Edge influence on vegetation at natural and anthropogenic edges of boreal forests in Canada and Fennoscandia

Although anthropogenic edges are an important consequence of timber harvesting, edges due to natural disturbances or landscape heterogeneity are also common. Forest edges have been well studied in temperate and tropical forests, but less so in less productive, disturbance-adapted boreal forests. We synthesized data on forest vegetation at edges of boreal forests and compared edge influence among edge types (fire, cut, lake/wetland; old vs. young), forest types (broadleaf vs. coniferous) and geographic regions. Our objectives were to quantify vegetation responses at edges of all types and to compare the strength and extent of edge influence among different types of edges and forests. Research was conducted using the same general sampling design in Alberta, Ontario and Quebec in Canada, and in Sweden and Finland. We conducted a meta-analysis for a variety of response variables including forest structure, deadwood abundance, regeneration, understorey abundance and diversity, and non-vascular plant cover. We also determined the magnitude and distance of edge influence (DEI) using randomization tests. Some edge responses (lower tree basal area, tree canopy and bryophyte cover; more logs; higher regeneration) were significant overall across studies. Edge influence on ground vegetation in boreal forests was generally weak, not very extensive (DEI usually < 20 m) and decreased with time. We found more extensive edge influence at natural edges, at younger edges and in broadleaf forests. The comparison among regions revealed weaker edge influence in Fennoscandian forests. Synthesis. Edges created by forest harvesting do not appear to have as strong, extensive or persistent influence on vegetation in boreal as in tropical or temperate forested ecosystems. We attribute this apparent resistance to shorter canopy heights, inherent heterogeneity in boreal forests and their adaptation to frequent natural disturbance. Nevertheless, notable differences between forest structure responses to natural (fire) and anthropogenic (cut) edges raise concerns about biodiversity implications of extensive creation of anthropogenic edges. By highlighting universal responses to edge influence in boreal forests that are significant irrespective of edge or forest type, and those which vary by edge type, we provide a context for the conservation of boreal forests. Edges created by forest harvesting do not appear to have as strong, extensive or persistent influence on vegetation in boreal as in tropical or temperate forested ecosystems. We attribute this apparent resistance to shorter canopy heights, inherent heterogeneity in boreal forests and their adaptation to frequent natural disturbance. Nevertheless, notable differences between forest structure responses to natural (fire) and anthropogenic (cut) edges raise concerns about biodiversity implications of extensive creation of anthropogenic edges. By highlighting universal responses to edge influence in boreal forests that are significant irrespective of edge or forest type, and those which vary by edge type, we provide a context for the conservation of boreal forests

Profit enhancing competitive pressure in vertically related industries

Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing

Stroke genetics informs drug discovery and risk prediction across ancestries

Previous genome-wide association studies (GWASs) of stroke - the second leading cause of death worldwide - were conducted predominantly in populations of European ancestry(1,2). Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis(3), and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach(4), we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry(5). Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries.</p

Dengue and Zika Virus Capsid Proteins Contain a Common PEX19-Binding Motif

Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have evolved sophisticated mechanisms to suppress the host immune system. For instance, flavivirus infections were found to sabotage peroxisomes, organelles with an important role in innate immunity. The current model suggests that the capsid (C) proteins of DENV and ZIKV downregulate peroxisomes, ultimately resulting in reduced production of interferons by interacting with the host protein PEX19, a crucial chaperone in peroxisomal biogenesis. Here, we aimed to explore the importance of peroxisomes and the role of C interaction with PEX19 in the flavivirus life cycle. By infecting cells lacking peroxisomes we show that this organelle is required for optimal DENV replication. Moreover, we demonstrate that DENV and ZIKV C bind PEX19 through a conserved PEX19-binding motif, which is also commonly found in cellular peroxisomal membrane proteins (PMPs). However, in contrast to PMPs, this interaction does not result in the targeting of C to peroxisomes. Furthermore, we show that the presence of C results in peroxisome loss due to impaired peroxisomal biogenesis, which appears to occur by a PEX19-independent mechanism. Hence, these findings challenge the current model of how flavivirus C might downregulate peroxisomal abundance and suggest a yet unknown role of peroxisomes in flavivirus biology

CPXV012 evolved a unique ER-lumenal sequence that is essential for TAP inhibition.

