Testes-specific hemoglobins in Drosophila evolved by a combination of sub- and neofunctionalization after gene duplication

<p>Abstract</p> <p>Background</p> <p>For a long time the presence of respiratory proteins in most insects has been considered unnecessary. However, in recent years it has become evident that globins belong to the standard repertoire of the insect genome. Like most other insect globins, the <it>glob1 </it>gene of <it>Drosophila melanogaster </it>displays a conserved expression pattern in the tracheae, the fat body and the Malpighian tubules.</p> <p>Results</p> <p>Here we show that the recently discovered <it>D. melanogaster </it>globin genes <it>glob2 </it>and <it>glob3 </it>both display an unusual male-specific expression in the reproductive tract during spermatogenesis. Both paralogs are transcribed at equivalent mRNA levels and largely overlap in their cellular expression patterns during spermatogenesis. Phylogenetic analyses showed that <it>glob2 </it>and <it>glob3 </it>reflect a gene duplication event that occurred in the ancestor of the <it>Sophophora </it>subgenus at least 40 million years ago. Therefore, flies of the <it>Drosophila </it>subgenus harbor only one <it>glob2/3</it>-like gene.</p> <p>Conclusions</p> <p>Phylogenetic and sequence analyses indicate an evolution of the <it>glob2 </it>and <it>glob3 </it>duplicates by a combination of sub- and neofunctionalization. Considering their restricted, testes-specific expression, an involvement of both globins in alleviating oxidative stress during spermatogenesis is conceivable.</p

Extensive transcriptional complexity during hypoxia-regulated expression of the myoglobin gene in cancer

Recently, the ectopic expression of myoglobin (MB) was reported in human epithelial cancer cell lines and breast tumor tissues, where MB expression increased with hypoxia. The better prognosis of MB-positive breast cancer patients suggested that the globin exerts a tumor-suppressive role, possibly by impairing mitochondrial activity in hypoxic breast carcinoma cells. To better understand MB gene regulation in cancer, we systematically investigated the architecture of the human MB gene, its transcripts and promoters. In silico analysis of transcriptome data from normal human tissues and cancer cell lines, followed by RACE-PCR verification, revealed seven novel exons in the MB gene region, most of which are untranslated exons located 5′-upstream of the coding DNA sequence (CDS). Sixteen novel alternatively spliced MB transcripts were detected, most of which predominantly occur in tumor tissue or cell lines. Quantitative RT-PCR analyses of MB expression in surgical breast cancer specimen confirmed the preferential usage of a hitherto unknown, tumor-associated MB promoter, which was functionally validated by luciferase reporter gene assays. In line with clinical observations of MB up-regulation in avascular breast tumors, the novel cancer-associated MB splice variants exhibited increased expression in tumor cells subjected to experimental hypoxia. The novel gene regulatory mechanisms unveiled in this study support the idea of a non-canonical role of MB during carcinogenesi

Duration of invasive mechanical ventilation prior to extracorporeal membrane oxygenation is not associated with survival in acute respiratory distress syndrome caused by coronavirus disease 2019

BACKGROUND: Duration of invasive mechanical ventilation (IMV) prior to extracorporeal membrane oxygenation (ECMO) affects outcome in acute respiratory distress syndrome (ARDS). In coronavirus disease 2019 (COVID-19) related ARDS, the role of pre-ECMO IMV duration is unclear. This single-centre, retrospective study included critically ill adults treated with ECMO due to severe COVID-19-related ARDS between 01/2020 and 05/2021. The primary objective was to determine whether duration of IMV prior to ECMO cannulation influenced ICU mortality. RESULTS: During the study period, 101 patients (mean age 56 [SD ± 10] years; 70 [69%] men; median RESP score 2 [IQR 1–4]) were treated with ECMO for COVID-19. Sixty patients (59%) survived to ICU discharge. Median ICU length of stay was 31 [IQR 20.7–51] days, median ECMO duration was 16.4 [IQR 8.7–27.7] days, and median time from intubation to ECMO start was 7.7 [IQR 3.6–12.5] days. Fifty-three (52%) patients had a pre-ECMO IMV duration of > 7 days. Pre-ECMO IMV duration had no effect on survival (p = 0.95). No significant difference in survival was found when patients with a pre-ECMO IMV duration of < 7 days (< 10 days) were compared to ≥ 7 days (≥ 10 days) (p = 0.59 and p = 1.0). CONCLUSIONS: The role of prolonged pre-ECMO IMV duration as a contraindication for ECMO in patients with COVID-19-related ARDS should be scrutinised. Evaluation for ECMO should be assessed on an individual and patient-centred basis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13613-022-00980-3

