The knockout of cytoglobin 1 in zebrafish (Danio rerio) alters lipid metabolism, iron homeostasis and oxidative stress response.

Cytoglobin (Cygb) is an evolutionary ancient heme protein with yet unclear physiological function(s). Mammalian Cygb is ubiquitously expressed in all tissues and is proposed to be involved in reactive oxygen species (ROS) detoxification, nitric oxide (NO) metabolism and lipid-based signaling processes. Loss-of-function studies in mouse associate Cygb with apoptosis, inflammation, fibrosis, cardiovascular dysfunction or oncogenesis. In zebrafish (Danio rerio), two cygb genes exist, cytoglobin 1 (cygb1) and cytoglobin 2 (cygb2). Both have different coordination states and distinct expression sites within zebrafish tissues. The biological roles of the cygb paralogs are largely uncharacterized. We used a CRISPR/Cas9 genome editing approach and generated a knockout of the penta-coordinated cygb1 for in vivo analysis. Adult male cygb1 knockouts develop phenotypic abnormalities, including weight loss. To identify the molecular mechanisms underlying the occurrence of these phenotypes and differentiate between function and effect of the knockout we compared the transcriptomes of cygb1 knockout at different ages to age-matched wild-type zebrafish. We found that immune regulatory and cell cycle regulatory transcripts (e.g. tp53) were up-regulated in the cygb1 knockout liver. Additionally, the expression of transcripts involved in lipid metabolism and transport, the antioxidative defense and iron homeostasis was affected in the cygb1 knockout. Cygb1 may function as an anti-inflammatory and cytoprotective factor in zebrafish liver, and may be involved in lipid-, iron-, and ROS-dependent signaling.

The roles of brain lipids and polar metabolites in the hypoxia tolerance of deep-diving pinnipeds.

Lipids make up more than half of the human brain's dry weight, yet the composition and function of the brain lipidome is not well characterized. Lipids not only provide the structural basis of cell membranes, but also take part in a wide variety of biochemical processes. In neurodegenerative diseases, lipids can facilitate neuroprotection and serve as diagnostic biomarkers. The study of organisms adapted to extreme environments may prove particularly valuable in understanding mechanisms that protect against stressful conditions and prevent neurodegeneration. The brain of the hooded seal (Cystophora cristata) exhibits a remarkable tolerance to low tissue oxygen levels (hypoxia). While neurons of most terrestrial mammals suffer irreversible damage after only short periods of hypoxia, in vitro experiments show that neurons of the hooded seal display prolonged functional integrity even in severe hypoxia. How the brain lipidome contributes to the hypoxia tolerance of marine mammals has been poorly studied. We performed an untargeted lipidomics analysis, which revealed that lipid species are significantly modulated in marine mammals compared with non-diving mammals. Increased levels of sphingomyelin species may have important implications for efficient signal transduction in the seal brain. Substrate assays also revealed elevated normoxic tissue levels of glucose and lactate, which suggests an enhanced glycolytic capacity. Additionally, concentrations of the neurotransmitters glutamate and glutamine were decreased, which may indicate reduced excitatory synaptic signaling in marine mammals. Analysis of hypoxia-exposed brain tissue suggests that these represent constitutive mechanisms rather than an induced response towards hypoxic conditions.

Transcriptomes of Clusterin- and S100B-transfected neuronal cells elucidate protective mechanisms against hypoxia and oxidative stress in the hooded seal (Cystophora cristata) brain.

