Transgenic pigs designed to express human α-galactosidase to avoid humoral xenograft rejection

The use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. However, the presence of xenoreactive antibodies in humans directed against swine Gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute reaction. To prevent hyperacute rejection, it is possible to change the swine genome by a human gene modifying the set of donor’s cell surface proteins. The gene construct pGal-GFPBsd containing the human gene encoding α-galactosidase enzyme under the promoter of EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a male pronucleus of the fertilised porcine oocyte. As a result, the founder male pig was obtained with the transgene mapping to chromosome 11p12. The polymerase chain reaction (PCR) analysis revealed and the Southern analysis confirmed transgene integration estimating the approximate number of transgene copies as 16. Flow cytometry analysis revealed a reduction in the level of epitope Gal on the cell surface of cells isolated from F0 and F1 transgenic animals. The complement-mediated cytotoxicity assay showed increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of α-galactosidase expression.     

No cumulative effect of 10 years of elevated [CO2] on perennial plant biomass components in the Mojave Desert

Elevated atmospheric CO₂ concentrations ([CO₂]) generally increase primary production of terrestrial ecosystems. Production responses to elevated [CO₂] may be particularly large in deserts, but information on their long‐term response is unknown. We evaluated the cumulative effects of elevated [CO₂] on primary production at the Nevada Desert FACE (free‐air carbon dioxide enrichment) Facility. Aboveground and belowground perennial plant biomass was harvested in an intact Mojave Desert ecosystem at the end of a 10‐year elevated [CO₂] experiment. We measured community standing biomass, biomass allocation, canopy cover, leaf area index (LAI), carbon and nitrogen content, and isotopic composition of plant tissues for five to eight dominant species. We provide the first long‐term results of elevated [CO₂] on biomass components of a desert ecosystem and offer information on understudied Mojave Desert species. In contrast to initial expectations, 10 years of elevated [CO₂] had no significant effect on standing biomass, biomass allocation, canopy cover, and C : N ratios of above‐ and belowground components. However, elevated [CO₂] increased short‐term responses, including leaf water‐use efficiency (WUE) as measured by carbon isotope discrimination and increased plot‐level LAI. Standing biomass, biomass allocation, canopy cover, and C : N ratios of above‐ and belowground pools significantly differed among dominant species, but responses to elevated [CO₂] did not vary among species, photosynthetic pathway (C₃ vs. C₄), or growth form (drought‐deciduous shrub vs. evergreen shrub vs. grass). Thus, even though previous and current results occasionally show increased leaf‐level photosynthetic rates, WUE, LAI, and plant growth under elevated [CO₂] during the 10‐year experiment, most responses were in wet years and did not lead to sustained increases in community biomass. We presume that the lack of sustained biomass responses to elevated [CO₂] is explained by inter‐annual differences in water availability. Therefore, the high frequency of low precipitation years may constrain cumulative biomass responses to elevated [CO₂] in desert environments.     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Adaptive Dynamics of Extortion and Compliance

Direct reciprocity is a mechanism for the evolution of cooperation. For the iterated prisoner’s dilemma, a new class of strategies has recently been described, the so-called zero-determinant strategies. Using such a strategy, a player can unilaterally enforce a linear relationship between his own payoff and the co-player’s payoff. In particular the player may act in such a way that it becomes optimal for the co-player to cooperate unconditionally. In this way, a player can manipulate and extort his co-player, thereby ensuring that the own payoff never falls below the co-player’s payoff. However, using a compliant strategy instead, a player can also ensure that his own payoff never exceeds the co-player’s payoff. Here, we use adaptive dynamics to study when evolution leads to extortion and when it leads to compliance. We find a remarkable cyclic dynamics: in sufficiently large populations, extortioners play a transient role, helping the population to move from selfish strategies to compliance. Compliant strategies, however, can be subverted by altruists, which in turn give rise to selfish strategies. Whether cooperative strategies are favored in the long run critically depends on the size of the population; we show that cooperation is most abundant in large populations, in which case average payoffs approach the social optimum. Our results are not restricted to the case of the prisoners dilemma, but can be extended to other social dilemmas, such as the snowdrift game. Iterated social dilemmas in large populations do not lead to the evolution of strategies that aim to dominate their co-player. Instead, generosity succeeds.     

Molecular basis of lateral force spectroscopy nano-diagnostics: computational unbinding of autism related chemokine MCP-1 from IgG antibody

Monocyte-chemoattractant protein-1 (MCP-1), also known as CCL2, is a potent chemoattractant of T cells and monocytes, involved in inflammatory and angio-proliferative brain and retinal diseases. Higher expression of MCP-1 is observed in metastatic tumors. Unusual levels of MCP-1 in the brain may be correlated with autism. Immunochemistry where atomic force microscope (AFM) tips functionalized with appropriate antibodies against MCP-1 are used could in principle support medical diagnostics. Useful signals from single molecule experiments may be generated if interaction forces are large enough. The chemokine-antibody unbinding force depends on a relative motion of the interacting fragments of the complex. In this paper the stability of the medically important MCP-1- immunoglobulin G antibody Fab fragment complex has been studied using steered molecular dynamics (SMD) computer simulations with the aim to model possible arrangements of nano-diagnostics experiments. Using SMD we confirm that molecular recognition in MCP1-IgG is based mainly on six pairs of residues: Glu39A - Arg98H, Lys56A - Asp52H, Asp65A - Arg32L, Asp68A - Arg32L, Thr32A - Glu55L, Gln61A - Tyr33H. The minimum external force required for mechanical dissociation of the complex depends on a direction of the force. The pulling of the MCP-1 antigen in the directions parallel to the antigen-antibody contact plane requires forces about 20 %–40 % lower than in the perpendicular one. Fortunately, these values are large enough that the fast lateral force spectroscopy may be used for effective nano-diagnostics purposes. We show that molecular modeling is a useful tool in planning AFM force spectroscopy experiments.