Funktionsaufklärung von Atmungsproteinen in Insekten

Globine sind kleine globuläre Proteine mit nahezu ubiquitärem Vorkommen in allen Tiergruppen. Sie weisen eine typische Sandwichstruktur auf, die in der Regel aus acht α-Helices mit einer zentralen prosthetischen Häm-Gruppe besteht und die Proteine zur Bindung gasförmiger Liganden befähigt. Die Funktionen der Globine reichen von O2-Transport und – Speicherung, über eine Beteiligung bei der Entgiftung reaktiver Sauerstoff- und Stickstoffspezies bis hin zu sensorischen physiologischen Aufgaben. Innerhalb der Klasse der Insekten schien das Vorhandensein von Globinen zunächst auf Insekten mit offensichtlich hypoxischen Habitaten beschränkt zu sein. Die Entdeckung des Globins glob1 in Drosophila melanogaster deutete jedoch eine sehr viel weitere Verbreitung der Globine in Insekten an, die sich durch die Identifizierung von Globingenen in einer Vielzahl von normoxisch lebenden Insekten, wie z.B. Apis mellifera oder Aedes aegypti bestätigte. D. melanogaster besitzt drei Globine, glob1, glob2 und glob3. Glob1 ist eng mit anderen intrazellulären Insektenglobinen verwandt, was zu der Annahme führte, dass es sich bei glob1 um das ursprüngliche und bei glob2 und glob3 um abgeleitete D. melanogaster Globine handelt. Glob1 wird in allen Entwicklungsstadien exprimiert, wobei die Hauptexpressionsorte der Fettkörper und das Tracheensystem sind. Die Transkription des glob1 startet von zwei alternativen Promotoren (Promotor I und II), wodurch in Kombination mit alternativem Splicing vier Transkriptvarianten (Isoform A-D) entstehen, deren Translation jedoch in einer Proteinvariante (glob1) resultiert. Hypoxische Bedingungen führen zu einer vermutlich HIF (=‚hypoxia-inducible factor‘) -vermittelten Abnahme der glob1 Genexpression, wohingegen Hyperoxie eine leichte Zunahme der glob1 mRNA Menge bewirkt. Der mithilfe des UAS/Gal4- Systems erzeugte, RNAi-vermittelte glob1 Knockdown führt zu einer schlechteren Überlebensrate adulter Fliegen unter hypoxischen Bedingungen, einer verkürzten Erholungszeit nach hypoxischem Stupor in Weibchen sowie zu einer erhöhten Resistenz gegenüber dem ROS (=‘reactive oxygen species‘) -generierenden Herbizid Paraquat in Larven und adulten Weibchen. Diese Beobachtungen sprechen für eine Funktion des Drosophila glob1 innerhalb der O2-Versorgung. Unter hyperoxischen Bedingungen hingegen wurde kein Unterschied zwischen Fliegen mit wildtypischer und manipulierter glob1-Expression festgestellt, wodurch eine Beteiligung des glob1 bei der Entgiftung reaktiver Sauerstoffspezies als mögliche Funktion vorerst ausscheidet. Bei glob2 und glob3 handelt es sich um duplizierte Gene. Auf phylogenetischen Rekonstruktionen basierend konnte die Entstehung der Globin-Duplikate auf ein Duplikationsereignis vor der Radiation des Subgenus Sophophora vor mindestens 40 Millionen Jahren zurückgeführt werden. Die durchgeführten Analysen zur molekularen Sequenzevolution der Globin-Duplikate deuten darauf hin, dass glob2 und glob3 nach der Duplikation eine Kombination aus Sub- und Neo-Funktionalisierungsprozessen durchlaufen haben. Glob2 und glob3 zeigen eine deckungsgleiche mRNA Expression, die auf die männliche Keimbahn beschränkt ist. Aufgrund des hohen Konservierungsgrads der für die Häm- und O2-Bindung essentiellen Aminosäuren kann von der Funktionalität beider Proteine ausgegangen werden. Die streng auf die männliche Keimbahn begrenzte Expression von glob2 und glob3 deutet auf eine Rolle der Globin-Duplikate innerhalb der Spermatogenese hin, die möglicherweise in einem Schutz der Spermatogenese vor oxidativem Stress besteht. Auch eine Beteiligung beim korrekten Ablauf der Spermien-Individualisierung, beispielsweise durch Regulation von Apoptoseprozessen wäre denkbar.Globins are small globular proteins with a ubiquitous distribution in nearly all kingdoms of life. They exhibit a typical sandwich structure, consisting of eight α-helices embedding a prosthetic heme group in their center which enables the proteins to bind gaseous ligands. Globins display a broad functional diversity ranging from O2-binding and –delivery, an involvement in detoxification of reactive oxygen and nitrogen species to sensory physiological functions. In the class Insecta, the occurrence of globins was initially believed to be restricted to insects living in hypoxic environments. The discovery of the hemoglobin glob1 in Drosophila melanogaster, however, indicated a far broader distribution in insects. This assumption in fact was verified by the identification of globin genes in a high number of insects living in normoxic habitats like for example Apis mellifera, Aedes aegypti or Bombyx mori. D. melanogaster harbours three globins named glob1, glob2 and glob3. Glob1 is closely related to other intracellular insect globins, leading to the assumption that glob1 might be a basal, conserved globin type, whereas the Drosophila-specific glob2 and glob3 might be more derived. Glob1 is expressed in all developmental stages, the main expression can be found in the tracheal system and the fat body. Transcription of glob1 starts from two alternative promoters (Promoter I and II), resulting in four alternative transcripts (Isoform A-D), all four being translated into the same protein. Hypoxic conditions induce a probably HIF (=hypoxia inducible factor) -mediated decrease of glob1 mRNA expression, whereas hyperoxia causes a slight increase in glob1 mRNA levels. The RNAi-mediated knockdown of glob1, achieved by applying the UAS/Gal4-system, resulted in diminished survival of adult flies exposed to hypoxic conditions, but also in beneficial effects like a shortened recovery time after hypoxic stupor and an increased resistance of larvae and female flies to the ROS (=reactive oxygen species) –generating herbicide Paraquat. These observations – although in part seemingly paradoxical –can be interpreted in terms of a function of glob1 in O2 supply. Experimental hyperoxia, however, did not reveal phenotypic differences in flies with manipulated glob1 expression compared to control flies. Thus, an involvement of glob1 in the detoxification of ROS appears unlikely at present. Glob2 and glob3 originate from a duplication event which could be dated based on phylogenetic reconstructions before the radiation of the subgenus Sophophora, at least 40 million years ago. Molecular evolutionary sequence analyses indicate that glob2 and glob3 evolved via a combination of sub- and neo-functionalization. Glob2 and glob3 exhibit a similar mRNA expression pattern, restricted to the male germline. This highly specific expression pattern indicates a function of the glob2/3 duplicates within spermatogenesis, e.g. by a protection of spermatogenesis from oxidative stress

