Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex

The purinergic receptor P2X(7) is part of a complex signaling mechanism participating in a variety of physiological and pathological processes. Depending on the activation scheme, P2X(7) receptors in vivo are non-selective cation channels or form large pores that can mediate apoptotic cell death. Expression of P2X(7)R in Xenopus oocytes results exclusively in formation of a non-selective cation channel. However, here we show that co-expression of P2X(7)R with pannexin1 in oocytes leads to the complex response seen in many mammalian cells, including cell death with prolonged ATP application. While the cation channel activity is resistant to carbenoxolone treatment, this gap junction and hemichannel blocking drug suppressed the currents induced by ATP in pannexin1/P2X(7)R co-expressing cells. Thus, pannexin1 appears to be the molecular substrate for the permeabilization pore (or death receptor channel) recruited into the P2X(7)R signaling complex.     

Simulating the embolization of blood vessels using magnetic microparticles and acupuncture needle in a magnetic field

Computer models were developed to simulate the capture and subsequent deposition of magnetic microparticles (MMPs) in a blood vessel adjacent to a ferromagnetic wire (e.g., acupuncture needle) magnetized by a uniform external magnetic field. Process parameter conditions were obtained to enable optimal capture of MMPs into the deposit. It was found that the maximum capture distance of the MMPs was within 0.5-2.0 mm when the particles were superparamagnetic and had large size (>1.0 microm) and relative large flow rates (2.5-5.0 cm/s) as in a healthy artery. It was also found that the deposits were asymmetrical and that their size was between 1.0 and 2.0 mm. For the case of lower flow rates as can be found in a tumor (<1.0 mm/s) and using small magnetite particles (0.25-2.0 microm) the maximum capture distance was larger, ranging between approximately 0.5 and 6.4 mm, depending on the blood flow rate, the radius of wire, and particle clustering. The range of embolization (deposition) in this later case was between 0.5 and 5.9 mm. The potential of this technique to generate MMPs deposits to embolize blood vessels inhibiting the blood supply and thus facilitating necrosis of tumors located deep within the patient (3-7 cm) is discussed.     

pH modulation of transport properties of alamethicin oligomers inserted in zwitterionic-based artificial lipid membranes

Electric features of biological membranes are major determinants of the function and physiological manifestation of membrane-penetrating peptides, and such features are prone to be modulated by the properties of the surrounding aqueous medium. In this work, we demonstrate that pH plays crucial roles in modulating electric characteristics of zwitterionic-based artificial lipid membranes. The effect of pH on electrical properties of such membranes was probed by evaluating the transport properties of embedded alamethicin oligomers over a wide range of pH values (i.e., 0.65, 2.08, 2.94, 7 and 10.1). Our data strongly support the paradigm of a pH-dependent variation of the surface and membrane dipole potential which, in conjunction with possible lateral pressure effects within the lipid membrane, lead to a non-monotonic modulation of the electrical conductance of alamethicin oligomers. As expected, pH modulation of transport properties through the alamethicin oligomer is more visible for narrower pores (that is, the 1st conductive state) with slightly better cation selectivity as compared to larger oligomers.     

Farming for pests? Local and landscape-scale effects of grassland management on rabbit densities

In recent decades in the UK, there has been an increasing trend in numbers of the European wild rabbit, a significant agricultural pest typically associated with grassland habitats. However, the relationship between rabbit abundance and grassland management, in particular grazing, has not been sufficiently explained. We studied rabbit densities in seven pasture-dominated sites in north-east England between autumn and spring in two consecutive years, and used generalised linear mixed models and generalised additive models to explore relationships between habitat and management variables and rabbit abundance at local (field) and landscape scales. At the local scale high rabbit densities were significantly associated with small fields and the very short, homogeneous swards created by intensive sheep grazing during autumn and winter. At the landscape scale, high rabbit numbers were associated with sites with most field margins and a predator removal policy. Our results indicate that landscape management may play a central role in explaining rabbit abundance and distribution in grasslands. We suggest that current pasture management may create favourable conditions for high rabbit densities, and consequently boost numbers of this significant pest species.     

High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent dye

We describe a development of a novel high-throughput phagocytosis assay based on a pH-sensitive cyanine dye, CypHer5E, which is maximally fluorescent in an acidic environment. This dye is ideally suited for the study of phagocytosis because of the acidic conditions generated in the intracellular phagocytic vesicles after particle uptake. Use of CypHer5E-labeled particles results in greatly reduced background from noninternalized particles and makes the assay more robust. Additionally, CypHer5E-labeled particles are resistant to fluorescence quenching observed in the aggressive and acidic environment of the phagosome with traditional dyes. The CypHer5E-based assay has been shown to work reliably in a variety of cell types, including primary human monocytes, primary human dendritic cells, primary human endothelial cells, human monocytic THP-1 cell line, and human/mouse hybrid macrophage cell line WBC264-9C. Inhibition of CypHer5E bead uptake by cytochalasin D was studied, and the 50% inhibition concentration (IC50) was determined. The assay was performed in 96- and 384-well formats, and it is appropriate for high-throughput cellular screening of processes and compounds affecting phagocytosis. The CypHer5E phagocytosis assay is superior to existing protocols because it allows easy distinction of true phagocytosis from particle adherence and can be used in microscopy-based measurement of phagocytosis.     