<p>(A) Sequence alignment of CPXV012 and its orthologs. Abbreviations and accession numbers are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004554#ppat.1004554.s008" target="_blank">Table S1</a>. GER91: The first 97 amino acids of the protein are aligned. The CPXV012 sequence of Brighton Red strain was used in this study (red box). The N- and C-terminal deletion constructs ^C8NΔ6-CPXV012 and ^C8CPXV012-CΔ5 are indicated as blue and green bars. (B) The last C-terminal, ER-lumenal residues of CPXV012 are essential for TAP inhibition. HeLa cells were transiently transfected with empty vector, full-length ^C8CPXV012, ^C8CPXV012-CΔ5, ^C8NΔ6-CPXV012, or BNLF2a^C8 in pIRES2-EGFP, respectively. MHC I surface expression was analyzed by flow cytometry. Only GFP-positive cells were analyzed. The dotted histogram represents the isotype control. Histograms for mock, BNLF2a^C8, ^C8CPXV012 full-length, and isotype transfection are from the same data set in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004554#ppat-1004554-g001" target="_blank">Fig. 1A</a>. (C) Evaluation of the histograms from B. Mean fluorescence intensity (MFI) was calculated for cells transfected with the indicated constructs. (D) Similar expression levels of the ^C8CPXV012 constructs and BNLF2a^C8 in cells analyzed by flow cytometry were confirmed by anti-C8 and anti-actin immunoblotting. (E) Inactive ^C8CPXV012-CΔ5 still binds to coreTAP1/2 heterodimers. Human coreTAP1^mVenus-C8 and coreTAP2^{mCerulean-StrepII} were coexpressed in HEK293T cells together with the ^C8CPXV012 variants as indicated. TAP1/2 heterodimeric complexes were tandem-affinity purified using streptavidin and anti-TAP1 (mAb 148.3) matrices. The HC10-antibody was used as negative control (mock). Input (solubilizate, 1/30 aliquot) and affinity purified complexes were analyzed by immunoblotting using C8- or TAP2-specific (mAb 435.3) antibodies, respectively. #, partially unfolded mCerulean.</p

HCMV-US6 and EBV-BNLF2a prevent the formation of CPXV012•TAP complexes.

<p>(A) Schematic presentation of the MHC I peptide-loading complex and the viral immune evasin US6 (type I membrane protein), BNLF2a (tail-anchored), and CPXV012 (type II). (B, C) ^flagCPXV012, TAP1, and TAP2 were coexpressed with either US6^myc (B) or BNLF2a^C8 (C) in <i>Sf</i>9 cells. Proteins were affinity-purified with myc-, flag-, or C8-specific antibodies (IP). The HC10-antibody was used as negative control (mock). Samples were analyzed by immunoblotting with the corresponding antibodies. An aliquot (1/20) of the crude membrane input is shown. *, glycosylated protein.</p

CPXV012 evolved a unique ER-lumenal sequence that is essential for TAP inhibition.

<p>(A) Sequence alignment of CPXV012 and its orthologs. Abbreviations and accession numbers are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004554#ppat.1004554.s008" target="_blank">Table S1</a>. GER91: The first 97 amino acids of the protein are aligned. The CPXV012 sequence of Brighton Red strain was used in this study (red box). The N- and C-terminal deletion constructs ^C8NΔ6-CPXV012 and ^C8CPXV012-CΔ5 are indicated as blue and green bars. (B) The last C-terminal, ER-lumenal residues of CPXV012 are essential for TAP inhibition. HeLa cells were transiently transfected with empty vector, full-length ^C8CPXV012, ^C8CPXV012-CΔ5, ^C8NΔ6-CPXV012, or BNLF2a^C8 in pIRES2-EGFP, respectively. MHC I surface expression was analyzed by flow cytometry. Only GFP-positive cells were analyzed. The dotted histogram represents the isotype control. Histograms for mock, BNLF2a^C8, ^C8CPXV012 full-length, and isotype transfection are from the same data set in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004554#ppat-1004554-g001" target="_blank">Fig. 1A</a>. (C) Evaluation of the histograms from B. Mean fluorescence intensity (MFI) was calculated for cells transfected with the indicated constructs. (D) Similar expression levels of the ^C8CPXV012 constructs and BNLF2a^C8 in cells analyzed by flow cytometry were confirmed by anti-C8 and anti-actin immunoblotting. (E) Inactive ^C8CPXV012-CΔ5 still binds to coreTAP1/2 heterodimers. Human coreTAP1^mVenus-C8 and coreTAP2^{mCerulean-StrepII} were coexpressed in HEK293T cells together with the ^C8CPXV012 variants as indicated. TAP1/2 heterodimeric complexes were tandem-affinity purified using streptavidin and anti-TAP1 (mAb 148.3) matrices. The HC10-antibody was used as negative control (mock). Input (solubilizate, 1/30 aliquot) and affinity purified complexes were analyzed by immunoblotting using C8- or TAP2-specific (mAb 435.3) antibodies, respectively. #, partially unfolded mCerulean.</p

