collagen molecules and water that form the mineralized collagen fibril (MCF). In this work, we developed a 3D random walk model to investigate the influence of bone nanostructure on water diffusion. We computed 1000 random walk trajectories of water molecules within the MCF geometric model. An important parameter to analyse transport behaviour in porous media is tortuosity, computed as the ratio between the effective path length and the straight-line distance between initial and final points. The diffusion coefficient is determined from the linear fit of the mean squared displacement of water molecules as a function of time. To achieve more insight into the diffusion phenomenon within MCF, we estimated the tortuosity and diffusivity at different quotes in the longitudinal direction of the model. Tortuosity is characterized by increasing values in the longitudinal direction. As expected, the diffusion coefficient decreases as tortuosity increases. Diffusivity outcomes confirm the findings achieved by experimental investigations. The computational model provides insights into the relation between the MCF structure and mass transport behaviour that may contribute to the improvement of bone-mimicking scaffolds

A new hexapeptide from the leader peptide of rMnSOD enters cells through the oestrogen receptor to deliver therapeutic molecules

A 24-amino acid leader peptide of a new human recombinant manganese superoxide dismutase can enter cells and carry molecules. Here, we demonstrated that six of the 24 amino acids penetrate cells through a particular gate represented by a specific amino acid sequence of the oestrogen receptor (ER). We analysed the internalization of the synthetic hexapeptide and the cytotoxic activity of the hexapeptide conjugated to cisplatin on a cell line panel. In most cell lines, the hexapeptide delivered an amount of cisplatin that was 2 to 8 times greater than that released by cisplatin when the drug was used alone. This increased delivery increases the therapeutic index of cisplatin and reduces side effects caused by a high dosage or long-term treatment times. We may consider this hexapeptide a new molecular carrier to deliver molecules with therapeutic activity into ER+ cells for diagnostic purposes and clinical or immune therapy

Reweighting twisted boundary conditions

Imposing twisted boundary conditions on the fermionic fields is a procedure extensively used when evaluating, for example, form factors on the lattice. Twisting is usually performed for one flavour and only in the valence, and this causes a breaking of unitarity. In this work we explore the possibility of restoring unitarity through the reweighting method. We first study some properties of the approach at tree level and then we stochastically evaluate ratios of fermionic determinants for different boundary conditions in order to include them in the gauge averages, avoiding in this way the expensive generation of new configurations for each choice of the twisting angle, θ. As expected the effect of reweighting is negligible in the case of large volumes but it is important when the volumes are small and the twisting angles are large. In particular we find a measurable effect for the plaquette and the pION correlation function in the case of θ = π/2 in a volume 16 X 8 3, and we observe a systematic upward shift in the pION dispersion relation. </p