BACKGROUND: The hooded seal (Cystophora cristata) exhibits impressive diving skills and can tolerate extended durations of asphyxia, hypoxia and oxidative stress, without suffering from irreversible neuronal damage. Thus, when exposed to hypoxia in vitro, neurons of fresh cortical and hippocampal tissue from hooded seals maintained their membrane potential 4-5 times longer than neurons of mice. We aimed to identify the molecular mechanisms underlying the intrinsic neuronal hypoxia tolerance. Previous comparative transcriptomics of the visual cortex have revealed that S100B and clusterin (apolipoprotein J), two stress proteins that are involved in neurological disorders characterized by hypoxic conditions, have a remarkably high expression in hooded seals compared to ferrets. When overexpressed in murine neuronal cells (HN33), S100B and clusterin had neuroprotective effects when cells were exposed to hypoxia. However, their specific roles in hypoxia have remained largely unknown. METHODS: In order to shed light on potential molecular pathways or interaction partners, we exposed HN33 cells transfected with either S100B, soluble clusterin (sCLU) or nuclear clusterin (nCLU) to normoxia, hypoxia and oxidative stress for 24 h. We then determined cell viability and compared the transcriptomes of transfected cells to control cells. Potential pathways and upstream regulators were identified via Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA). RESULTS: HN33 cells transfected with sCLU and S100B demonstrated improved glycolytic capacity and reduced aerobic respiration at normoxic conditions. Additionally, sCLU appeared to enhance pathways for cellular homeostasis to counteract stress-induced aggregation of proteins. S100B-transfected cells sustained lowered energy-intensive synaptic signaling. In response to hypoxia, hypoxia-inducible factor (HIF) pathways were considerably elevated in nCLU- and sCLU-transfected cells. In a previous study, S100B and sCLU decreased the amount of reactive oxygen species and lipid peroxidation in HN33 cells in response to oxidative stress, but in the present study, these functional effects were not mirrored in gene expression changes. CONCLUSIONS: sCLU and S100B overexpression increased neuronal survival by decreasing aerobic metabolism and synaptic signaling in advance to hypoxia and oxidative stress conditions, possibly to reduce energy expenditure and the build-up of deleterious reactive oxygen species (ROS). Thus, a high expression of CLU isoforms and S100B is likely beneficial during hypoxic conditions.

Transcriptomes Suggest That Pinniped and Cetacean Brains Have a High Capacity for Aerobic Metabolism While Reducing Energy-Intensive Processes Such as Synaptic Transmission.

The mammalian brain is characterized by high energy expenditure and small energy reserves, making it dependent on continuous vascular oxygen and nutritional supply. The brain is therefore extremely vulnerable to hypoxia. While neurons of most terrestrial mammals suffer from irreversible damage after only short periods of hypoxia, neurons of the deep-diving hooded seal (Cystophora cristata) show a remarkable hypoxia-tolerance. To identify the molecular mechanisms underlying the intrinsic hypoxia-tolerance, we excised neurons from the visual cortices of hooded seals and mice (Mus musculus) by laser capture microdissection. A comparison of the neuronal transcriptomes suggests that, compared to mice, hooded seal neurons are endowed with an enhanced aerobic metabolic capacity, a reduced synaptic transmission and an elevated antioxidant defense. Publicly available whole-tissue brain transcriptomes of the bowhead whale (Balaena mysticetus), long-finned pilot whale (Globicephala melas), minke whale (Balaenoptera acutorostrata) and killer whale (Orcinus orca), supplemented with 2 newly sequenced long-finned pilot whales, suggest that, compared to cattle (Bos taurus), the cetacean brain also displays elevated aerobic capacity and reduced synaptic transmission. We conclude that the brain energy balance of diving mammals is preserved during diving, due to reduced synaptic transmission that limits energy expenditure, while the elevated aerobic capacity allows efficient use of oxygen to restore energy balance during surfacing between dives.

Elevated antioxidant defence in the brain of deep-diving pinnipeds.

While foraging, marine mammals undertake repetitive diving bouts. When the animal surfaces, reperfusion makes oxygen readily available for the electron transport chain, which leads to increased production of reactive oxygen species and risk of oxidative damage. In blood and several tissues, such as heart, lung, muscle and kidney, marine mammals generally exhibit an elevated antioxidant defence. However, the brain, whose functional integrity is critical to survival, has received little attention. We previously observed an enhanced expression of several antioxidant genes in cortical neurons of hooded seals (Cystophora cristata). Here, we studied antioxidant gene expression and enzymatic activity in the visual cortex, cerebellum and hippocampus of harp seals (Pagophilus groenlandicus) and hooded seals. Moreover, we tested several genes for positive selection. We found that antioxidants in the first line of defence, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione (GSH) were constitutively enhanced in the seal brain compared to mice (Mus musculus), whereas the glutaredoxin and thioredoxin systems were not. Possibly, the activity of the latter systems is stress-induced rather than constitutively elevated. Further, some, but not all members, of the glutathione-s-transferase (GST) family appear more highly expressed. We found no signatures of positive selection, indicating that sequence and function of the studied antioxidants are conserved in pinnipeds.

