Symbiont-specific responses to environmental cues in a threesome lichen symbiosis.

Photosymbiodemes are a special case of lichen symbiosis where one lichenized fungus engages in symbiosis with two different photosynthetic partners, a cyanobacterium and a green alga, to develop two distinctly looking photomorphs. We compared gene expression of thallus sectors of the photosymbiodeme-forming lichen Peltigera britannica containing cyanobacterial photobionts with thallus sectors with both green algal and cyanobacterial photobionts and investigated differential gene expression at different temperatures representing mild and putatively stressful conditions. First, we quantified photobiont-mediated differences in fungal gene expression. Second, because of known ecological differences between photomorphs, we investigated symbiont-specific responses in gene expression to temperature increases. Photobiont-mediated differences in fungal gene expression could be identified, with upregulation of distinct biological processes in the different morphs, showing that interaction with specific symbiosis partners profoundly impacts fungal gene expression. Furthermore, high temperatures expectedly led to an upregulation of genes involved in heat shock responses in all organisms in whole transcriptome data and to an increased expression of genes involved in photosynthesis in both photobiont types at 15 and 25°C. The fungus and the cyanobacteria exhibited thermal stress responses already at 15°C, the green algae mainly at 25°C, demonstrating symbiont-specific responses to environmental cues and symbiont-specific ecological optima.

Alternative electron transport pathways contribute to tolerance to high light stress in lichenized algae.

The photosynthetic apparatus of lichen photobionts has been well-characterized by chlorophyll fluorescence analysis (e.g., by pulse amplitude modulation [PAM]), which provides a proxy of the activity of photosystem II (PSII) and its antenna. However, such kinetics are unable to directly characterize photosystem I (PSI) activity and the associated alternative electron pathways that may be involved in photoprotection. Instead, PSI can be probed in vivo by near-infrared absorption, measured at the same time as standard chlorophyll fluorescence (e.g., using the WALZ Dual PAM). Here, we used the Dual PAM to investigate cyclic electron flow and photoprotection in a range of mostly temperate lichens sampled from shaded to more open microhabitats. Sun species displayed lower acceptor side limitation of PSI (Y[NA]) early in illumination when compared to shade species, indicative of higher flavodiiron-mediated pseudocyclic electron flow. In response to high irradiance, some lichens accumulate melanin, and Y[NA] was lower and NAD(P)H dehydrogenase (NDH-2)-type cyclic flow was higher in melanised than pale forms. Furthermore, non-photochemical quenching (NPQ) was higher and faster relaxing in shade than sun species, while all lichens displayed high rates of photosynthetic cyclic electron flow. In conclusion, our data suggest that (1) low acceptor side limitation of PSI is important for sun-exposed lichens; (2) NPQ helps shade species tolerate brief exposure to high irradiance; and (3) cyclic electron flow is a prominent feature of lichens regardless of habitat, although NDH-2-type flow is associated with high light acclimation.

Gene expression responses to thermal shifts in the endangered lichen Lobaria pulmonaria.

Anthropogenic climate change has led to unprecedented shifts in temperature across many ecosystems. In a context of rapid environmental changes, acclimation is an important process as it may influence the capacity of organisms to survive under novel thermal conditions. Mechanisms of acclimation could involve upregulation of stress response genes involved in protein folding, DNA damage repair and the regulation of signal transduction genes, along with a simultaneous downregulation of genes involved in growth or the cell cycle, in order to maintain cellular functions and equilibria. We transplanted Lobaria pulmonaria lichens originating from different forests to determine the relative effects of long-term acclimation and genetic factors on the variability in expression of mycobiont and photobiont genes. We found a strong response of the mycobiont and photobiont to high temperatures, regardless of sample origin. The green-algal photobiont had an overall lower response than the mycobiont. Gene expression of both symbionts was also influenced by acclimation to transplantation sites and by genetic factors. L. pulmonaria seems to have evolved powerful molecular pathways to deal with environmental fluctuations and stress and can acclimate to new habitats by transcriptomic convergence. Although L. pulmonaria has the molecular machinery to counteract short-term thermal stress, survival of lichens such as L. pulmonaria depends mostly on their long-term positive carbon balance, which can be compromised by higher temperatures and reduced precipitation, and both these outcomes have been predicted for Central Europe in connection with global climate change.

Evolutionary lessons from California plant phylogeography.

