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The highly motile and versatile protozoan pathogen Trypanosoma brucei undergoes a complex life cycle in the tsetse fly. Here we introduce the host insect as an expedient model environment for microswimmer research, as it allows examination of microbial motion within a diversified, secluded and yet microscopically tractable space. During their week-long journey through the different microenvironments of the fly´s interior organs, the incessantly swimming trypanosomes cross various barriers and confined surroundings, with concurrently occurring major changes of parasite cell architecture. Multicolour light sheet fluorescence microscopy provided information about tsetse tissue topology with unprecedented resolution and allowed the first 3D analysis of the infection process. High-speed fluorescence microscopy illuminated the versatile behaviour of trypanosome developmental stages, ranging from solitary motion and near-wall swimming to collective motility in synchronised swarms and in confinement. We correlate the microenvironments and trypanosome morphologies to high-speed motility data, which paves the way for cross-disciplinary microswimmer research in a naturally evolved environment.
Social immunity is a key factor for honeybee health, including behavioral defense strategies such as the collective use of antimicrobial plant resins (propolis). While laboratory data repeatedly show significant propolis effects, field data are scarce, especially at the colony level. Here, we investigated whether propolis, as naturally deposited in the nests, can protect honeybees against ectoparasitic mites Varroa destructor and associated viruses, which are currently considered the most serious biological threat to European honeybee subspecies, Apis mellifera, globally. Propolis intake of 10 field colonies was manipulated by either reducing or adding freshly collected propolis. Mite infestations, titers of deformed wing virus (DWV) and sacbrood virus (SBV), resin intake, as well as colony strength were recorded monthly from July to September 2013. We additionally examined the effect of raw propolis volatiles on mite survival in laboratory assays. Our results showed no significant effects of adding or removing propolis on mite survival and infestation levels. However, in relation to V. destructor, DWV titers increased significantly less in colonies with added propolis than in propolis-removed colonies, whereas SBV titers were similar. Colonies with added propolis were also significantly stronger than propolis-removed colonies. These findings indicate that propolis may interfere with the dynamics of V. destructor-transmitted viruses, thereby further emphasizing the importance of propolis for honeybee health.
Central place foragers are faced with the challenge to learn the position of their nest entrance in its surroundings, in order to find their way back home every time they go out to search for food. To acquire navigational information at the beginning of their foraging career, Cataglyphis noda performs learning walks during the transition from interior worker to forager. These small loops around the nest entrance are repeatedly interrupted by strikingly accurate back turns during which the ants stop and precisely gaze back to the nest entrance—presumably to learn the landmark panorama of the nest surroundings. However, as at this point the complete navigational toolkit is not yet available, the ants are in need of a reference system for the compass component of the path integrator to align their nest entrance-directed gazes. In order to find this directional reference system, we systematically manipulated the skylight information received by ants during learning walks in their natural habitat, as it has been previously suggested that the celestial compass, as part of the path integrator, might provide such a reference system. High-speed video analyses of distinct learning walk elements revealed that even exclusion from the skylight polarization pattern, UV-light spectrum and the position of the sun did not alter the accuracy of the look back to the nest behavior. We therefore conclude that C. noda uses a different reference system to initially align their gaze directions. However, a comparison of neuroanatomical changes in the central complex and the mushroom bodies before and after learning walks revealed that exposure to UV light together with a naturally changing polarization pattern was essential to induce neuroplasticity in these high-order sensory integration centers of the ant brain. This suggests a crucial role of celestial information, in particular a changing polarization pattern, in initially calibrating the celestial compass system.