The More, the Merrier? Multiple Myoglobin Genes in Fish Species, Especially in Gray Bichir (Polypterus senegalus) and Reedfish (Erpetoichthys calabaricus).

The members of the globin superfamily are a classical model system to investigate gene evolution and their fates as well as the diversity of protein function. One of the best-known globins is myoglobin (Mb), which is mainly expressed in heart muscle and transports oxygen from the sarcolemma to the mitochondria. Most vertebrates harbor a single copy of the myoglobin gene, but some fish species have multiple myoglobin genes. Phylogenetic analyses indicate an independent emergence of multiple myoglobin genes, whereby the origin is mostly the last common ancestor of each order. By analyzing different transcriptome data sets, we found at least 15 multiple myoglobin genes in the polypterid gray bichir (Polypterus senegalus) and reedfish (Erpetoichthys calabaricus). In reedfish, the myoglobin genes are expressed in a broad range of tissues but show very different expression values. In contrast, the Mb genes of the gray bichir show a rather scattered expression pattern; only a few Mb genes were found expressed in the analyzed tissues. Both, gray bichir and reedfish possess lungs which enable them to inhabit shallow and swampy waters throughout tropical Africa with frequently fluctuating and low oxygen concentrations. The myoglobin repertoire probably reflects the molecular adaptation to these conditions. The sequence divergence, the substitution rate, and the different expression pattern of multiple myoglobin genes in gray bichir and reedfish imply different functions, probably through sub- and neofunctionalization during evolution.

Giant lungfish genome elucidates the conquest of land by vertebrates.

Lungfishes belong to lobe-fined fish (Sarcopterygii) that, in the Devonian period, 'conquered' the land and ultimately gave rise to all land vertebrates, including humans1-3. Here we determine the chromosome-quality genome of the Australian lungfish (Neoceratodus forsteri), which is known to have the largest genome of any animal. The vast size of this genome, which is about 14× larger than that of humans, is attributable mostly to huge intergenic regions and introns with high repeat content (around 90%), the components of which resemble those of tetrapods (comprising mainly long interspersed nuclear elements) more than they do those of ray-finned fish. The lungfish genome continues to expand independently (its transposable elements are still active), through mechanisms different to those of the enormous genomes of salamanders. The 17 fully assembled lungfish macrochromosomes maintain synteny to other vertebrate chromosomes, and all microchromosomes maintain conserved ancient homology with the ancestral vertebrate karyotype. Our phylogenomic analyses confirm previous reports that lungfish occupy a key evolutionary position as the closest living relatives to tetrapods4,5, underscoring the importance of lungfish for understanding innovations associated with terrestrialization. Lungfish preadaptations to living on land include the gain of limb-like expression in developmental genes such as hoxc13 and sall1 in their lobed fins. Increased rates of evolution and the duplication of genes associated with obligate air-breathing, such as lung surfactants and the expansion of odorant receptor gene families (which encode proteins involved in detecting airborne odours), contribute to the tetrapod-like biology of lungfishes. These findings advance our understanding of this major transition during vertebrate evolution.

Cell Culture Experiments Reveal that High S100B and Clusterin Levels may Convey Hypoxia-tolerance to the Hooded Seal (Cystophora cristata) Brain.

While the brain of most mammals suffers from irreversible damage after only short periods of low oxygen levels (hypoxia), marine mammals are excellent breath-hold divers that have adapted to hypoxia. In addition to physiological adaptations, such as large oxygen storing capacity and strict oxygen economy during diving, the neurons of the deep-diving hooded seal (Cystophora cristata) have an intrinsic tolerance to hypoxia. We aim to understand the molecular basis of this neuronal hypoxia tolerance. Previously, transcriptomics of the cortex of the hooded seal have revealed remarkably high expression levels of S100B and clusterin (apolipoprotein J) when compared to the ferret, a non-diving carnivore. Both genes have much-debated roles in hypoxia and oxidative stress. Here, we evaluated the effects of S100B and of two isoforms of clusterin (soluble and nucleus clusterin) on the survival, metabolic activity and the amount of reactive oxygen species (ROS) in HN33 neuronal mouse cells exposed to hypoxia and oxidative stress. S100B and soluble clusterin had neuroprotective effects, with reduced ROS-levels and retention of normoxic energy status of cells during both stress conditions. The protective effects of nucleus clusterin were restricted to hypoxia. S100B and clusterin showed purifying selection in marine and terrestrial mammals, indicating a functional conservation across species. Immunofluorescence revealed identical cellular distributions of S100B and clusterin in mice, ferrets and hooded seals, further supporting the functional conservation. Taken together, our data suggest that the neuroprotective effects of all three proteins are exclusively facilitated by their increased expression in the brain of the hooded seal.