Phylogeography documents the spatial distribution of genetic lineages that result from demographic processes, such as population expansion, population contraction, and gene movement, shaped by climate fluctuations and the physical landscape. Because most phylogeographic studies have used neutral markers, the role of selection may have been undervalued. In this paper, we contend that plants provide a useful evolutionary lesson about the impact of selection on spatial patterns of neutral genetic variation, when the environment affects which individuals can colonize new sites, and on adaptive genetic variation, when environmental heterogeneity creates divergence at specific loci underlying local adaptation. Specifically, we discuss five characteristics found in plants that intensify the impact of selection: sessile growth form, high reproductive output, leptokurtic dispersal, isolation by environment, and the potential to evolve longevity. Collectively, these traits exacerbate the impact of environment on movement between populations and local selection pressures-both of which influence phylogeographic structure. We illustrate how these unique traits shape these processes with case studies of the California endemic oak, Quercus lobata, and the western North American lichen, Ramalina menziesii Obviously, the lessons we learn from plant traits are not unique to plants, but they highlight the need for future animal, plant, and microbe studies to incorporate its impact. Modern tools that generate genome-wide sequence data are now allowing us to decipher how evolutionary processes affect the spatial distribution of different kinds of genes and also to better model future spatial distribution of species in response to climate change.

Discovery of long-distance gamete dispersal in a lichen-forming ascomycete.

Accurate estimates of gamete and offspring dispersal range are required for the understanding and prediction of spatial population dynamics and species persistence. Little is known about gamete dispersal in fungi, especially in lichen-forming ascomycetes. Here, we estimate the dispersal functions of clonal propagules, gametes and ascospores of the epiphytic lichen Lobaria pulmonaria. We use hierarchical Bayesian parentage analysis, which integrates genetic and ecological information from multiannual colonization and dispersal source data collected in a large, old-growth forest landscape. The effective dispersal range of gametes is several hundred metres to kilometres from potential paternal individuals. By contrast, clonal propagules disperse only tens of metres, and ascospores disperse over several thousand metres. Our study reveals the dispersal distances of individual reproductive units; clonal propagules, gametes and ascospores, which is of great importance for a thorough understanding of the spatial dynamics of ascomycetes. Sexual reproduction occurs between distant individuals. However, whereas gametes and ascospores disperse over long distances, the overall rate of colonization of trees is low. Hence, establishment is the limiting factor for the colonization of new host trees by the lichen in old-growth landscapes.

Antiendothelial αvß3 Antibodies Are a Major Cause of Intracranial Bleeding in Fetal/Neonatal Alloimmune Thrombocytopenia.

OBJECTIVE: Fetal/neonatal alloimmune thrombocytopenia is a severe bleeding disorder, which can result in intracranial hemorrhage (ICH), leading to death or neurological sequelae. In whites, maternal anti-human platelet antigen-1a (HPA-1a) antibodies are responsible for the majority of cases. No predictive factors for ICH are available to guide prophylactic treatment during pregnancy. In this study, we investigated antibodies from mothers with ICH-positive fetal/neonatal alloimmune thrombocytopenia and with ICH-negative fetal/neonatal alloimmune thrombocytopenia to identify serological and functional differences between the groups. APPROACH AND RESULTS: In an antigen capture assay, we observed a stronger binding of +ICH antibodies to endothelial cell (EC)-derived αvß3. By absorption experiments, we subsequently identified anti-HPA-1a antibodies of anti-αvß3 specificity in the +ICH but not in the -ICH cohort. Only the anti-αvß3 subtype, but not the anti-ß3 subtype, induced EC apoptosis of HPA-1a-positive ECs by caspase-3/7 activation, and mediated by reactive oxygen species. In addition, only the anti-αvß3 subtype, but not the anti-ß3 subtype, interfered with EC adhesion to vitronectin and with EC tube formation. CONCLUSIONS: We conclude that the composition of the anti-HPA-1a antibody subtype(s) of the mother may determine whether ICH occurs. Analysis of anti-HPA-1a antibodies of the anti-αvß3 subtype in maternal serum has potential in the diagnostic prediction of ICH development and may allow for modification of prophylactic treatment in fetal/neonatal alloimmune thrombocytopenia.

Choline Transporter-Like Protein-2: New von Willebrand Factor-Binding Partner Involved in Antibody-Mediated Neutrophil Activation and Transfusion-Related Acute Lung Injury.

