A consistent phylogenetic backbone for the fungi

The kingdom of fungi provides model organisms for biotechnology, cell biology, genetics, and life sciences in general. Only when their phylogenetic relationships are stably resolved, can individual results from fungal research be integrated into a holistic picture of biology. However, and despite recent progress, many deep relationships within the fungi remain unclear. Here, we present the first phylogenomic study of an entire eukaryotic kingdom that uses a consistency criterion to strengthen phylogenetic conclusions. We reason that branches (splits) recovered with independent data and different tree reconstruction methods are likely to reflect true evolutionary relationships. Two complementary phylogenomic data sets based on 99 fungal genomes and 109 fungal expressed sequence tag (EST) sets analyzed with four different tree reconstruction methods shed light from different angles on the fungal tree of life. Eleven additional data sets address specifically the phylogenetic position of Blastocladiomycota, Ustilaginomycotina, and Dothideomycetes, respectively. The combined evidence from the resulting trees supports the deep-level stability of the fungal groups toward a comprehensive natural system of the fungi. In addition, our analysis reveals methodologically interesting aspects. Enrichment for EST encoded data-a common practice in phylogenomic analyses-introduces a strong bias toward slowly evolving and functionally correlated genes. Consequently, the generalization of phylogenomic data sets as collections of randomly selected genes cannot be taken for granted. A thorough characterization of the data to assess possible influences on the tree reconstruction should therefore become a standard in phylogenomic analyses.     

The Paf1 Complex Subunit Rtf1 Buffers Cells Against the Toxic Effects of [PSI+] and Defects in Rkr1-Dependent Protein Quality Control in Saccharomyces cerevisiae

The Rtf1 subunit of the Paf1 complex is required for specific histone modifications, including histone H2B lysine 123 monoubiquitylation. In Saccharomyces cerevisiae, deletion of RTF1 is lethal in the absence of Rkr1, a ubiquitin-protein ligase involved in the destruction of nonstop proteins, which arise from mRNAs lacking stop codons or translational readthrough into the poly(A) tail. We performed a transposon-based mutagenesis screen to identify suppressors of rtf1Δ rkr1Δ lethality and found that a mutation in the gene encoding the protein chaperone Hsp104 rescued viability. Hsp104 plays a role in prion propagation, including the maintenance of [PSI(+)], which contributes to the synthesis of nonstop proteins. We demonstrate that rtf1Δ and rkr1Δ are synthetically lethal only in the presence of [PSI(+)]. The deletion, inactivation, and overexpression of HSP104 or the overexpression of prion-encoding genes URE2 and LSM4 clear [PSI(+)] and rescue rtf1Δ rkr1Δ lethality. In addition, the presence of [PSI(+)] decreases the fitness of rkr1Δ strains. We investigated whether the loss of RTF1 exacerbates an overload in nonstop proteins in rkr1Δ [PSI(+)] strains but, using reporter plasmids, found that rtf1Δ decreases nonstop protein levels, indicating that excess nonstop proteins may not be the cause of synthetic lethality. Instead, our data suggest that the loss of Rtf1-dependent histone modifications increases the burden on quality control pathways in cells lacking Rkr1 and containing [PSI(+)].     

Novel Humanized and Highly Efficient Bispecific Antibodies Mediate Killing of Prostate Stem Cell Antigen-Expressing Tumor Cells by CD8+ and CD4+ T Cells

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3-anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8(+) and CD4(+) T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4(+) T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.     

STOICHIOMETRIC RESPONSES OF PHYTOPLANKTON SPECIES TO THE INTERACTIVE EFFECT OF NUTRIENT SUPPLY RATIOS AND GROWTH RATES1

Three species of phytoplankton, Rhodomonas sp., Phaeodactylum tricornutum Bohlin, and Isochrysis galbana Parke, were cultivated in semicontinuous culture to analyze the response of carbon (C):nitrogen (N):phosphorus (P) stoichiometry to the interactive effect of five N:P supply ratios and four growth rates (dilution rates). The relationship between cellular N and P quotas and growth rates fits well to both the Droop and Ågren’s functions for all species. We observed excess uptake of both N and P in the three species. N:P biomass ratios showed a significant positive relationship with N:P supply ratios across the entire range of growth rates, and N:P biomass ratios converged to an intermediate value independent of N:P supply ratios at higher growth rates. The effect of growth rates on N:P biomass ratios was positive at lower N:P supply ratios, but negative at higher N:P supply ratios for both Rhodomonas sp. and I. galbana, while for P. tricornutum this effect was negative at all N:P supply ratios. A significant interactive effect of N:P supply ratios and growth rates on N:P biomass ratios was found in both Rhodomonas sp. and P. tricornutum, but not in I. galbana. Our results suggest that Ågren’s functions may explain the underlying biochemical principle for the Droop model. The parameters in the Droop and Ågren’s functions can be useful indications of algal succession in the phytoplankton community in changing oceans.     