The Globin Gene Family in Arthropods: Evolution and Functional Diversity.

Globins are small heme-proteins that reversibly bind oxygen. Their most prominent roles in vertebrates are the transport and storage of O2 for oxidative energy metabolism, but recent research has suggested alternative, non-respiratory globin functions. In the species-rich and ecologically highly diverse taxon of arthropods, the copper-containing hemocyanin is considered the main respiratory protein. However, recent studies have suggested the presence of globin genes and their proteins in arthropod taxa, including model species like Drosophila. To systematically assess the taxonomic distribution, evolution and diversity of globins in arthropods, we systematically searched transcriptome and genome sequence data and found a conserved, widespread occurrence of three globin classes in arthropods: hemoglobin-like (HbL), globin X (GbX), and globin X-like (GbXL) protein lineages. These globin types were previously identified in protostome and deuterostome animals including vertebrates, suggesting their early ancestry in Metazoa. The HbL genes show multiple, lineage-specific gene duplications in all major arthropod clades. Some HbL genes (e.g., Glob2 and 3 of Drosophila) display particularly fast substitution rates, possibly indicating the evolution of novel functions, e.g., in spermatogenesis. In contrast, arthropod GbX and GbXL globin genes show high evolutionary stability: GbXL is represented by a single-copy gene in all arthropod groups except Brachycera, and representatives of the GbX clade are present in all examined taxa except holometabolan insects. GbX and GbXL both show a brain-specific expression. Most arthropod GbX and GbXL proteins, but also some HbL variants, include sequence motifs indicative of potential N-terminal acylation (i.e., N-myristoylation, 3C-palmitoylation). All arthropods except for the brachyceran Diptera harbor at least one such potentially acylated globin copy, confirming the hypothesis of an essential, conserved globin function associated with the cell membrane. In contrast to other animals, the fourth ancient globin lineage, represented by neuroglobin, appears to be absent in arthropods, and the putative arthropod orthologs of the fifth metazoan globin lineage, androglobin, lack a recognizable globin domain. Thus, the remarkable evolutionary stability of some globin variants is contrasted by occasional dynamic gene multiplication or even loss of otherwise strongly conserved globin lineages in arthropod phylogeny.

Adhesion between P. falciparum infected erythrocytes and human endothelial receptors follows alternative binding dynamics under flow and febrile conditions.

Characterizing the adhesive dynamics of Plasmodium falciparum infected erythrocytes (IEs) to different endothelial cell receptors (ECRs) in flow is a big challenge considering available methods. This study investigated the adhesive dynamics of IEs to five ECRs (CD36, ICAM-1, P-selectin, CD9, CSA) using simulations of in vivo-like flow and febrile conditions. To characterize the interactions between ECRs and knobby and knobless IEs of two laboratory-adapted P. falciplarum isolates, cytoadhesion analysis over time was performed using a new tracking bioinformatics method. The results revealed that IEs performed rolling adhesion exclusively over CD36, but exhibited stationary binding to the other four ECRs. The absence of knobs affected rolling adhesion both with respect to the distance travelled by IEs and their velocity. Knobs played a critical role at febrile temperatures by stabilizing the binding interaction. Our results clearly underline the complexity of the IE-receptor interaction and the importance of knobs for the survival of the parasite at fever temperatures, and lead us to propose a new hypothesis that could open up new strategies for the treatment of malaria.

Transcriptome analysis reveals a high aerobic capacity in the whale brain.