OBJECTIVE: In contrast to other antibodies involved in transfusion-related acute lung injury, anti-HNA-3a antibodies are incapable of inducing direct neutrophil activation and seem to interact with endothelial cells (ECs) primarily. In animal studies, anti-HNA-3a-mediated transfusion-related acute lung injury could be precipitated in the absence of neutrophils, but was stronger when neutrophils were present. In a different context the target protein of these antibodies, choline transporter-like protein-2 (CTL-2), was reported to interact with a protein of the inner ear carrying 2 von Willebrand factor (VWF) A-domains. These observations prompted us to investigate whether VWF might be involved in anti-HNA-3a-mediated neutrophil activation, and whether signaling via CD11b/CD18 is involved, as in various other experimental settings. APPROACH AND RESULTS: Cell adhesion demonstrated specific binding of CTL-2 to VWF. Immunoprecipitation analysis of CTL-2/CD11b/CD18 coexpressing cells indicated that anti-HNA-3a colocalizes CTL-2 and CD11b/CD18 when VWF is present. Functional studies revealed that anti-HNA-3a-mediated neutrophil agglutination is an active, protein kinase C-dependent and partially Fc-dependent process. Agglutination and the production of reactive oxygen species seem to require the formation of a trimolecular complex between the target antigen (CTL-2), CD11b/CD18 and VWF. In line with these observations, anti-HNA-3a induced less severe transfusion-related acute lung injury and less neutrophil recruitment to the alveolar space in VWF knockout mice. CONCLUSIONS: We introduce CTL-2 as a new binding partner for VWF. Interaction of neutrophils with VWF via CTL-2 allows anti-HNA-3a to induce signal transduction via CD11b/CD18, which leads to neutrophil activation and agglutination. In transfusion-related acute lung injury, this mechanism may further aggravate endothelial leakage.

Rapid characterization of hybridomas producing monoclonal antibodies against platelet ß3 integrin using ELIspot.

Generally, B-cell responses against human platelet antigens are assessed by the serological detection of specific platelet antibodies, mostly against ß3 integrin. However, this approach seems to be of low sensitivity, since platelet autoantibodies against αIIbß3 are detected in only 50% of all patients with immune thrombocytopenia (ITP). In this study, a novel B-cell ELIspot method was established to characterize the specificity of mouse monoclonal antibodies (moabs) against human ß3 integrin. Moabs produced by hybridomas were immobilized on membrane and bound antibodies were visualized as spots using biotinylated recombinant proteins αIIbß3 or αvß3 and the enzyme labeled streptavidin-substrate system. Three hybridomas, Gi5, Gi16 and AP3, designated previously as anti-αIIbß3, anti-αIIb and anti-ß3, respectively, were investigated. Hybridoma producing moab against CD177 was used as the negative control. Whereas AP3 reacted with αIIbß3 and αvß3, Gi5 only formed spots with αIIbß3. Titration analysis showed that the number of spots correlated significantly with the number of seeded cells. Approximately 15 antibody producing hybridoma cells could be identified among 103 nonproducing B-cells. Furthermore, superior correlation with the total number of IgG producing cells was obtained. Analysis of the third hybridoma, Gi16 (anti-αIIb), showed only few spots with αIIbß3, indicating that this hybridoma contained different clones (producer and non-producer). Significant increased number of spots could be identified after re-cloning of these clones by limiting dilution method. Our results demonstrate that this B-cell ELIspot assay can be used for the identification of a small number of hybridoma cells producing moabs against ß3 integrin, verification of their monoclonality, productivity and for determining their specificity in the early state of workup steps. In the future, this approach may be useful to define B-cell clones in patients who developed platelet antibodies against different ß3-integrins and to differentiate their diversities.