CPXV012 blocks ATP binding to TAP1 and TAP2.

<p>(A) CPXV012 inhibits ATP binding to TAP. TAP1, TAP2, and ^C8CPXV012 were coexpressed in <i>Sf</i>9 cells as indicated. TAP1/TAP2 or TAP1/TAP2/^C8CPXV012 complexes were affinity purified using anti-TAP1 (mAb 148.3) or anti-C8 antibodies, respectively. Dynabead immobilized proteins were pre-incubated with 15 µM 8-azido-ATP[γ]biotin in the presence or absence of 5 mM ATP for 5 min on ice and subsequently subjected to UV irradiation. Dynabead immobilized proteins were then separated by SDS-PAGE and detected by immunoblotting with the antibodies as indicated. Biotinylated proteins were visualized using extravidin-HRP conjugate. In the presence (open bars) and absence of ^C8CPXV012 (filled bars), the amount of ATP cross-linked TAP was normalized to the TAP2 protein expression levels. (B) CPXV012 inhibits ATP binding sites to TAP1 and TAP2. TAP1, ^TsnTAP2, and ^flagCPXV012 were coexpressed and crude membranes were incubated with 8-azido-ATP[γ]biotin (15 µM) in the presence or absence of ATP (5 mM) for 5 min on ice. After UV irradiation, proteins were immunoprecipitated with TAP1 (mAb 148.3) or flag-specific antibodies (IP). The HC10-antibody was used as negative control (mock). Immunoprecipitated samples were then separated by SDS-PAGE and analyzed by immunoblotting with extravidin-HRP or the corresponding antibodies. The amount of TAP photo cross-linked by 8-azido-ATP in the absence (filled bars) or presence (open bars) of ^flagCPXV012 was normalized to TAP1 or ^TsnTAP2 protein expression levels, respectively.</p

The ER-lumenal CPXV012 fragment is sufficient for trans-inhibition of antigen translocation.

<p>(A) CPXV012 sequence and synthetic peptides derived from the C-terminal ER-lumenal end of CPXV012. (B) The C-terminal 10 residues of CPXV012 are sufficient for inhibition of the peptide-stimulated ATP hydrolysis of TAP. Purified coreTAP (0.2 µM) was incubated with ATP (1 mM) and high-affinity substrate peptide R9LQK (1 µM) in the presence and absence of C-terminal CPXV012 peptides (20 µM) as indicated. Release of inorganic phosphate was quantified and normalized to the coreTAP-dependent hydrolysis in the presence of R9LQK. Each data point represents the mean of triplicate measurements. Error bars show S.D. (C) The ATP hydrolysis activity of purified TAP (0.2 µM) was measured in the presence of ATP (1 mM), substrate peptide R9LQK (0.5 µM), and increasing concentrations of CPXV012 10mer-peptide. By fitting of the data, a half-maximum inhibition value (IC₅₀) of 72±20 µM was determined. (D) 10mer CPXV012 fragment blocks peptide translocation <i>in vitro</i>. Transport of RRYC^(F)KSTEL peptide (1 µM; C^(F), fluorescein-labeled cysteine) by reconstituted coreTAP was analyzed in the presence and absence of ATP (3 mM) in combination with 100 µM unlabeled peptide R9LQK or CPXV012 10mer-peptide added to or entrapped in liposomes (external and lumenal, respectively). (E) Inhibition mechanism of CPXV012. TAP transports peptides until a critical concentration inside the ER lumen induces trans-inhibition of the transport complex.</p