Frugivory on Persea lingue in temperate Chilean forests: interactions between fruit availability and habitat fragmentation across multiple spatial scales

Habitat degradation and fragmentation are expected to reduce seed dispersal rates by reducing fruit availability as well as the movement and abundance of frugivores. These deleterious impacts may also interact with each other at different spatial scales, leading to nonlinear effects of fruit abundance on seed dispersal. In this study we assessed whether the degradation and fragmentation of southern Chilean forests had the potential to restrict seed dispersal the lingue (Persea lingue) tree, a fleshy-fruited tree species. Of five frugivore bird species, the austral thrush (Turdus falcklandii) and the fire-eyed diucon (Xolmis pyrope) were the only legitimate seed dispersers as well as being the most abundant species visiting lingue trees. The results showed little or no direct effect of habitat fragmentation on seed dispersal estimates, possibly because the assemblage of frugivore birds was comprised habitat-generalist species. Instead, the number of fruits removed per focal tree exhibited an enhanced response to crop size, but only in the more connected fragments. In the fruit-richer fragment networks, there was an increased fragment-size effect on the proportion of fruits removed in comparison to fruit-poor networks in which the fragment size effect was spurious. We suggest that such nonlinear effects are widespread in fragmented forest regions, resulting from the link between the spatial scales over which frugivores sample resources and the spatial heterogeneity in fruiting resources caused by habitat fragmentation and degradation.     

Identification of NCF2/p67phox as a novel p53 target gene

Analysis of microarrays performed in p53-, TAp63α- and ΔNp63α-inducible SaOs-2 cell lines allowed the identification of NCF2 mRNA upregulation in response to p53 induction. NCF2 gene encodes for p67phox, the cytosolic subunit of the NADPH oxidase enzyme complex. The recruitment of p67phox to the cell membrane causes the activation of the NADPH oxidase complex followed by the generation of NADP+ and superoxide from molecular oxygen. The presence of three putative p53 binding sites on the NCF2 promoter was predicted, and the subsequent luciferase and chromatin immunoprecipitation assays showed the activation of NCF2 promoter by p53 and its direct binding in vivo to at least one of the sites, thus confirming the hypothesis. NCF2 upregulation was also confirmed by real-time PCR in several cell lines after p53 activation. NCF2 knockdown by siRNA results in a significant reduction of ROS production and stimulates cell death, suggesting a protective function of Nox2-generated ROS in cells against apoptosis. These results provide insight into the redox-sensitive signaling mechanism that mediates cell survival involving p53 and its novel target NCF2/p67phox.     

Epigenetic control of early neurodegenerative events in diabetic retinopathy by the histone deacetylase SIRT6

Diabetic retinopathy (DR ) is one of the common complications associated with diabetes mellitus and the leading cause of blindness worldwide. Recent research has demonstrated that DR is not only a microvascular disease but may be a result of neurodegenerative processes. Moreover, glucose‐induced neuron and glial cell damage may occur shortly after the onset of diabetes which makes the disease hard to diagnose at early stages. SIRT 6, a NAD ‐dependent sirtuin deacylase, modulates aging, energy metabolism, and neurodegeneration. In previous studies we showed that SIRT 6 deficiency causes major retinal transmission defects, changes in the expression of glycolytic genes, and elevated levels of apoptosis. Given the importance of glucose availability for retinal function and the critical role of SIRT 6 in modulating glycolysis, we aimed to analyze SIRT 6 participation in the molecular machinery that regulates the development of experimental DR . Using non‐obese diabetic mice, we determined by western blot that 2 weeks after the onset of the disease, high glucose concentrations induced retinal increase in a neovascularization promoting factor (vascular endothelial growth factor, VEGF ), and the loss of a neuroprotective factor (brain‐derived neurotrophic factor, BDNF) associated with reduced levels of SIRT 6 and increased acetylation levels of its substrates (H3K9 and H3K56) suggesting a deregulation of key neural factors. Noteworthy, retinas from CNS conditionally deleted SIRT 6 mice showed a resemblance to diabetic retinas exhibiting lower protein levels of BDNF factor and increased protein levels of VEGF . Moreover, cultured Müller glial cells subjected to high glucose concentrations exhibited decreased levels of SIRT 6 and increased levels of H3K56 acetylation. In addition, the increment of VEGF levels induced by high glucose was reverted by the over‐expression of SIRT 6 in this cell type. Accordingly, siRNA experiments showed that, when SIRT 6 was silenced, VEGF levels increased. Our findings suggest that epigenetically regulated neurodegenerative events may occur at an early diabetic stage prior to the characteristic proliferative and vascular changes observed at a later diabetic stage.