Alloantibody against new platelet alloantigen (Lap(a)) on glycoprotein IIb is responsible for a case of fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by the destruction of platelets (PLTs) in the fetus or newborn by maternal PLT antibodies that crossed the placenta during pregnancy. STUDY DESIGN AND METHODS: In this study, we aim to elucidate the properties of a new PLT alloantigen (Lap(a)) that is associated with a severe case of FNAIT. Analysis of maternal serum with phenotyped PLTs by monoclonal antibody-specific immobilization of platelet antigens showed positive reaction against PLT glycoprotein (GP)IIb/IIIa and HLA Class I expressed on paternal PLTs. RESULTS: In contrast to GPIIIa-reactive anti-HPA-1a, anti-Lap(a) alloantibodies precipitated predominantly GPIIb. Indeed, a point mutation G>C at Position 2511 located in Exon 25 of the ITGA2B gene was found in Lap(a)-positive donors. This mutation causes an amino exchange Gln>His at Position 806 located in the calf-2 domain of GPIIb. Lap(a)-positive individuals were not found in 300 random blood donors. Our expression study showed that anti-Lap(a) alloantibodies reacted with stable transfected HEK293 cells expressing the mutated GPIIb isoform (His806). CHO cells carrying this isoform, however, failed to react with anti-Lap(a) alloantibodies, indicating that Lap(a) epitopes depend on the Gln806 His mutation and the carbohydrate composition of the GPIIb. This mutation did not hamper the binding of anti-HPA-3a, which recognizes a point mutation (Ile843 Ser) located in calf-2 domain. Finally, we found that Lap(a) and some HPA-3a epitopes are sensitive to O-glycanase. CONCLUSIONS: This study not only underlines the relevance of rare HPAs on the pathomechanism of FNAIT, but also helps to understand the pitfalls of serologic assays to detect anti-GPIIb alloantibodies.

Phylogeography of Ramalina menziesii, a widely distributed lichen-forming fungus in western North America.

The complex topography and climate history of western North America offer a setting where lineage formation, accumulation and migration have led to elevated inter- and intraspecific biodiversity in many taxa. Here, we study Ramalina menziesii, an epiphytic lichenized fungus with a range encompassing major ecosystems from Baja California to Alaska to explore the predictions of two hypotheses: (i) that the widespread distribution of R. menziesii is due to a single migration episode from a single lineage and (ii) that the widespread distribution is due to the formation and persistence of multiple lineages structured throughout the species' range. To obtain evidence for these predictions, we first construct a phylogenetic tree and identify multiple lineages structured throughout the species' range--some ancient ones that are localized and other more recent lineages that are widely distributed. Second, we use an isolation with migration model to show that sets of ecoregion populations diverged from each other at different times, demonstrating the importance of historical and current barriers to gene flow. Third, we estimated migration rates among ecoregions and find that Baja California populations are relatively isolated, that inland California ecoregion populations do not send out emigrants and that migration out of California coastal and Pacific Northwest populations into inland California ecoregions is high. Such intraspecific geographical patterns of population persistence and dispersal both contribute to the wide range of this genetically diverse lichen fungus and provide insight into the evolutionary processes that enhance species diversity of the California Floristic Province.

Microclimatic differentiation of gene pools in the Lobaria pulmonaria symbiosis in a primeval forest landscape.

Population genetics of the tree-colonizing lichen Lobaria pulmonaria were studied in the largest primeval beech forest of Europe, covering 10 000 ha. During an intensive survey of the area, we collected 1522 thallus fragments originating from 483 trees, which were genotyped with eight mycobiont- and 14 photobiont-specific microsatellite markers. The mycobiont and photobiont of L. pulmonaria were found to consist of two distinct gene pools, which are co-existing within small areas of 3-180 ha in a homogeneous beech forest. The small-scale distribution pattern of the symbiotic gene pools show habitat partitioning of lineages associated with either floodplains or mountain forests. Using approximate Bayesian computation (ABC), we dated the divergence of the two fungal gene pools of L. pulmonaria as the Early Pleistocene. Both fungal gene pools survived the Pleistocene glacial cycles in the Carpathians, although possibly in climatically different refugia. Fungal diversification prior to these cycles and the selection of photobionts with different altitudinal distributions explain the current sympatric, but ecologically differentiated habitat partitioning of L. pulmonaria. In addition, the habitat preferences of the mycobiont are determined by other factors and are rather independent of those of the photobiont at the landscape level. The distinct gene pools should be considered evolutionarily significant units and deserve specific conservation priorities in the future, for example gene pool A, which is a Pliocene relict.

Ecological specialization in Trebouxia (Trebouxiophyceae) photobionts of Ramalina menziesii (Ramalinaceae) across six range-covering ecoregions of western North America.