Defense against biotic or abiotic stresses is one of the benefits of living in symbiosis. Leaf-cutting ants, which live in an obligate mutualism with a fungus, attenuate thermal and desiccation stress of their partner through behavioral responses, by choosing suitable places for fungus-rearing across the soil profile. The underground environment also presents hypoxic (low oxygen) and hypercapnic (high carbon dioxide) conditions, which can negatively influence the symbiont. Here, we investigated whether workers of the leaf-cutting ant Acromyrmex lundii use the CO\(_{2}\) concentration as an orientation cue when selecting a place to locate their fungus garden, and whether they show preferences for specific CO\(_{2}\) concentrations. We also evaluated whether levels preferred by workers for fungus-rearing differ from those selected for themselves. In the laboratory, CO\(_{2}\) preferences were assessed in binary choices between chambers with different CO\(_{2}\) concentrations, by quantifying number of workers in each chamber and amount of relocated fungus. Leaf-cutting ants used the CO\(_{2}\) concentration as a spatial cue when selecting places for fungus-rearing. A. lundii preferred intermediate CO\(_{2}\) levels, between 1 and 3%, as they would encounter at soil depths where their nest chambers are located. In addition, workers avoided both atmospheric and high CO\(_{2}\) levels as they would occur outside the nest and at deeper soil layers, respectively. In order to prevent fungus desiccation, however, workers relocated fungus to high CO\(_{2}\) levels, which were otherwise avoided. Workers’ CO\(_{2}\) preferences for themselves showed no clear-cut pattern. We suggest that workers avoid both atmospheric and high CO\(_{2}\) concentrations not because they are detrimental for themselves, but because of their consequences for the symbiotic partner. Whether the preferred CO\(_{2}\) concentrations are beneficial for symbiont growth remains to be investigated, as well as whether the observed preferences for fungus-rearing influences the ants’ decisions where to excavate new chambers across the soil profile.
Nest ventilation in the leaf-cutting ant Atta vollenweideri is driven via a wind-induced mechanism. On their nests, workers construct small turrets that are expected to facilitate nest ventilation. We hypothesized that the construction and structural features of the turrets would depend on the colony’s current demands for ventilation and thus might be influenced by the prevailing environmental conditions inside the nest. Therefore, we tested whether climate-related parameters, namely airflow, air humidity and CO\(_{2}\) levels in the outflowing nest air influenced turret construction in Atta vollenweideri. In the laboratory, we simulated a semi-natural nest arrangement with fungus chambers, a central ventilation tunnel providing outflow of air and an aboveground building arena for turret construction. In independent series, different climatic conditions inside the ventilation tunnel were experimentally generated, and after 24 hours, several features of the built turret were quantified, i.e., mass, height, number and surface area (aperture) of turret openings. Turret mass and height were similar in all experiments even when no airflow was provided in the ventilation tunnel. However, elevated CO\(_{2}\) levels led to the construction of a turret with several minor openings and a larger total aperture. This effect was statistically significant at higher CO\(_{2}\) levels of 5% and 10% but not at 1% CO\(_{2}\). The construction of a turret with several minor openings did not depend on the strong differences in CO\(_{2}\) levels between the outflowing and the outside air, since workers also built permeated turrets even when the CO\(_{2}\) levels inside and outside were both similarly high. We propose that the construction of turrets with several openings and larger opening surface area might facilitate the removal of CO\(_{2}\) from the underground nest structure and could therefore be involved in the control of nest climate in leaf-cutting ants.
The instructive component of waggle dance communication has been shown to increase resource uptake of Apis mellifera colonies in highly heterogeneous resource environments, but an assessment of its relevance in temperate landscapes with different levels of resource heterogeneity is currently lacking. We hypothesized that the advertisement of resource locations via dance communication would be most relevant in highly heterogeneous landscapes with large spatial variation of floral resources. To test our hypothesis, we placed 24 Apis mellifera colonies with either disrupted or unimpaired instructive component of dance communication in eight Central European agricultural landscapes that differed in heterogeneity and resource availability. We monitored colony weight change and pollen harvest as measure of foraging success. Dance disruption did not significantly alter colony weight change, but decreased pollen harvest compared to the communicating colonies by 40%. There was no general effect of resource availability on nectar or pollen foraging success, but the effect of landscape heterogeneity on nectar uptake was stronger when resource availability was high. In contrast to our hypothesis, the effects of disrupted bee communication on nectar and pollen foraging success were not stronger in landscapes with heterogeneous compared to homogenous resource environments. Our results indicate that in temperate regions intra-colonial communication of resource locations benefits pollen foraging more than nectar foraging, irrespective of landscape heterogeneity. We conclude that the so far largely unexplored role of dance communication in pollen foraging requires further consideration as pollen is a crucial resource for colony development and health.
