Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonance

The biological function of protein, DNA, and RNA molecules often depends on relative movements of domains with dimensions of a few nanometers. This length scale can be accessed by distance measurements between spin labels if pulsed electron paramagnetic resonance (EPR) techniques such as electron-electron double resonance (ELDOR) and double-quantum EPR are used. The approach does not require crystalline samples and is well suited to biomacromolecules with an intrinsic flexibility as distributions of distances can be measured. Furthermore, oligomerization or complexation of biomacromolecules can also be studied, even if it is incomplete. The sensitivity of the technique and the reliability of the measured distance distribution depend on careful optimization of the experimental conditions and procedures for data analysis. Interpretation of spin-to-spin distance distributions in terms of the structure of the biomacromolecules furthermore requires a model for the conformational distribution of the spin labels.     

The closure of the smallest ideal of an ultrafilter semigroup

Let S be a discrete semigroup, let β S be the Stone-Čech compactification of S, and let T be a closed subsemigroup of β S. We characterize ultrafilters from the smallest ideal K(T) of T and from its closure c ℓ  K(T). We show that, for a large class of closed subsemigroups of β S, c ℓ  K(T) is not an ideal of T. This class includes the subsemigroups 0⁺⊂βℝ _d and ℍ _κ ⊂β(⊕ _κ ℤ₂).     

Radicals of some semigroup algebras

In this paper we seek to determine the Jacobson radical of certain algebras based on semigroups, and in particular on the semigroups (βS,□), where S is a cancellative, countable, abelian semigroup and βS is its Stone–?ech semigroup compactification. In particular, we wish to determine the radical of ? ^?1(β?).     

A simple model of bit whirl for deep drilling applications

A simple analytical model of bit whirl is presented. In contrast to the previous works, which consider bottom hole assembly mass imbalance to model bit whirl, the model here takes into account the history-dependent boundary conditions at the bit–rock interface as well as the bit geometry. In particular, an analytical expression for the bit–rock interaction is derived. It is shown that the bit geometry affects the bit–rock interaction only through three dimensionless parameters. Depending on the value of these parameters the system can be stable or undergo forward or backward whirl.     

Rotamer libraries of spin labelled cysteines for protein studies

Studies of structure and dynamics of proteins using site-directed spin labelling rely on explicit modelling of spin label conformations. The large computational effort associated with such modelling with molecular dynamics (MD) simulations can be avoided by a rotamer library approach based on a coarse-grained representation of the conformational space of the spin label. We show here that libraries of about 200 rotamers, obtained by iterative projection of a long MD trajectory of the free spin label onto a set of canonical dihedral angles, provide a representation of the underlying trajectory adequate for EPR distance measurements. Rotamer analysis was performed on selected X-ray structures of spin labelled T4 lysozyme mutants to characterize the spin label rotamer ensemble on a single protein site. Furthermore, predictions based on the rotamer library approach are shown to be in nearly quantitative agreement with electron paramagnetic resonance (EPR) distance data on the Na(+)/H(+) antiporter NhaA and on the light-harvesting complex LHCII whose structures are known from independent cryo electron microscopy and X-ray studies, respectively. Suggestions for the selection of labelling sites in proteins are given, limitations of the approach discussed, and requirements for further development are outlined.     

Photosubstitution in a trisheteroleptic ruthenium complex inhibits conjunctival melanoma growth in a zebrafish orthotopic xenograft model

In vivo data are rare but essential for establishing the clinical potential of ruthenium-based photoactivated chemotherapy (PACT) compounds, a new family of phototherapeutic drugs that are activated via ligand photosubstitution. Here a novel trisheteroleptic ruthenium complex [Ru(dpp)(bpy)(mtmp)](PF₆)₂ ([2](PF₆)₂, dpp = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2′-bipyridine, mtmp = 2-methylthiomethylpyridine) was synthesized and its light-activated anticancer properties were validated in cancer cell monolayers, 3D tumor spheroids, and in embryonic zebrafish cancer models. Upon green light irradiation, the non-toxic mtmp ligand is selectively cleaved off, thereby releasing a phototoxic ruthenium-based photoproduct capable notably of binding to nuclear DNA and triggering DNA damage and apoptosis within 24–48 h. In vitro, fifteen minutes of green light irradiation (21 mW cm⁻², 19 J cm⁻², 520 nm) were sufficient to generate high phototherapeutic indexes (PI) for this compound in a range of cancer cell lines including lung (A549), prostate (PC3Pro4), conjunctival melanoma (CRMM1, CRMM2, CM2005.1) and uveal melanoma (OMM1, OMM2.5, Mel270) cancer cell lines. The therapeutic potential of [2](PF₆)₂ was further evaluated in zebrafish embryo ectopic (PC3Pro4) or orthotopic (CRMM1, CRMM2) tumour models. The ectopic model consisted of red fluorescent PC3Pro4-mCherry cells injected intravenously (IV) into zebrafish, that formed perivascular metastatic lesions at the posterior ventral end of caudal hematopoietic tissue (CHT). By contrast, in the orthotopic model, CRMM1- and CRMM2-mCherry cells were injected behind the eye where they developed primary lesions. The maximally-tolerated dose (MTD) of [2](PF₆)₂ was first determined for three different modes of compound administration: (i) incubating the fish in prodrug-containing water (WA); (ii) injecting the prodrug intravenously (IV) into the fish; or (iii) injecting the prodrug retro-orbitally (RO) into the fish. To test the anticancer efficiency of [2](PF₆)₂, the embryos were treated 24 h after engraftment at the MTD. Optimally, four consecutive PACT treatments were performed on engrafted embryos using 60 min drug-to-light intervals and 90 min green light irradiation (21 mW cm⁻², 114 J cm⁻², 520 nm). Most importantly, this PACT protocol was not toxic to the zebrafish. In the ectopic prostate tumour models, where [2](PF₆)₂ showed the highest photoindex in vitro (PI > 31), the PACT treatment did not significantly diminish the growth of primary lesions, while in both conjunctival melanoma orthotopic tumour models, where [2](PF₆)₂ showed more modest photoindexes (PI ∼ 9), retro-orbitally administered PACT treatment significantly inhibited growth of the engrafted tumors. Overall, this study represents the first demonstration in zebrafish cancer models of the clinical potential of ruthenium-based PACT, here against conjunctival melanoma.

A new tris-heteroleptic photoactivated chemotherapy ruthenium complex induces apoptosis upon green light activation in a zebrafish orthothopic conjunctival melanoma xenograft model.