• Premise of the study: Many lichens exhibit extensive ranges spanning several ecoregions. It has been hypothesized that this wide ecological amplitude is facilitated by fungal association with locally adapted photobiont strains.• Methods: We studied the identity and geographic distribution of photobionts of the widely distributed North American lichen Ramalina menziesii based on rbcL (chloroplast DNA) and nuclear ribosomal ITS DNA sequences. To test for ecological specialization, we associate photobiont genotypes with local climate and phorophyte.• Key results: Of the photobiont lineages of R. menziesii, 94% belong to a clade including Trebouxia decolorans. The remaining are related to T. jamesii. The photobionts showed (1) significant structure according to ecoregion and phorophyte species and (2) genetic associations with phorophyte species and climate.• Conclusions: Geography, climate, and ecological specialization shape genetic differentiation of lichen photobionts. One great advantage of independent dispersal of the fungus is symbiotic association with locally adapted photobiont strains.

Anti-human neutrophil antigen-3a induced transfusion-related acute lung injury in mice by direct disturbance of lung endothelial cells.

OBJECTIVE: Antibodies against human neutrophil antigen-3a (HNA-3a) located on choline transporter-like protein 2 induce severe transfusion-related acute lung injury (TRALI). This study aims to identify the mechanism implicated in anti-HNA-3a-mediated TRALI. APPROACH AND RESULTS: Our analysis shows that anti-HNA-3a recognizes 2 choline transporter-like protein 2 isoforms (P1 and P2) on human microvascular endothelial cells from lung blood vessels but reacts only with the P1 isoform on neutrophils. Direct treatment of HNA-3a-positive endothelial cells with anti-HNA-3a, but not with anti-HNA-3b, leads to reactive oxygen species production, increased albumin influx, and decreased endothelial resistance associated with the formation of actin stress filaments and loosening of junctional vascular endothelium-cadherin. In a novel in vivo mouse model, TRALI was documented by significant increase in lung water content, albumin concentration, and neutrophil numbers in the bronchoalveolar lavage on injection of human anti-HNA-3a in lipopolysaccharides-treated, as well as nontreated mice. Interestingly, although neutrophil depletion alleviated severity of lung injury, it failed to prevent TRALI in this model. Infusion of anti-HNA-3a F(ab')2 fragments caused moderate TRALI. Finally, mice lacking nicotinamide adenine dinucleotide phosphate oxidase (NOX2(y/-)) were protected from anti-HNA-3a-mediated TRALI. CONCLUSIONS: These data demonstrate the initiation of endothelial barrier dysfunction in vitro and in vivo by direct binding of anti-HNA-3a on endothelial cells. It seems, however, that the presence of neutrophils aggravates barrier dysfunction. This novel mechanism of TRALI primarily mediated by endothelial cell dysfunction via choline transporter-like protein 2 may help to define new treatment strategies to decrease TRALI-related mortality.

Rapid enzyme-linked immunosorbent assay for the detection of antibodies against human neutrophil antigens -1a, -1b, and -1c.

BACKGROUND: Several methods exist for the detection of neutrophil antibodies; most of them, however, require fresh neutrophils. In this study, an enzyme-linked immunosorbent assay (ELISA) using recombinant HNA-1 antigens (rHNAs) was developed to detect HNA-1a, -1b, and -1c alloantibodies in serum samples. STUDY DESIGN AND METHODS: Soluble rHNA-1a, -1b, and -1c were isolated from culture supernatant of transfected insect cells. Purified rHNA antigens were immobilized on microtiter wells using antibody against V5-Tag protein. Sera were added, and bound antibodies were detected by enzyme-labeled secondary antibodies. In parallel, monoclonal antibody-immobilized granulocyte antigen (MAIGA) was performed with two different monoclonal antibodies (MoAbs) against FcγRIIIb (3G8 and BW209). RESULTS: Fifteen MAIGA-positive sera containing HNA-1a alloantibodies were tested in ELISA. Thirteen of 15 (86.7%) MAIGA-positive sera captured by MoAbs 3G8 and/or BW209 reacted specifically with rHNA-1a. Four (26.7%) HNA-1a sera showed additional reaction with rHNA-1c. When anti-HNA-1b alloantibodies were analyzed in ELISA, 13 of 15 (86.7%) showed specific positive reaction with rHNA-1b, and 12 of 15 (80.0%) cross-reacted with rHNA-1c. Two HNA-1c sera reacted specifically with rHNA-1c. Immunoprecipitation analysis of all ELISA-negative HNA-1a and -1b sera did not show any specific band indicating false-positive reaction of these sera in MAIGA assay. CONCLUSIONS: These results suggested that rapid ELISA using recombinant neutrophil antigens may provide a valuable method for rapid screening of human alloantibodies against HNA-1a, -1b, and -1c in patients with neutropenia and in blood donors.