Predators of highly defensive prey likely develop cost-reducing adaptations. The ant Megaponera analis is a specialized termite predator, solely raiding termites of the subfamily Macrotermitinae (in this study, mostly colonies of Pseudocanthotermes sp.) at their foraging sites. The evolutionary arms race between termites and ants led to various defensive mechanisms in termites (for example, a caste specialized in fighting predators). Because M. analis incurs high injury/mortality risks when preying on termites, some risk-mitigating adaptations seem likely to have evolved. We show that a unique rescue behavior in M. analis, consisting of injured nestmates being carried back to the nest, reduces combat mortality. After a fight, injured ants are carried back by their nestmates; these ants have usually lost an extremity or have termites clinging to them and are able to recover within the nest. Injured ants that are forced experimentally to return without help, die in 32% of the cases. Behavioral experiments show that two compounds, dimethyl disulfide and dimethyl trisulfide, present in the mandibular gland reservoirs, trigger the rescue behavior. A model accounting for this rescue behavior identifies the drivers favoring its evolution and estimates that rescuing enables maintenance of a 28.7% larger colony size. Our results are the first to explore experimentally the adaptive value of this form of rescue behavior focused on injured nestmates in social insects and help us to identify evolutionary drivers responsible for this type of behavior to evolve in animals.
Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.
Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C\(_{6}\) and long-chain C\(_{16}\)-ceramides and azido-functionalized ceramide analogs against pathogenic Neisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C\(_{6}\)-ceramide were active against Neisseria meningitidis and N. gonorrhoeae, whereas they were inactive against Escherichia coli and Staphylococcus aureus. Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω–azido-C\(_{6}\)-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω–azido-C\(_{6}\)-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenic Neisseriae.
Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes.
Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.
Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) are two different species of betaherpesviruses that integrate into sub-telomeric ends of human chromosomes, for which different prevalence rates of integration have been reported. It has been demonstrated that integrated viral genome is stable and is fully retained. However, study of chromosomally integrated viral genome in individuals carrying inherited HHV-6 (iciHHV-6) showed unexpected number of viral DR copies. Hence, we created an in vitro infection model and studied retention of full or partial viral genome over a period of time. We observed an exceptional event where cells retained viral direct repeats (DRs) alone in the absence of the full viral genome. Finally, we found evidence for non-telomeric integration of HHV-6A DR in both cultured cells and in an iciHHV-6 individual. Our results shed light on several novel features of HHV-6A chromosomal integration and provide valuable information for future screening techniques.
For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival.
Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.
Animal circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. Here we dissect the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. We also show that the central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals.
Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n = 9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n = 5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC\(_{50}\)< 3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents.
Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.
A fundamental problem in deciding between mutually exclusive options is that the decision needs to be categorical although the properties of the options often differ but in grade. We developed an experimental handle to study this aspect of behavior organization. Larval Drosophila were trained such that in one set of animals odor A was rewarded, but odor B was not (A+/B), whereas a second set of animals was trained reciprocally (A/B+). We then measured the preference of the larvae either for A, or for B, or for “morphed” mixtures of A and B, that is for mixtures differing in the ratio of the two components. As expected, the larvae showed higher preference when only the previously rewarded odor was presented than when only the previously unrewarded odor was presented. For mixtures of A and B that differed in the ratio of the two components, the major component dominated preference behavior—but it dominated less than expected from a linear relationship between mixture ratio and preference behavior. This suggests that a minor component can have an enhanced impact in a mixture, relative to such a linear expectation. The current paradigm may prove useful in understanding how nervous systems generate discrete outputs in the face of inputs that differ only gradually.