The brain of diving mammals is repeatedly exposed to low oxygen conditions (hypoxia) that would have caused severe damage to most terrestrial mammals. Some whales may dive for >2 h with their brain remaining active. Many of the physiological adaptations of whales to diving have been investigated, but little is known about the molecular mechanisms that enable their brain to survive sometimes prolonged periods of hypoxia. Here, we have used an RNA-Seq approach to compare the mRNA levels in the brains of whales with those of cattle, which serves as a terrestrial relative. We sequenced the transcriptomes of the brains from cattle (Bos taurus), killer whale (Orcinus orca), and long-finned pilot whale (Globicephala melas). Further, the brain transcriptomes of cattle, minke whale (Balaenoptera acutorostrata) and bowhead whale (Balaena mysticetus), which were available in the databases, were included. We found a high expression of genes related to oxidative phosphorylation and the respiratory electron chain in the whale brains. In the visual cortex of whales, transcripts related to the detoxification of reactive oxygen species were more highly expressed than in the visual cortex of cattle. These findings indicate a high oxidative capacity in the whale brain that might help to maintain aerobic metabolism in periods of reduced oxygen availability during dives.

Genetic and functional diversity of the multiple lungfish myoglobins.

It is known that the West African lungfish (Protopterus annectens) harbours multiple myoglobin (Mb) genes that are differentially expressed in various tissues and that the Mbs differ in their abilities to confer tolerance towards hypoxia. Here, we show that other lungfish species (Protopterus dolloi, Protopterus aethiopicus and Lepidosiren paradoxa) display a similar diversity of Mb genes and have orthologous Mb genes. To investigate the functional diversification of these genes, we studied the structures, O2 binding properties and nitrite reductase enzymatic activities of recombinantly expressed P. annectens Mbs (PanMbs). CD spectroscopy and small-angle X-ray scattering revealed the typical globin-fold in all investigated recombinant Mbs, indicating a conserved structure. The highest O2 affinity was measured for PanMb2 (P50  = 0.88 Torr at 20 °C), which is mainly expressed in the brain, whereas the muscle-specific PanMb1 has the lowest O2 affinity (P50  = 3.78 Torr at 20 °C), suggesting that tissue-specific O2 requirements have resulted in the emergence of distinct Mb types. Two of the mainly neuronally expressed Mbs (PanMb3 and PanMb4b) have the highest nitrite reductase rates. These data show different O2 binding and enzymatic properties of lungfish Mbs, reflecting multiple subfunctionalisation and neofunctionalisation events that occurred early in the evolution of lungfish. Some Mbs may have also taken over the functions of neuroglobin and cytoglobin, which are widely expressed in vertebrates but appear to be missing in lungfish.

Twenty years of the 'Preparation for Oxidative Stress' (POS) theory: Ecophysiological advantages and molecular strategies.

Freezing, dehydration, salinity variations, hypoxia or anoxia are some of the environmental constraints that many organisms must frequently endure. Organisms adapted to these stressors often reduce their metabolic rates to maximize their chances of survival. However, upon recovery of environmental conditions and basal metabolic rates, cells are affected by an oxidative burst that, if uncontrolled, leads to (oxidative) cell damage and eventually death. Thus, a number of adapted organisms are able to increase their antioxidant defenses during an environmental/functional hypoxic transgression; a strategy that was interpreted in the 1990s as a "preparation for oxidative stress" (POS). Since that time, POS mechanisms have been identified in at least 83 animal species representing different phyla including Cnidaria, Nematoda, Annelida, Tardigrada, Echinodermata, Arthropoda, Mollusca and Chordata. Coinciding with the 20th anniversary of the postulation of the POS hypothesis, we compiled this review where we analyze a selection of examples of species showing POS-mechanisms and review the most recent advances in understanding the underlying molecular mechanisms behind those strategies that allow animals to survive in harsh environments.

Globin E is a myoglobin-related, respiratory protein highly expressed in lungfish oocytes.

Globins are a classical model system for the studies of protein evolution and function. Recent studies have shown that - besides the well-known haemoglobin and myoglobin - additional globin-types occur in vertebrates that serve different functions. Globin E (GbE) was originally identified as an eye-specific protein of birds that is distantly related to myoglobin. GbE is also present in turtles and the coelacanth but appeared to have been lost in other vertebrates. Here, we show that GbE additionally occurs in lungfish, the closest living relatives of the tetrapods. Each lungfish species harbours multiple (≥5) GbE gene copies. Surprisingly, GbE is exclusively and highly expressed in oocytes, with mRNA levels that exceed that of myoglobin in the heart. Thus, GbE is the first known oocyte-specific globin in vertebrates. No GbE transcripts were found in the ovary or egg transcriptomes of other vertebrates, suggesting a lungfish-specific function. Spectroscopic analysis and kinetic studies of recombinant GbE1 of the South American lungfish Lepidosiren paradoxa revealed a typical pentacoordinate globin with myoglobin-like O2-binding kinetics, indicating similar functions. Our findings suggest that the multiple copies of GbE evolved to enhance O2-supply in the developing embryo of lungfish, analogous to the embryonic and fetal haemoglobins of other vertebrates. In evolution, GbE must have changed its expression site from oocytes to eyes, or vice versa.