Identification and proteomic analysis of distinct UBE3A/E6AP protein complexes

The E6AP ubiquitin ligase catalyzes the high-risk human papillomaviruses' E6-mediated ubiquitylation of p53, contributing to the neoplastic progression of cells infected by these viruses. Defects in the activity and the dosage of E6AP are linked to Angelman syndrome and to autism spectrum disorders, respectively, highlighting the need for precise control of the enzyme. With the exception of HERC2, which modulates the ubiquitin ligase activity of E6AP, little is known about the regulation or function of E6AP normally. Using a proteomic approach, we have identified and validated several new E6AP-interacting proteins, including HIF1AN, NEURL4, and mitogen-activated protein kinase 6 (MAPK6). E6AP exists as part of several different protein complexes, including the proteasome and an independent high-molecular-weight complex containing HERC2, NEURL4, and MAPK6. In examining the functional consequence of its interaction with the proteasome, we found that UBE3C (another proteasome-associated ubiquitin ligase), but not E6AP, contributes to proteasomal processivity in mammalian cells. We also found that E6 associates with the HERC2-containing high-molecular-weight complex through its binding to E6AP. These proteomic studies reveal a level of complexity for E6AP that has not been previously appreciated and identify a number of new cellular proteins through which E6AP may be regulated or functioning.     

Modelling the potential impact of climate variability and change on species regeneration potential in the temperate forests of South‐Eastern Australia

The sensitivity of early plant regeneration to environmental change makes regeneration a critical stage for understanding species response to climate change. We investigated the spatial and temporal response of eucalypt trees in the Central Highland region of south eastern Australia to high and low climate change scenarios. We developed a novel mechanistic model incorporating germination processes, TACA‐GEM, to evaluate establishment probabilities of five key eucalypt species, Eucalyptus pauciflora, Eucalyptus delegatensis, Eucalyptus regnans, Eucalyptus nitens and Eucalyptus obliqua. Changes to regeneration potential at landscape and site levels were calculated to determine climate thresholds. Model results demonstrated that climate change is likely to impact plant regeneration. We observed increases and decreases in regeneration potential depending on the ecosystem, indicating that some species will increase in abundance in some forest types, whilst other forest types will become inhabitable. In general, the dry forest ecosystems were most impacted, whilst the wet forests were least impacted. We also observed that species with seed dormancy mechanisms, like E. pauciflora and E. delegatensis, are likely to be at higher risk than those without. Landscape‐ and site‐level analysis revealed heterogeneity in species response at different scales. On a landscape scale, a 4.3 °C mean temperature increase and 22% decline in precipitation (predicted for 2080) is predicted to be a threshold for large spatial shifts in species regeneration niches across the study region, while a 2.6 °C increase and 15% decline in precipitation (predicted for 2050) will likely result in local site‐level shifts. Site‐level analysis showed that considerable declines in regeneration potential for E. delegatensis, E. pauciflora and E. nitens were modelled to occur in some ecosystems by 2050. While overall model performance and accuracy was good, better understanding of effects from extreme events and other underlying processes on regeneration will improve modelling and development of species conservation strategies.     

DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions

The histone deacetylase inhibitor (HDACi) LBH589 has been verified as an effective anticancer agent. The identification and characterization of new targets for LBH589 action would further enhance our understanding of the molecular mechanisms involved in HDACi therapy. The role of the tumor suppressor death-associated protein kinase (DAPK) in LBH589-induced cytotoxicity has not been investigated to date. Stable DAPK knockdown (shRNA) and DAPK overexpressing (DAPK+++) cell lines were generated from HCT116 wildtype colon cancer cells. LBH589 inhibited cell proliferation, reduced the long-term survival, and up-regulated and activated DAPK in colorectal cancer cells. Moreover, LBH589 significantly suppressed the growth of colon tumor xenografts and in accordance with the in vitro studies, increased DAPK levels were detected immunohistochemically. LBH589 induced a DAPK-dependent autophagy as assessed by punctuate accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62 protein. LBH589-induced autophagy seems to be predominantly caused by DAPK protein interactions than by its kinase activity. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage of caspase 3 and PARP but didn’t rescue the cells from LBH589-induced cell death in crystal violet staining suggesting both caspase-dependent as well as caspase-independent apoptosis pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-mediated apoptosis in a DAPK-dependent manner. Altogether our data suggest that DAPK induces autophagy in response to HDACi-treatment. In autophagy deficient cells, DAPK plays an essential role in committing cells to HDACi-induced apoptosis.