Annexin A1: a central player in the anti-inflammatory and neuroprotective role of microglia

The brain microenvironment is continuously monitored by microglia with the detection of apoptotic cells or pathogens being rapidly followed by their phagocytosis to prevent inflammatory responses. The protein annexin A1 (ANXA1) is key to the phagocytosis of apoptotic leukocytes during peripheral inflammatory resolution, but the pathophysiological significance of its expression in the CNS that is restricted almost exclusively to microglia is unclear. In this study, we test the hypothesis that ANXA1 is important in the microglial clearance of apoptotic neurons in both noninflammatory and inflammatory conditions. We have identified ANXA1 to be sparingly expressed in microglia of normally aged human brains and to be more strongly expressed in Alzheimer's disease. Using an in vitro model comprising microglial and neuronal cell lines, as well as primary microglia from wild-type and ANXA1 null mice, we have identified two distinct roles for microglial ANXA1: 1) controlling the noninflammatory phagocytosis of apoptotic neurons and 2) promoting resolution of inflammatory microglial activation. In particular, we showed that microglial-derived ANXA1 targets apoptotic neurons, serving as both an "eat me" signal and a bridge between phosphatidylserine on the dying cell and formyl peptide receptor 2 on the phagocytosing microglia. Moreover, inflammatory activation of microglia impairs their ability to discriminate between apoptotic and nonapoptotic cells, an ability restored by exogenous ANXA1. We thus show that ANXA1 is fundamental for brain homeostasis, and we suggest that ANXA1 and its peptidomimetics can be novel therapeutic targets in neuroinflammation.     

Partial 28S rDNA sequences and the antiquity of hydrothermal vent endemic gastropods

A molecular phylogenetic investigation of the hypothesized antiquity of the hydrothermal vent endemic Neomphalina (Mollusca; Gastropoda) is reported. Sequences of two domains of the gene encoding for 28S ribosomal RNA were acquired for 3 outgroup and 32 gastropod genera. Use of the likelihood ratio test indicated complex substitution patterns for these domains and taxa, corresponding to a general time-reversible model with among-site rate variation. Phylogenetic analyses were performed using this model under maximum likelihood criteria. The data lacked resolution of gastropod radiations of the Paleozoic and all three of the outgroup sequences were randomized relative to the ingroup. Acceleration of evolutionary rates had additionally randomized the sequences of the Patellogastropoda relative to the other Gastropoda. The data resolved radiations of the Mesozoic and supported monophyly of the sampled Neritopsina, Vetigastropoda, Neomphalina, Caenogastropoda (including Campanile and the Architaenioglossa), and Heterobranchia (Valvata + Euthyneura), although several results were not significantly different from nonmonophyletic alternatives. Mesozoic origins of the hydrothermal vent endemic Neomphalina are preliminarily supported and implications for the hydrothermal vent refugia hypothesis discussed. Issues related to phylogenetic resolution of the Gastropoda are additionally discussed.     

Iron-dependent hydrogenases of Entamoeba histolytica and Giardia lamblia: activity of the recombinant entamoebic enzyme and evidence for lateral gene transfer

Entamoeba histolytica and Spironucleus barkhanus have genes that encode short iron-dependent hydrogenases (Fe-hydrogenases), even though these protists lack hydrogenosomes. To understand better the biochemistry of the protist Fe-hydrogenases, we prepared a recombinant E. histolytica short Fe-hydrogenase and measured its activity in vitro. A Giardia lamblia gene encoding a short Fe-hydrogenase was identified from shotgun genomic sequences, and RT-PCR showed that cultured entamoebas and giardias transcribe short Fe-hydrogenase mRNAs. A second E. histolytica gene, which encoded a long Fe-hydrogenase, was identified from shotgun genomic sequences. Phylogenetic analyses suggested that the short Fe-hydrogenase genes of entamoeba and diplomonads share a common ancestor, while the long Fe-hydrogenase gene of entamoeba appears to have been laterally transferred from a bacterium. These results are discussed in the context of competing ideas for the origins of genes encoding fermentation enzymes of these protists.     

MAD1 and p27(KIP1) cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity

To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27(KIP1). Generation of Mad1/p27(KIP1) double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27(KIP1), including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27(KIP1) double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27(KIP1) single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27(KIP1) operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.     

Evidence for lateral transfer of genes encoding ferredoxins,nitroreductases, NADH oxidase,and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica

Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica.     