Congruent genetic structure in the lichen-forming fungus Lobaria pulmonaria and its green-algal photobiont.

The extent of codispersal of symbionts is one of the key factors shaping genetic structures of symbiotic organisms. Concordant patterns of genetic structure are expected in vertically transmitted symbioses, whereas horizontal transmission generally uncouples genetic structures unless the partners are coadapted. Here, we compared the genetic structures of mutualists, the lichen-forming fungus Lobaria pulmonaria and its primary green-algal photobiont, Dictyochloropsis reticulata. We performed analysis of molecular variance and variogram analysis to compare genetic structures between symbiosis partners. We simulated the expected number of multilocus-genotype recurrences to reveal whether the distribution of multilocus genotypes of either species was concordant with panmixia. Simulations and tests of linkage disequilibrium provided compelling evidence for the codispersal of mutualists. To test whether genotype associations between symbionts were consistent with randomness, as expected under horizontal transmission, we simulated the recurrence of fungal-algal multilocus genotype associations expected by chance. Our data showed nonrandom associations of fungal and algal genotypes. Either vertical transmission or horizontal transmission coupled with coadaptation between symbiont genotypes may have created these nonrandom associations. This study is among the first to show codispersal and highly congruent genetic structures in the partners of a lichen mutualism.

Implication of transfected cell lines for the detection of alloantibodies against human neutrophil antigen-3.

BACKGROUND: Alloantibodies against human neutrophil antigen-3 (HNA-3) are responsible for the fatalities reported in transfusion-related acute lung injury. Consequently, reliable detection of these alloantibodies is mandatory to improve blood transfusion safety. In this study, we developed stable cell lines for the detection of HNA-3 antibodies. STUDY DESIGN AND METHODS: HEK293T were transfected with HNA-3a or HNA-3b constructs and sorted by flow cytometry according to high surface expression. Transfected cells were tested with sera containing HNA-3 antibodies in flow cytometry and antibody capture assay (ACA). The results were compared with granulocyte agglutination test and granulocyte immunofluorescence test. RESULTS: In flow cytometry, 12 of 14 HNA-3a sera reacted specifically with HNA-3aa cells. One serum sample showed positive reaction with HNA-3bb cells. All HNA-3b sera recognized HNA-3bb cells. No reaction was observed with broad reactive antibodies against HLA Class I. In ACA, all HNA-3a sera (12/12) showed positive reactivity with HNA-3aa cells with no cross-reactivity with HNA-3bb cells. Again, all HNA-3b sera reacted with HNA-3bb cells only. Furthermore, genotyping of 249 individuals detected a new HNA-3 allele caused by a nucleotide substitution C>T at Position 457 leading to L(153)F mutation in choline transporter-like protein-2. This mutation impairs polymerase chain reaction with sequence-specific primers based HNA-3a typing. However, analysis with cells expressing F(153) isoform showed that this mutation did not alter the binding of HNA-3 antibodies. CONCLUSIONS: This study demonstrated that HEK293T cells expressing stable recombinant HNA-3 are suitable for the detection of HNA-3 alloantibodies allowing reliable screening of blood products.

Neutrophil transmigration mediated by the neutrophil-specific antigen CD177 is influenced by the endothelial S536N dimorphism of platelet endothelial cell adhesion molecule-1.

The human neutrophil-specific adhesion molecule CD177 (also known as the NB1 alloantigen) becomes upregulated on the cell surface in a number of inflammatory settings. We recently showed that CD177 functions as a novel heterophilic counterreceptor for the endothelial junctional protein PECAM-1 (CD31), an interaction that is mediated by membrane-proximal PECAM-1 IgD 6, which is known to harbor an S(536)N single nucleotide polymorphism of two major isoforms V(98)N(536)G(643) and L(98)S(536)R(643) and a yet-to-be-determined region on CD177. In vitro transendothelial migration experiments revealed that CD177(+) neutrophils migrated significantly faster through HUVECs expressing the LSR, compared with the VNG, allelic variant of PECAM-1 and that this correlated with the decreased ability of anti-PECAM-1 Ab of ITIM tyrosine phosphorylation in HUVECs expressing the LSR allelic variant relative to the VNG allelic variant. Moreover, engagement of PECAM-1 with rCD177-Fc (to mimic heterophilic CD177 binding) suppressed Ab-induced tyrosine phosphorylation to a greater extent in cells expressing the LSR isoform compared with the VNG isoform, with a corresponding increased higher level of beta-catenin phosphorylation. These data suggest that heterophilic PECAM-1/CD177 interactions affect the phosphorylation state of PECAM-1 and endothelial cell junctional integrity in such a way as to facilitate neutrophil transmigration in a previously unrecognized allele-specific manner.