Peripheral CD8+ T cell proliferation is prognostic for patients with advanced thoracic malignancies

There is a complex interplay between the immune system and a developing tumor that is manifest in the way that the balance of T cell subsets in the local tumor environment reflects clinical outcome. Tumor infiltration by CD8⁺ T cells and regulatory T cells (Treg) is associated with improved and reduced survival, respectively, in many cancer types. However, little is known of the prognostic value of immunological parameters measured in peripheral blood. In this study, peripheral CD8⁺ T cells and Treg from 43 patients with malignant mesothelioma or advanced non-small-cell lung cancer scheduled to commence palliative chemotherapy were assessed by flow cytometry and evaluated for association with patient survival. Patients had a higher proportion of peripheral Treg, proliferating CD8⁺ T cells and CD8⁺ T cells with an activated effector phenotype compared with age-matched healthy controls. Higher proportions of Treg and proliferating CD8⁺ T cells were both associated with poor survival in univariate analyses (hazard ratio [HR] 3.81, 95 % CI 1.69–8.57; p < 0.01 and HR 2.86, 95 % CI 1.26–6.50; p < 0.05, respectively). CD8⁺ T cell proliferation was independently predictive of reduced survival in multivariate analysis (HR 2.58, 95 % CI 1.01–6.61; p < 0.05). These findings suggest that peripheral CD8⁺ T cell proliferation can be a useful prognostic marker in patients with thoracic malignancies planned for palliative chemotherapy.     

Activation of TRPV4 channel in pancreatic INS-1E beta cells enhances glucose-stimulated insulin secretion via calcium-dependent mechanisms

Transient receptor potential channel vanilloid type 4 (TRPV4) is a Ca²⁺- and Mg²⁺-permeable cation channel that influences oxidative metabolism and insulin sensitivity. The role of TRPV4 in pancreatic beta cells is largely unknown. Here, we characterize the role of TRPV4 in controlling intracellular Ca²⁺ and insulin secretion in INS-1E beta cells. Osmotic, thermal or pharmacological activation of TRPV4 caused a rapid rise of intracellular Ca²⁺ and enhanced glucose-stimulated insulin secretion. In the presence of the TRPV channel blocker ruthenium red (RuR) or after suppression of TRPV4 protein production, TRPV4 activators failed to increase [Ca²⁺]_i and insulin secretion in INS-1E cells.     

Variations and trends of birch pollen seasons during 15 years (1996–2010) in relation to weather conditions in Poznań (western Poland)

Birch (Betula) pollen seasons were examined in relation to meteorological conditions in Poznań (1996–2010). Birch pollen grains were collected using a volumetric spore trap. An alternate biennial cycle of birch pollen season intensity was noticed in Poznań. The main factors influencing birch pollen season intensity were average daily minimum temperatures during the second fortnight of May and the month of June one year before pollination as well as the intensity of the pollen season of the previous year. Most of the pollen grains are recorded during the first week of the season; the number of pollen grains recorded at this time is positively correlated with mean maximum temperature and negatively correlated with daily rainfall. The significant effect of rainfall in reducing the season pollen index was noticed only during weak pollen seasons (season pollen index <?mean). In addition, mean daily maximum temperature during the first two weeks of the birch pollen season markedly influences its duration. No significant trends in duration and intensity of the pollen season were recorded, however, a slight tendency towards early pollination was observed (?0.4 days/year, p?=?0.310).     

Historical Biogeography and Diversification of Truffles in the Tuberaceae and Their Newly Identified Southern Hemisphere Sister Lineage

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae – the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (∼156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.     

The role of rock mining for maintaining Dauco carotae-Crepidetum rhoeadifoliae Hejný et Grüll in Hejný et al. 1979 — a new to Poland plant association

This work presents the Dauco carotae-Crepidetum rhoeadifoliae plant association, which is new to Poland. The association has been observed in industrial reclamation areas in the vicinity of carbonate mineral excavation sites in the central part of the Opole region. In the vast majority of cases, plots of this association developed in reclaimed areas. The majority of diagnostic species for the association was found within surveyed plots, including Verbascum thapsus, V. densiflorum and Bryum argenteum. Taxa characteristic of the alliance were also constantly present, i.e. Daucus carota, Melilotus alba, M. officinalis, Echium vulgare and Erysimum hieracifolium. This association belongs to the rarest syntaxa in Poland included in the Dauco-Melilotion alliance of ruderal communities with a predominance of hemicryptophytes, therophytes and perennials. The main diagnostic species — Crepis rhoeadifolia, belongs to very rare elements of Polish flora. It has been observed only in the southern part of the country in approx. 20 sites. Crepis rhoeadifolia had not been observed in Silesia for approx. 40 years, which is why it was considered to be an extinct taxon in this region. Rediscovering of the species allowed for diagnosing the Dauco-Crepidetum rhoeadifoliae association. This association is an example of a pioneer phytocenosis of, most likely, anthropogenic origin in Silesia.