CHROMOSOMAL STUDIES OF SUBGENUS TRIDENTATAE OF ARTEMISIA: EVIDENCE FOR AUTOPOLYPLOIDY

The sagebrushes (subgenus Tridentatae of Artemisia—new combination presented in the text) are western North America's most widespread and populous shrub group. Chromosome counts from 120 populations confirm the base chromosome number at x = 9 with numerous 2n = 2x = 18 diploids and 2n = 4x = 36 tetraploids. Few higher polyploids are known, and aneuploidy and supernumerary chromosomes are rare. All 11 Tridentatae species are now known cytologically. All but the narrowly endemic A. argillosa are known from at least three locations: A. arbuscula (2n = 18, 36), A. bigelovii (2n = 18, 36), A. cana (2n = 18, 36), A. longiloba (2n = 18, 36), A. nova (2n = 18, 36), A. pygmaea (2n = 18, 36), A. rigida (2n = 18, 36), A. rothrockii (2n = 18, 36, 54, ca. 72), A. tridentata (2n = 18, 36, 54), and A. tripartita (2n = 18, 36). The chromosome number of A. argillosa, reported here for the first time, is 2n = 36. Chromosome numbers of eight subspecies also have been determined. The subgenus is characterized by autopolyploidy as indicated by morphologically indistinguishable 2x and 4x plants, a few mixed ploidy populations, consistent formation of IVs in 4x PMCs, a relatively uniform 2x karyotype, and a 4x karyotype, which is approximately twice the 2x one. Karyotypic differences, if they exist at all, are on a populational level rather than a systematic taxonomic level. The Tridentatae have apparently rapidly differentiated in situ in North America under the stimulus of recurring aridic cycles since late Tertiary or early Quaternary. They likely derive from more primitive circumboreal stock originating from the Eurasian homeland of Artemisia. The differentiation of myriad forms of Tridentatae was seemingly achieved through genic rather than genomic means. Karyotypic analysis supports a position within Tridentatae of A. rigida, A. bigelovii, and A. pygmaea.     

Resistance Responses of Potato to Vesicular-Arbuscular Mycorrhizal Fungi under Varying Abiotic Phosphorus Levels

In mycorrhizal symbioses, susceptibility of a host plant to infection by fungi is influenced by environmental factors, especially the availability of soil phosphorus. This study describes morphological and biochemical details of interactions between a vesicular-arbuscular mycorrhizal (VAM) fungus and potato (Solanum tuberosum L. cv Russet Burbank) plants, with a particular focus on the physiological basis for P-induced resistance of roots to infection. Root infection by the VAM fungus Glomus fasciculatum ([Thaxt. sensu Gerdemann] Gerdemann and Trappe) was extensive for plants grown with low abiotic P supply, and plant biomass accumulation was enhanced by the symbiosis. The capacity of excised roots from P-deficient plants to produce ethylene in the presence or absence of exogenous 1-amino cyclopropane-1-carboxylic acid (ACC) was markedly reduced by VAM infection. This apparent inhibition of ACC oxidase (ACC_ox) activity was localized to areas containing infected roots, as demonstrated in split-root studies. Furthermore, leachate from VAM roots contained a potent water-soluble inhibitor of ethylene generation from exogenous ACC by nonmycorrhizal (NM) roots. The leachate from VAM-infected roots had a higher concentration of phenolics, relative to that from NM roots. Moreover, the rates of ethylene formation and phenolic concentration in leachates from VAM roots were inversely correlated, suggesting that this inhibitor may be of a phenolic nature. The specific activity of extracellular peroxidase recovered in root leachates was not stimulated by VAM infection, although activity on a fresh weight basis was significantly enhanced, reflecting the fact that VAM roots had higher protein content than NM roots. Polyphenol oxidase activity of roots did not differ between NM and VAM roots. These results characterize the low resistance response of P-deficient plants to VAM infection. When plants were grown with higher abiotic P supply, the relative benefit of the VAM symbiosis to plant growth decreased and root infection was lower. The in vivo ACC_ox activity was also greater in roots of plants grown on high levels of P compared with those grown on low levels, although the influence of VAM infection was partially to counteract the nutritional effect of P on ACC_ox activity. Similar to ACC_ox activity, extracellular peroxidase activity of roots increased linearly with increasing abiotic P supply, thus indicating a greater potential for resistance to VAM infection. These findings suggest that VAM fungi may alter phenolic metabolism of roots so as to hinder ethylene production and the root's ability to invoke a defense response. Raising the abiotic P supply to plants at least partially restores the capacity of roots to produce ethylene and may, in this way, increase the root's resistance to VAM infection.     

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.