Knockdown of Drosophila hemoglobin suggests a role in O2 homeostasis

Almost all insects are equipped with a tracheal system, which appears to be sufficient for O2 supply even in phases of high metabolic activity. Therefore, with the exception of a few species dwelling in hypoxic habitats, specialized respiratory proteins had been considered unnecessary in insects. The recent discovery and apparently universal presence of intracellular hemoglobins in insects has remained functionally unexplained. The fruitfly Drosophila melanogaster harbors three different globin genes (referred to as glob1-3). Glob1 is the most highly expressed globin and essentially occurs in the tracheal system and the fat body. To better understand the functions of insect globins, the levels of glob1 were modulated in Drosophila larvae and adults by RNAi-mediated knockdown and transgenic over-expression. No effects on the development were observed in flies with manipulated glob1 levels. However, the knockdown of glob1 led to a significantly reduced survival rate of adult flies under hypoxia (5% and 1.5% O2). Surprisingly, the glob1 knockdown flies also displayed increased resistance towards the reactive oxygen species-forming agent paraquat, which may be explained by a restricted availability of O2 resulting in decreased formation of harmful O2(-). In summary, our results suggest an important functional role of glob1 in O2 homeostasis, possibly by enhancing O2 supply

Characterization of the hemoglobin of the backswimmer Anisops deanei (Hemiptera)