Background
Artificial rearing of honey bee larvae is an established method which enables to fully standardize the rearing environment and to manipulate the supplied diet to the brood. However, there are no studies which compare learning performance or neuroanatomic differences of artificially-reared (in-lab) bees in comparison with their in-hive reared counterparts.
Methods
Here we tested how different quantities of food during larval development affect body size, brain morphology and learning ability of adult honey bees. We used in-lab rearing to be able to manipulate the total quantity of food consumed during larval development. After hatching, a subset of the bees was taken for which we made 3D reconstructions of the brains using confocal laser-scanning microscopy. Learning ability and memory formation of the remaining bees was tested in a differential olfactory conditioning experiment. Finally, we evaluated how bees reared with different quantities of artificial diet compared to in-hive reared bees.
Results
Thorax and head size of in-lab reared honey bees, when fed the standard diet of 160 µl or less, were slightly smaller than hive bees. The brain structure analyses showed that artificially reared bees had smaller mushroom body (MB) lateral calyces than their in-hive counterparts, independently of the quantity of food they received. However, they showed the same total brain size and the same associative learning ability as in-hive reared bees. In terms of mid-term memory, but not early long-term memory, they performed even better than the in-hive control.
Discussion
We have demonstrated that bees that are reared artificially (according to the Aupinel protocol) and kept in lab-conditions perform the same or even better than their in-hive sisters in an olfactory conditioning experiment even though their lateral calyces were consistently smaller at emergence. The applied combination of experimental manipulation during the larval phase plus subsequent behavioral and neuro-anatomic analyses is a powerful tool for basic and applied honey bee research.
In early spring, red wood ants Formica polyctena are often observed clustering on the nest surface in large numbers basking in the sun. It has been hypothesized that sun-basking behaviour may contribute to nest heating because of both heat carriage into the nest by sunbasking workers, and catabolic heat production from the mobilization of the workers’ lipid reserves. We investigated sun-basking behaviour in laboratory colonies of F. polyctena exposed to an artificial heat source. Observations on identified individuals revealed that not all ants bask in the sun. Sun-basking and non-sun-basking workers did not differ in body size nor in respiration rates. The number of sun-basking ants and the number of their visits to the hot spot depended on the temperature of both the air and the hot spot. To investigate whether sun basking leads to a physiological activation linked with increased lipolysis, we measured respiration rates of individual workers as a function of temperature, and compared respiration rates of sun-basking workers before and two days after they were allowed to expose themselves to a heat source over 10 days, at self-determined intervals. As expected for ectothermic animals, respiration rates increased with increasing temperatures in the range 5 to 35˚C. However, the respiration rates of sun-basking workers measured two days after a long-term exposure to the heat source were similar to those before sun basking, providing no evidence for a sustained increase of the basal metabolic rates after prolonged sun basking. Based on our measurements, we argue that self-heating of the nest mound in early spring has therefore to rely on alternative heat sources, and speculate that physical transport of heat in the ant bodies may have a significant effect.
Bee pollination increases yield quantity and quality of cash crops in Burkina Faso, West Africa
(2017)
Mutualistic biotic interactions as among flowering plants and their animal pollinators are a key component of biodiversity. Pollination, especially by insects, is a key element in ecosystem functioning, and hence constitutes an ecosystem service of global importance. Not only sexual reproduction of plants is ensured, but also yields are stabilized and genetic variability of crops is maintained, counteracting inbreeding depression and facilitating system resilience. While experiencing rapid environmental change, there is an increased demand for food and income security, especially in sub-Saharan communities, which are highly dependent on small scale agriculture. By combining exclusion experiments, pollinator surveys and field manipulations, this study for the first time quantifies the contribution of bee pollinators to smallholders’ production of the major cash crops, cotton and sesame, in Burkina Faso. Pollination by honeybees and wild bees significantly increased yield quantity and quality on average up to 62%, while exclusion of pollinators caused an average yield gap of 37% in cotton and 59% in sesame. Self-pollination revealed inbreeding depression effects on fruit set and low germination rates in the F1-generation. Our results highlight potential negative consequences of any pollinator decline, provoking risks to agriculture and compromising crop yields in sub-Saharan West Africa.