Diversity, evolution, and function of myriapod hemocyanins.

BACKGROUND: Hemocyanin transports O2 in the hemolymph of many arthropod species. Such respiratory proteins have long been considered unnecessary in Myriapoda. As a result, the presence of hemocyanin in Myriapoda has long been overlooked. We analyzed transcriptome and genome sequences from all major myriapod taxa - Chilopoda, Diplopoda, Symphyla, and Pauropoda - with the aim of identifying hemocyanin-like proteins. RESULTS: We investigated the genomes and transcriptomes of 56 myriapod species and identified 46 novel full-length hemocyanin subunit sequences in 20 species of Chilopoda, Diplopoda, and Symphyla, but not Pauropoda. We found in Cleidogona sp. (Diplopoda, Chordeumatida) a hemocyanin-like sequence with mutated copper-binding centers, which cannot bind O2. An RNA-seq approach showed markedly different hemocyanin mRNA levels from ~ 6 to 25,000 reads per kilobase per million reads. To evaluate the contribution of hemocyanin to O2 transport, we specifically studied the hemocyanin of the centipede Scolopendra dehaani. This species harbors two distinct hemocyanin subunits with low expression levels. We showed cooperative O2 binding in the S. dehaani hemolymph, indicating that hemocyanin supports O2 transport even at low concentration. Further, we demonstrated that hemocyanin is > 1500-fold more highly expressed in the fertilized egg than in the adult. CONCLUSION: Hemocyanin was most likely the respiratory protein in the myriapod stem-lineage, but multiple taxa may have independently lost hemocyanin and thus the ability of efficient O2 transport. In myriapods, hemocyanin is much more widespread than initially appreciated. Some myriapods express hemocyanin only at low levels, which are, nevertheless, sufficient for O2 supply. Notably, also in myriapods, a non-respiratory protein similar to insect storage hexamerins evolved from the hemocyanin.

Functional diversification of sea lamprey globins in evolution and development.

Agnathans have a globin repertoire that markedly differs from that of jawed (gnathostome) vertebrates. The sea lamprey (Petromyzon marinus) harbors at least 18 hemoglobin, two myoglobin, two globin X, and one cytoglobin genes. However, agnathan hemoglobins and myoglobins are not orthologous to their cognates in jawed vertebrates. Thus, blood-based O2 transport and muscle-based O2 storage proteins emerged twice in vertebrates from a tissue-globin ancestor. Notably, the sea lamprey displays three switches in hemoglobin expression in its life cycle, analogous to hemoglobin switching in vertebrates. To study the functional changes associated with the evolution and ontogenesis of distinct globin types, we determined O2 binding equilibria, type of quaternary assembly, and nitrite reductase enzymatic activities of one adult (aHb5a) and one embryonic/larval hemoglobin (aHb6), myoglobin (aMb1) and cytoglobin (Cygb) of the sea lamprey. We found clear functional differentiation among globin types expressed at different developmental stages and in different tissues. Cygb and aMb1 have high O2 affinity and nitrite reductase activity, while the two hemoglobins display low O2 affinity and nitrite reductase activity. Cygb and aHb6 but not aHb5a show cooperative O2 binding, correlating with increased stability of dimers, as shown by gel filtration and molecular modeling. The high O2-affinity and the lack of cooperativity confirm the identity of the sea lamprey aMb1 as O2 storage protein of the muscle. The dimeric structure and O2-binding properties of sea lamprey and mammalian Cygb were very similar, suggesting a conservation of function since their divergence around 500million years ago.

Divergent roles of the Drosophila melanogaster globins.