While O-binding hemoglobin-like proteins are present in many insects, prominent amounts of hemoglobin have only been found in a few species. Backswimmers of the genera Anisops and Buenoa (Notonectidae) have high concentrations of hemoglobin in the large tracheal cells of the abdomen. Oxygen from the hemoglobin is delivered to a gas bubble and controls the buoyant density, which enables the bugs to maintain their position without swimming and to remain stationary in the mid-water zone where they hunt for prey. We have obtained the cDNA sequences of three Anisops deanei hemoglobin chains by RT-PCR and RACE techniques. The deduced amino acid sequences show an unusual insertion of a single amino acid in the conserved helix E, but this does not affect protein stability or ligand binding kinetics. Recombinant A. deanei hemoglobin has an oxygen affinity of P = 2.4 kPa (18 torr) and reveals the presence of a dimeric fraction or two different conformations. The absorption spectra demonstrate that the Anisops hemoglobin is a typical pentacoordinate globin. Phylogenetic analyses show that the backswimmer hemoglobins evolved within Heteroptera and most likely originated from an intracellular hemoglobin with divergent function

V-ATPase/mTOR Signaling Regulates Megalin-Mediated Apical Endocytosis

mTOR kinase is a master growth regulator that can be stimulated by multiple signals, including amino acids and the lysosomal small GTPase Rheb. Recent studies have proposed an important role for the V-ATPase in the sensing of amino acids in the lysosomal lumen. Using the Drosophila wing as a model epithelium, we show here that the V-ATPase is required for Rheb-dependent epithelial growth. We further uncover a positive feedback loop for the control of apical protein uptake that depends on V-ATPase/mTOR signaling. This feedback loop includes Rheb-dependent transcriptional regulation of the multiligand receptor Megalin, which itself is required for Rheb-induced endocytosis. In addition, we provide evidence that long-term mTOR inhibition with rapamycin in mice causes reduction of Megalin levels and proteinuria in the proximal tubular epithelium of the kidney. Thus, our findings unravel a homeostatic mechanism that allows epithelial cells to promote protein uptake under normal conditions and to prevent uptake in lysosomal stress conditions

Inadequate Energy Delivery Is Frequent among COVID-19 Patients Requiring ECMO Support and Associated with Increased ICU Mortality

Background: Patients receiving extracorporeal membrane oxygenation (ECMO) support are at high risk for malnutrition. There are currently no general nutrition guidelines for coronavirus disease 2019 (COVID-19) patients during ECMO therapy. Methods: We conducted a retrospective analysis of COVID-19 patients requiring venovenous ECMO support at a large tertiary hospital center. Nutrition goals were calculated using 25 kcal/kg body weight (BW)/day. Associations between nutrition support and outcome were evaluated using Kaplan–Meier and multivariable Cox regression analyses. Results: Overall, 102 patients accounted for a total of 2344 nutrition support days during ECMO therapy. On 40.6% of these days, nutrition goals were met. Undernutrition was found in 40.8%. Mean daily calorie delivery was 73.7% of calculated requirements, mean daily protein delivery was 0.7 g/kg BW/d. Mean energy intake of ≥70% of calculated targets was associated with significantly lower ICU mortality independently of age, disease severity at ECMO start and body mass index (adjusted hazard ratio: 0.372, p = 0.007). Conclusions: Patients with a mean energy delivery of ≥70% of calculated targets during ECMO therapy had a better ICU survival compared to patients with unmet energy goals. These results indicate that adequate nutritional support needs to be a major priority in the treatment of COVID-19 patients requiring ECMO support

A new experimental approach to test why biodiversity effects strengthen as ecosystems age

Previous experimental studies found strengthening relationships between biodiversity and ecosystem functioning (BEF) over time. Simultaneous temporal changes of abiotic and biotic conditions, such as in the composition of soil communities, soil carbon and nutrient concentrations, plant community assembly or selection processes, are currently discussed as potential drivers for strengthening BEF relationships. Despite the popularity of these explanations, experimental tests of underlying mechanisms of strengthening BEF relationships over time are scarce, and confounding influences of calendar year cannot be ruled out unless ecosystems of different age are compared in the same calendar years. To address this critical gap of knowledge, we reestablished the plant communities of a long-term biodiversity experiment that had started in 2002 (the Jena Experiment) with new seeds and old or new soil again in 2016. Comparing these treatments with the original communities set up in 2002, we tested whether old communities had stronger plant diversity effects on plant productivity than young ones and if this depended on soil- or plant-related processes. Our first results show that in old communities, the effect of plant diversity on productivity was indeed stronger than in young communities and that this could not be explained by the age of the soil only. However, we found significant effects of soil on the composition of soil organisms, which might be relevant for other ecosystem functions and may have stronger effects over time. Our new experimental approach enables us to test which mechanisms cause strengthening BEF relationships for many different ecosystem functions independent of the study year