Division of labor is a hallmark of social insects. In the honeybee (Apis mellifera) each sterile female worker performs a series of social tasks. The most drastic changes in behavior occur when a nurse bee, who takes care of the brood and the queen in the hive, transitions to foraging behavior. Foragers provision the colony with pollen, nectar or water. Nurse bees and foragers differ in numerous behaviors, including responsiveness to gustatory stimuli. Differences in gustatory responsiveness, in turn, might be involved in regulating division of labor through differential sensory response thresholds. Biogenic amines are important modulators of behavior. Tyramine and octopamine have been shown to increase gustatory responsiveness in honeybees when injected into the thorax, thereby possibly triggering social organization. So far, most of the experiments investigating the role of amines on gustatory responsiveness have focused on the brain. The potential role of the fat body in regulating sensory responsiveness and division of labor has large been neglected. We here investigated the role of the fat body in modulating gustatory responsiveness through tyramine signaling in different social roles of honeybees. We quantified levels of tyramine, tyramine receptor gene expression and the effect of elevating fat body tyramine titers on gustatory responsiveness in both nurse bees and foragers. Our data suggest that elevating the tyramine titer in the fat body pharmacologically increases gustatory responsiveness in foragers, but not in nurse bees. This differential effect of tyramine on gustatory responsiveness correlates with a higher natural gustatory responsiveness of foragers, with a higher tyramine receptor (Amtar1) mRNA expression in fat bodies of foragers and with lower baseline tyramine titers in fat bodies of foragers compared to those of nurse bees. We suggest that differential tyramine signaling in the fat body has an important role in the plasticity of division of labor through changing gustatory responsiveness.
Sleep is a highly conserved and essential behaviour in many species, including the fruit fly Drosophila melanogaster. In the wild, sensory signalling encoding environmental information must be integrated with sleep drive to ensure that sleep is not initiated during detrimental conditions. However, the molecular and circuit mechanisms by which sleep timing is modulated by the environment are unclear. Here we introduce a novel behavioural paradigm to study this issue. We show that in male fruit flies, onset of the daytime siesta is delayed by ambient temperatures above 29°C. We term this effect Prolonged Morning Wakefulness (PMW). We show that signalling through the TrpA1 thermo-sensor is required for PMW, and that TrpA1 specifically impacts siesta onset, but not night sleep onset, in response to elevated temperatures. We identify two critical TrpA1-expressing circuits and show that both contact DN1p clock neurons, the output of which is also required for PMW. Finally, we identify the circadian blue-light photoreceptor CRYPTOCHROME as a molecular regulator of PMW, and propose a model in which the Drosophila nervous system integrates information encoding temperature, light, and time to dynamically control when sleep is initiated. Our results provide a platform to investigate how environmental inputs co-ordinately regulate sleep plasticity.
Hangover links nuclear RNA signaling to cAMP regulation via the phosphodiesterase 4d ortholog dunce
(2017)
The hangover gene defines a cellular stress pathway that is required for rapid ethanol tolerance in Drosophila melanogaster. To understand how cellular stress changes neuronal function, we analyzed Hangover function on a cellular and neuronal level. We provide evidence that Hangover acts as a nuclear RNA binding protein and we identified the phosphodiesterase 4d ortholog dunce as a target RNA. We generated a transcript-specific dunce mutant that is impaired not only in ethanol tolerance but also in the cellular stress response. At the neuronal level, Dunce and Hangover are required in the same neuron pair to regulate experience-dependent motor output. Within these neurons, two cyclic AMP (cAMP)-dependent mechanisms balance the degree of tolerance. The balance is achieved by feedback regulation of Hangover and dunce transcript levels. This study provides insight into how nuclear Hangover/RNA signaling is linked to the cytoplasmic regulation of cAMP levels and results in neuronal adaptation and behavioral changes.