Features of crossing-over in virus-infected tomato

The evidence of increased crossing over rate in tomato hybrids infected with TAV (Tomato aspermy virus), PVX (Potato virus X), TMV (Tobacco mosaic virus), TMV+PVX indicates the recombinogenic effect of viral infection. Cytological studies of the early diakinesis in healthy and virus-infected tomato revealed significant changes in chiasma number and position. The most significant changes were established for bivalents with two interstitial chiasmata and with one terminal and one interstitial. The data obtained indicate redistribution of the chiasmata position and induction of additional exchanges. The virus-induced recombination is segment-specific and depends on the host plant genotype, virus infection and the interaction between them.     

RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells

The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double-strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway. We show that formation of ubiquitin foci as well as cell survival after DNA damage depends on both RNF8 and RNF168. However, we find that RNF8 and RNF168 knockout cell lines respond differently to treatment with camptothecin indicating that they do not function in a strictly linear manner. Surprisingly, we show that HERC2 is required neither for survival nor for ubiquitin foci formation after DNA damage in DT40. Moreover, the E3 ubiquitin ligase activity of HERC2 is not redundant to that of RNF8 or RNF168.     

Single-Molecule Investigation of the Influence Played by Lipid Rafts on Ion Transport and Dynamic Features of the Pore-Forming Alamethicin Oligomer

In this experimental work we employed single-molecule electrical recordings on alamethicin oligomers inserted in lipid bilayers made of brain sphingomyelin (bSM), palmitoyloleoylphosphatidylcholine (POPC) and cholesterol (chol) to unravel novel aspects regarding lipid raft interactions with pore-forming peptides. We probed the effect of lipid rafts on electrical properties of inserted alamethicin oligomers, and our data convincingly prove that the single-channel electrical conductance of various subconductance states of the alamethicin oligomer (1) increases in the presence of raft-containing ternary lipid mixtures (POPC-chol-bSM) compared to cases when bilayers were made of POPC-chol and POPC and (2) decreases in the presence of raft-containing ternary lipid mixtures compared to nonraft ternary mixtures which favor the fluid and liquid ordered phases alone. Our data demonstrate that the presence of lipid rafts leads to a slower association kinetics of alamethicin oligomers, seemingly reflecting a slower lateral diffusion process of such peptide aggregates compared to the case of nonraft, binary lipid mixtures. Furthermore, we show that the electrical capacitance of ternary lipid mixtures (POPC-chol-bSM) decreases in the presence of raft domains by comparison to nonraft binary phases (POPC-chol) or POPC alone, and this could constitute an additional mechanism via which macroscopic electrical manifestations of eukaryotic cells are modulated by the coexistence of gel and fluid domains of the plasma membrane.     

Late Campanian to late Maastrichtian bryozoans encrusting on belemnite rostra from the Aktolagay Plateau in western Kazakhstan

Twenty species belonging to fifteen genera of cyclostome and cheilostome bryozoans encrusting belemnite rostra are described from the late Campanian to Maastrichtian of the Aktolagay Plateau, in western Kazakhstan. Due to the moderate to poor preservation of the material, only four cheilostome species are identified down to the species level: Wilbertopora? besoktiensis (Voigt, 1967), ‘Aechmellina’ stenostoma Voigt, 1930, and two new species, ‘Aechmellina’ viskovae and Cheethamia aktolagayensis. All remaining species are left in open nomenclature. Type material of Wilbertopora? besoktiensis from the early Maastrichtian of the Mangyshlak Peninsula in Kazakhstan, has been re-examined. Palaeobiogeographical and implications are discussed. Cheilostomes slightly dominated over cyclostomes in the Aktolagay Plateau fauna encrusting on belemnites in terms of diversity. The dominant colony forms observed were spots and sheets.     

Ancient DNA reveals differences in behaviour and sociality between brown bears and extinct cave bears

Ancient DNA studies have revolutionized the study of extinct species and populations, providing insights on phylogeny, phylogeography, admixture and demographic history. However, inferences on behaviour and sociality have been far less frequent. Here, we investigate the complete mitochondrial genomes of extinct Late Pleistocene cave bears and middle Holocene brown bears that each inhabited multiple geographically proximate caves in northern Spain. In cave bears, we find that, although most caves were occupied simultaneously, each cave almost exclusively contains a unique lineage of closely related haplotypes. This remarkable pattern suggests extreme fidelity to their birth site in cave bears, best described as homing behaviour, and that cave bears formed stable maternal social groups at least for hibernation. In contrast, brown bears do not show any strong association of mitochondrial lineage and cave, suggesting that these two closely related species differed in aspects of their behaviour and sociality. This difference is likely to have contributed to cave bear extinction, which occurred at a time in which competition for caves between bears and humans was likely intense and the ability to rapidly colonize new hibernation sites would have been crucial for the survival of a species so dependent on caves for hibernation as cave bears. Our study demonstrates the potential of ancient DNA to uncover patterns of behaviour and sociality in ancient species and populations, even those that went extinct many tens of thousands of years ago.