High Diversity of Type I Polyketide Genes in Bacidia rubella as Revealed by the Comparative Analysis of 23 Lichen Genomes.

Fungi involved in lichen symbioses produce a large array of secondary metabolites that are often diagnostic in the taxonomic delimitation of lichens. The most common lichen secondary metabolites-polyketides-are synthesized by polyketide synthases, particularly by Type I PKS (TI-PKS). Here, we present a comparative genomic analysis of the TI-PKS gene content of 23 lichen-forming fungal genomes from Ascomycota, including the de novo sequenced genome of Bacidia rubella. Firstly, we identify a putative atranorin cluster in B. rubella. Secondly, we provide an overview of TI-PKS gene diversity in lichen-forming fungi, and the most comprehensive Type I PKS phylogeny of lichen-forming fungi to date, including 624 sequences. We reveal a high number of biosynthetic gene clusters and examine their domain composition in the context of previously characterized genes, confirming that PKS genes outnumber known secondary substances. Moreover, two novel groups of reducing PKSs were identified. Although many PKSs remain without functional assignments, our findings highlight that genes from lichen-forming fungi represent an untapped source of novel polyketide compounds.

Phylogeographic analyses of an epiphytic foliose lichen show multiple dispersal events westward from the Hengduan Mountains of Yunnan into the Himalayas.

Lobaria pindarensis is an endemic species of the Himalayas and the Hengduan Mountains. Little information is available on the phylogeography genetics and colonization history of this species or how its distribution patterns changed in response to the orographic history of the Himalayas and Hengduan Mountains. Based on samples covering a major part of the species' distribution range, we used 443 newly generated sequences of nine loci for molecular coalescent analyses in order to reconstruct the evolutionary history of L. pindarensis, and to reconstruct the species' ancestral phylogeographic distributions using Bayesian binary MCMC analyses. The results suggest that current populations originated from the Yunnan region of the Hengduan Mountains in the middle Pliocene, and that the Himalayas of Bhutan were colonized by a lineage that diverged from Yunnan ca. 2.72 Ma. The analysis additionally indicates that the Nepal and Xizang areas of the Himalayas were colonized from Yunnan as well, and that there was later a second dispersal event from Yunnan to Bhutan. We conclude that the change in climate and habitat related to the continuous uplift of the Himalayas and the Hengduan Mountains in the late Pliocene and middle Pleistocene influenced the geographic distribution pattern of L. pindarensis.

NO2 air pollution drives species composition, but tree traits drive species diversity of urban epiphytic lichen communities.

Lichens serve as important bioindicators of air pollution in cities. Here, we studied the diversity of epiphytic lichens in the urban area of Munich, Bavaria, southern Germany, to determine which factors influence species composition and diversity. Lichen diversity was quantified in altogether 18 plots and within each, five deciduous trees were investigated belonging to on average three tree species (range 1-5). Of the 18 plots, two were sampled in control areas in remote areas of southern Germany. For each lichen species, frequency of occurrence was determined in 10 quadrats of 100 cm2 on the tree trunk. Moreover, the cover percentage of bryophytes was determined and used as a variable to represent potential biotic competition. We related our diversity data (species richness, Shannon index, evenness, abundance) to various environmental variables including tree traits, i.e. bark pH levels and species affiliation and air pollution data, i.e. NO2 and SO2 concentrations measured in the study plots. The SO2 levels measured in our study were generally very low, while NO2 levels were rather high in some plots. We found that the species composition of the epiphytic lichen communities was driven mainly by NO2 pollution levels and all of the most common species in our study were nitrophilous lichens. Low NO2 but high SO2 values were associated with high lichen evenness. Tree-level lichen diversity and abundance were mainly determined by tree traits, not air pollution. These results confirm that ongoing NO2 air pollution within cities is a major threat to lichen diversity, with non-nitrophilous lichens likely experiencing the greatest risk of local extinctions in urban areas in the future. Our study moreover highlights the importance of large urban green spaces for species diversity. City planners need to include large green spaces when designing urban areas, both to improve biodiversity and to promote human health and wellbeing.