A precise and rapid adjustment of fluxes through metabolic pathways is crucial for organisms to prevail in changing environmental conditions. Based on this reasoning, many guiding principles that govern the evolution of metabolic networks and their regulation have been uncovered. To this end, methods from dynamic optimization are ideally suited since they allow to uncover optimality principles behind the regulation of metabolic networks. We used dynamic optimization to investigate the influence of toxic intermediates in connection with the efficiency of enzymes on the regulation of a linear metabolic pathway. Our results predict that transcriptional regulation favors the control of highly efficient enzymes with less toxic upstream intermediates to reduce accumulation of toxic downstream intermediates. We show that the derived optimality principles hold by the analysis of the interplay between intermediate toxicity and pathway regulation in the metabolic pathways of over 5000 sequenced prokaryotes. Moreover, using the lipopolysaccharide biosynthesis in Escherichia coli as an example, we show how knowledge about the relation of regulation, kinetic efficiency and intermediate toxicity can be used to identify drug targets, which control endogenous toxic metabolites and prevent microbial growth. Beyond prokaryotes, we discuss the potential of our findings for the development of antifungal drugs.
The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and replication fork progression at particular G-rich motifs. This motif can form G-quadruplex (G4) structures in vitro. G4 are stable DNA structures that are known to impede replication fork progression. In the absence of Mms1, genome stability is at risk at these G-rich/G4 regions as demonstrated by gross chromosomal rearrangement assays. Mms1 binds throughout the cell cycle to these G-rich/G4 regions and supports the binding of Pif1 DNA helicase. Based on these data we propose a mechanistic model in which Mms1 binds to specific G-rich/G4 motif located on the lagging strand template for DNA replication and supports Pif1 function, DNA replication and genome integrity.
The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.
Multiple myeloma (MM) represents a haematological cancer characterized by the pathological hyper proliferation of antibody-producing B-lymphocytes. Patients typically suffer from kidney malfunction and skeletal disorders. In the context of MM, the transforming growth factor β (TGFβ) member Activin A was recently identified as a promoter of both accompanying symptoms. Because studies have shown that bone morphogenetic protein (BMP)-2-mediated activities are counteracted by Activin A, we analysed whether BMP2, which also binds to the Activin A receptors ActRII and ActRIIB but activates the alternative SMAD-1/5/8 pathway, can be used to antagonize Activin A activities, such as in the context of MM. Therefore three BMP2 derivatives were generated with modified binding activities for the type II (ActRIIB) and/or type I receptor (BMPRIA) showing either increased or decreased BMP2 activity. In the context of MM these BMP2 muteins show two functionalities since they act as a) an anti-proliferative/apoptotic agent against neoplastic B-cells, b) as a bone-formation promoting growth factor. The molecular basis of both activities was shown in two different cellular models to clearly rely on the properties of the investigated BMP2 muteins to compete for the binding of Activin A to the Activin type II receptors. The experimental outcome suggests new therapeutic strategies using BMP2 variants in the treatment of MM-related pathologies.
Cytotoxic T lymphocytes are effector CD8\(^{+}\) T cells that eradicate infected and malignant cells. Here we show that the transcription factor NFATc1 controls the cytotoxicity of mouse cytotoxic T lymphocytes. Activation of Nfatc1\(^{-/-}\) cytotoxic T lymphocytes showed a defective cytoskeleton organization and recruitment of cytosolic organelles to immunological synapses. These cells have reduced cytotoxicity against tumor cells, and mice with NFATc1-deficient T cells are defective in controlling Listeria infection. Transcriptome analysis shows diminished RNA levels of numerous genes in Nfatc1\(^{-/-}\) CD8\(^{+}\) T cells, including Tbx21, Gzmb and genes encoding cytokines and chemokines, and genes controlling glycolysis. Nfatc1\(^{-/-}\), but not Nfatc2\(^{-/-}\) CD8\(^{+}\) T cells have an impaired metabolic switch to glycolysis, which can be restored by IL-2. Genome-wide ChIP-seq shows that NFATc1 binds many genes that control cytotoxic T lymphocyte activity. Together these data indicate that NFATc1 is an important regulator of cytotoxic T lymphocyte effector functions.