     

North Atlantic minke whale (Balaenoptera acutorostrata) feeding habits and migrations evaluated by stable isotope analysis of baleen

Isotopic analyses of the incrementally growing baleen in Mysticeti have been used to learn about their feeding and movement patterns. Using methods previously applied to Pacific minke whales, stable δ¹⁵N and δ¹³C isotope values were measured along the baleen plates of male and female minke whales from two locations in the Northeast Atlantic. The sample sizes used in this study are comparable to those previously used in the literature, and, although limited in size, the evidence suggests differences in isotopic signatures between whales caught at different locations. Both the δ¹⁵N and δ¹³C data suggest whales at the higher latitude site of Svalbard have a narrower diet than the whales from Lofoten/Vesterålen in Norway. Across all whales, the δ¹⁵N data indicate the whales primarily prey on fish for much of the year, only switching to zooplankton during the spring bloom. The δ¹³C data fail to confirm whether the whales migrate over long distances.     

Biodistribution of a Bispecific Single-chain Diabody and Its Half-life Extended Derivatives

Small recombinant antibody molecules such as bispecific single-chain diabodies (scDb) possessing a molecular mass of ∼55 kDa are rapidly cleared from circulation. We have recently extended the plasma half-life of scDb applying various strategies including PEGylation, N-glycosylation and fusion to an albumin-binding domain (ABD) from streptococcal protein G. Here, we further analyzed the influence of these modifications on the biodistribution of a scDb directed against carcinoembryonic antigen (CEA) and CD3 capable of retargeting T cells to CEA-expressing tumor cells. We show that a prolonged circulation time results in an increased accumulation in CEA⁺ tumors, which was most pronounced for scDb-ABD and PEGylated scDb. Interestingly, tumor accumulation of the scDb-ABD fusion protein was ∼2-fold higher compared with PEGylated scDb, although both molecules exhibit similar plasma half-lives and similar affinities for CEA. Comparing half-lives in neonatal Fc receptor (FcRn) wild-type and FcRn heavy chain knock-out mice the contribution of the FcRn to the long plasma half-life of scDb-ABD was confirmed. The half-life of scDb-ABD was ∼2-fold lower in the knock-out mice, while no differences were observed for PEGylated scDb. Binding of the scDb derivatives to target and effector cells was not or only marginally affected by the modifications, although, compared with scDb, a reduced cytotoxic activity was observed for scDb-ABD, which was further reduced in the presence of albumin. In summary, these findings demonstrate that the extended half-life of a bispecific scDb translates into improved accumulation in antigen-positive tumors but that modifications might also affect scDb-mediated cytotoxicity.Bispecific single-chain diabodies (scDb)² are recombinant molecules composed of the variable heavy and light chain domains of two antibodies connected by three linkers in the order V_HA-V_LB-V_HB-V_LA (1). These domains assemble into molecules with a compact structure and molecular masses of ∼55 kDa. Bispecific single-chain diabodies have been developed for various applications including the retargeting of cytotoxic T lymphocytes to tumor cells for cellular cancer therapy (2).Although scDb are capable of efficiently retargeting effector cells to tumor cells the small size leads to their rapid elimination after i.v. injection. The terminal half-life of these molecules in mice is only in the range of 5–6 h, compared with several days for whole IgG molecules (3, 4). The fast clearance of such small molecules from circulation hampers therapeutic applications, e.g. requiring infusions or repeated injections to maintain a therapeutically effective dose over a prolonged period of time (5). For example, a bispecific tandem scFv directed against CD19 and CD3 (blinatumomab) having a similar size as an scDb molecule had to be given as an 8-week infusion (maximum dose 60 μg/m² per day) in a clinical phase I trial for the treatment of B cell lymphoma patients (6).To extend plasma half-lives of therapeutic proteins and thus to improve pharmacokinetics and pharmacodynamics, several strategies can be applied (7). Strategies such as conjugation of polyethylene glycol chains (PEGylation) or production of hyperglycosylated variants through introduction of additional N-glycosylation sites primarily aim at increasing the hydrodynamic volume of the molecule, thus reducing renal filtration and degradation. Some of these strategies further implement FcRn-mediated recycling processes, e.g. fusion to the IgG Fc region and fusion or binding to serum albumin.We recently applied several of these strategies to improve the plasma half-life of a scDb molecule. These strategies included site-directed conjugation of a 40-kDa PEG chain (PEGylated scDb, scDb-A′-PEG_40k), production of N-glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites (scDb-ABC_1–7), a scDb-human serum albumin fusion protein (scDb-HSA), and a scDb fused to an albumin-binding domain from streptococcal protein G (scDb-ABD) (3, 4, 8). In these studies we showed that N-glycosylation only moderately increased half-life, while a strong improvement was observed for the PEGylated scDb, scDb-HSA, and scDb-ABD.In the present study we further analyzed the biodistribution of unmodified scDb as well as three of the scDb derivatives (PEGylated scDb, N-glycosylated scDb, scDb-ABD) in tumor-bearing mice. We show that the modified scDb molecules exhibit a reduced renal clearance and that an extended half-life leads to an increased accumulation in antigen-positive tumors. The strongest improvement was observed for scDb-ABD. Using FcRn knock-out mice we confirmed that FcRn-mediated recycling contributes to the long half-life of scDb-ABD. Affinities of the scDb derivatives for target and effector cells were not or only marginally affected by the modifications, although, compared with scDb, a reduced cytotoxic activity was observed for scDb-ABD, which was further reduced in the presence of albumin. These findings demonstrate that half-life extension of scDb results in increased tumor accumulation but that modifications might also affect scDb-mediated cytotoxicity.     