In contrast to long-held assumptions, the gene repertoire of most insects includes hemoglobins. Analyses of the genome of the fruitfly Drosophila melanogaster identified three distinct hemoglobin genes (glob1, glob2, and glob3). While glob1 is predominantly associated with the tracheal system and fat body, glob2 and glob3 are almost exclusively expressed in the testis. The physiological role of globins in Drosophila is uncertain. Here, we studied the functions of the three globins in a cell culture system. Drosophila Schneider 2 (S2) cells were stably transfected with each of the three globins and the empty vector as control. Under hypoxia (1% atmospheric O2), only glob1 overexpression enhanced the activity of mitochondrial oxidases and the ATP content. However, the positive effect of glob1 expression disappeared after 24h hypoxia, suggesting metabolic adaptations of the S2 cells. glob2 and glob3 had no positive effect on hypoxia-survival. After application of oxidative stress by H2O2, glob2 dramatically enhanced the viability of S2 cells. Evaluation of the intracellular localization of the globins using specific antibodies and green fluorescent protein-fusion constructs suggested that glob1 and glob2 most likely reside in the cytoplasm, while glob3 is associated with structures that may represent parts of the intracellular transport machinery. In silico analyses of public RNA-Seq data from different developmental stages provided that glob1 is co-expressed with genes of the aerobic energy metabolism, while glob2 and glob3 expression can be related to spermatogenesis and reproduction. Together, the results indicate divergent functions of the Drosophila globins: glob1 may play a role in the O2-dependent metabolism while glob2 may protect spermatogenesis from reactive oxygen species.

Characterisation of Plasmodium falciparum populations selected on the human endothelial receptors P-selectin, E-selectin, CD9 and CD151.

The ability of the parasite Plasmodium falciparum to evade the immune system and be sequestered within human small blood vessels is responsible for severe forms of malaria. The sequestration depends on the interaction between human endothelial receptors and P. falciparum erythrocyte membrane protein 1 (PfEMP1) exposed on the surface of the infected erythrocytes (IEs). In this study, the transcriptomes of parasite populations enriched for parasites that bind to human P-selectin, E-selectin, CD9 and CD151 receptors were analysed. IT4_var02 and IT4_var07 were specifically expressed in IT4 parasite populations enriched for P-selectin-binding parasites; eight var genes (IT4_var02/07/09/13/17/41/44/64) were specifically expressed in isolate populations enriched for CD9-binding parasites. Interestingly, IT4 parasite populations enriched for E-selectin- and CD151-binding parasites showed identical expression profiles to those of a parasite population exposed to wild-type CHO-745 cells. The same phenomenon was observed for the 3D7 isolate population enriched for binding to P-selectin, E-selectin, CD9 and CD151. This implies that the corresponding ligands for these receptors have either weak binding capacity or do not exist on the IE surface. Conclusively, this work expanded our understanding of P. falciparum adhesive interactions, through the identification of var transcripts that are enriched within the selected parasite populations.

Transcriptome Analysis Identifies Key Metabolic Changes in the Hooded Seal (Cystophora cristata) Brain in Response to Hypoxia and Reoxygenation.

The brain of diving mammals tolerates low oxygen conditions better than the brain of most terrestrial mammals. Previously, it has been demonstrated that the neurons in brain slices of the hooded seal (Cystophora cristata) withstand hypoxia longer than those of mouse, and also tolerate reduced glucose supply and high lactate concentrations. This tolerance appears to be accompanied by a shift in the oxidative energy metabolism to the astrocytes in the seal while in terrestrial mammals the aerobic energy production mainly takes place in neurons. Here, we used RNA-Seq to compare the effect of hypoxia and reoxygenation in vitro on brain slices from the visual cortex of hooded seals. We saw no general reduction of gene expression, suggesting that the response to hypoxia and reoxygenation is an actively regulated process. The treatments caused the preferential upregulation of genes related to inflammation, as found before e.g. in stroke studies using mammalian models. Gene ontology and KEGG pathway analyses showed a downregulation of genes involved in ion transport and other neuronal processes, indicative for a neuronal shutdown in response to a shortage of O2 supply. These differences may be interpreted in terms of an energy saving strategy in the seal's brain. We specifically analyzed the regulation of genes involved in energy metabolism. Hypoxia and reoxygenation caused a similar response, with upregulation of genes involved in glucose metabolism and downregulation of the components of the pyruvate dehydrogenase complex. We also observed upregulation of the monocarboxylate transporter Mct4, suggesting increased lactate efflux. Together, these data indicate that the seal brain responds to the hypoxic challenge by a relative increase in the anaerobic energy metabolism.