Population genomics of prokaryotes has been studied in depth in only a small number of primarily pathogenic bacteria, as genome sequences of isolates of diverse origin are lacking for most species. Here, we conducted a large‐scale survey of population structure in prevalent human gut microbial species, sampled from their natural environment, with a culture‐independent metagenomic approach. We examined the variation landscape of 71 species in 2,144 human fecal metagenomes and found that in 44 of these, accounting for 72% of the total assigned microbial abundance, single‐nucleotide variation clearly indicates the existence of sub‐populations (here termed subspecies). A single subspecies (per species) usually dominates within each host, as expected from ecological theory. At the global scale, geographic distributions of subspecies differ between phyla, with Firmicutes subspecies being significantly more geographically restricted. To investigate the functional significance of the delineated subspecies, we identified genes that consistently distinguish them in a manner that is independent of reference genomes. We further associated these subspecies‐specific genes with properties of the microbial community and the host. For example, two of the three Eubacterium rectale subspecies consistently harbor an accessory pro‐inflammatory flagellum operon that is associated with lower gut community diversity, higher host BMI, and higher blood fasting insulin levels. Using an additional 676 human oral samples, we further demonstrate the existence of niche specialized subspecies in the different parts of the oral cavity. Taken together, we provide evidence for subspecies in the majority of abundant gut prokaryotes, leading to a better functional and ecological understanding of the human gut microbiome in conjunction with its host.
proGenomes: a resource for consistent functional and taxonomic annotations of prokaryotic genomes
(2017)
The availability of microbial genomes has opened many new avenues of research within microbiology. This has been driven primarily by comparative genomics approaches, which rely on accurate and consistent characterization of genomic sequences. It is nevertheless difficult to obtain consistent taxonomic and integrated functional annotations for defined prokaryotic clades. Thus, we developed proGenomes, a resource that provides user-friendly access to currently 25 038 high-quality genomes whose sequences and consistent annotations can be retrieved individually or by taxonomic clade. These genomes are assigned to 5306 consistent and accurate taxonomic species clusters based on previously established methodology. proGenomes also contains functional information for almost 80 million protein-coding genes, including a comprehensive set of general annotations and more focused annotations for carbohydrate-active enzymes and antibiotic resistance genes. Additionally, broad habitat information is provided for many genomes. All genomes and associated information can be downloaded by user-selected clade or multiple habitat-specific sets of representative genomes. We expect that the availability of high-quality genomes with comprehensive functional annotations will promote advances in clinical microbial genomics, functional evolution and other subfields of microbiology. proGenomes is available at http://progenomes.embl.de.
Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identifed the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efciently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
Multifunctional enzyme, type-1 (MFE1) is a monomeric enzyme with a 2E-enoyl-CoA hydratase and a 3S-hydroxyacyl-CoA dehydrogenase (HAD) active site. Enzyme kinetic data of rat peroxisomal MFE1 show that the catalytic efficiencies for converting the short-chain substrate 2E-butenoyl-CoA into acetoacetyl-CoA are much lower when compared with those of the homologous monofunctional enzymes. The mode of binding of acetoacetyl-CoA (to the hydratase active site) and the very similar mode of binding of NAD\(^+\) and NADH (to the HAD part) are described and compared with those of their monofunctional counterparts. Structural comparisons suggest that the conformational flexibility of the HAD and hydratase parts of MFE1 are correlated. The possible importance of the conformational flexibility of MFE1 for its biocatalytic properties is discussed.