Parthenolide and DMAPT induce cell death in primitive CML cells through reactive oxygen species

Tyrosine kinase inhibitors (TKI) have become a first‐line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34⁺lin^?) cells. We found that both agents induced cell death in CML, while having little effect on the equivalent normal hematopoietic cells. PTL and DMAPT caused an increase in reactive oxygen species (ROS) levels and inhibited NF‐κB activation. PTL and DMAPT inhibited cell proliferation and induced cell cycle arrest in G₀ and G₂ phases. Furthermore, we found cell cycle inhibition to correlate with down‐regulation of cyclin D1 and cyclin A. In summary, our study shows that PTL and DMAPT have a strong inhibitory effect on CML cells. Given that cell cycle arrest was not dependent on ROS induction, we speculate that this effect could be a direct consequence of NF‐κB inhibition and if this mechanism was to be evaded, PTL and DMAPT induced cell death would be potentiated.     

Sponge cell aggregation: checkpoints in development indicate a high level of organismal complexity

Studies of regeneration provide insight across many scales of animal biology from the processes of cellular communication to the ecology of whole populations. Sponges are highly regenerative animals, with studies showing adults can both recover large portions of their body after predation or damage due to storms, and even reform whole individuals, via an aggregation stage, from dissociated tissues. While sponges are clearly highly regenerative, few studies actually show dissociated cells forming functional individuals. As sponges often serve as model organisms for studying the development and function of traits in metazoans, determining the universality and mechanics of their regeneration potential is important. We tested the capacity of members of seven sponge species from temperate freshwater and marine environments, from a range of taxonomic positions, and with different habits, to form functional sponges after dissociation. Development to a functional sponge progressed through a series of checkpoints: the sorting of cells and removal of debris; adhesion to a substrate and differentiation of cells; organization of cells into tissues; and regionalization of tissues. Two of the seven species tested, Spongilla lacustris and Haliclona cf. permollis, progressed through all four checkpoints, while the remaining five species progressed to various levels of development before aggregates disintegrated. Our findings highlight three important conclusions: (1) The ability of aggregates to differentiate into functional sponges is not as widespread as previously thought; (2) The species‐specific ability of aggregates to develop to functional sponges appears to be an adaptive trait; and (3) The progression of development in aggregates through checkpoints, which in later development involves formation of tissues and regionalization of tissues, highlights the complexity of the sponge body plan and suggests fundamental rules in development shared across metazoans.