Epicardium-derived cells (EPDC) and atrial stromal cells (ASC) display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73,CD90, CD44, CD105), cardiac markers (Gata4, Tbx5, troponin T) and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM), cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin) known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases.
Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding, and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system.
Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency.
Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mice display increased cell densities in the DR at embryonic stages E13.5, E17.5, and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5.
Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that Cdh13 deficiency affects the cell density of the developing DR and the posterior innervation of the prefrontal cortex (PFC), and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.
Background:
Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence.
Results:
Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region.
Conclusions:
Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.
Some members of the physiological human microbiome occasionally cause life-threatening disease even in immunocompetent individuals. A prime example of such a commensal pathogen is Neisseria meningitidis, which normally resides in the human nasopharynx but is also a leading cause of sepsis and epidemic meningitis. Using N. meningitidis as model organism, we tested the hypothesis that virulence of commensal pathogens is a consequence of within host evolution and selection of invasive variants due to mutations at contingency genes, a mechanism called phase variation. In line with the hypothesis that phase variation evolved as an adaptation to colonize diverse hosts, computational comparisons of all 27 to date completely sequenced and annotated meningococcal genomes retrieved from public databases showed that contingency genes are indeed enriched for genes involved in host interactions. To assess within-host genetic changes in meningococci, we further used ultra-deep whole-genome sequencing of throat-blood strain pairs isolated from four patients suffering from invasive meningococcal disease. We detected up to three mutations per strain pair, affecting predominantly contingency genes involved in type IV pilus biogenesis. However, there was not a single (set) of mutation(s) that could invariably be found in all four pairs of strains. Phenotypic assays further showed that these genetic changes were generally not associated with increased serum resistance, higher fitness in human blood ex vivo or differences in the interaction with human epithelial and endothelial cells in vitro. In conclusion, we hypothesize that virulence of meningococci results from accidental emergence of invasive variants during carriage and without within host evolution of invasive phenotypes during disease progression in vivo.
\(Ambra1\) is linked to autophagy and neurodevelopment. Heterozygous \(Ambra1\) deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of \(AMBRA1\) for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal \(AMBRA1\) genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower \(AMBRA1\) mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890 (A versus G allele) may affect its stability through modification of folding, as predicted by \(in\) \(silico\) analysis. Searching for further autism-relevant characteristics in \(Ambra1^{+/−}\) mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an \(in\) \(vivo\) readout of neuronal excitation–inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of \(AMBRA1/Ambra1\) partial loss-of-function genotypes for female autistic traits. Moreover, they suggest \(Ambra1\) heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism.
Timing seasonal events, like reproduction or diapause, is crucial for the survival of many species. Global change causes phenologies worldwide to shift, which requires a mechanistic explanation of seasonal time measurement. Day length (photoperiod) is a reliable indicator of winter arrival, but it remains unclear how exactly species measure day length. A reference for time of day could be provided by a circadian clock, by an hourglass clock, or, as some newer models suggest, by a damped circadian clock. However, damping of clock outputs has so far been rarely observed. To study putative clock outputs of Acyrthosiphon pisum aphids, we raised individual nymphs on coloured artificial diet, and measured rhythms in metabolic activity in light-dark illumination cycles of 16:08 hours (LD) and constant conditions (DD). In addition, we kept individuals in a novel monitoring setup and measured locomotor activity. We found that A. pisum is day-active in LD, potentially with a bimodal distribution. In constant darkness rhythmicity of locomotor behaviour persisted in some individuals, but patterns were mostly complex with several predominant periods. Metabolic activity, on the other hand, damped quickly. A damped circadian clock, potentially driven by multiple oscillator populations, is the most likely explanation of our results.
Plants initially accepted by foraging leaf-cutting ants are later avoided if they prove unsuitable for their symbiotic fungus. Plant avoidance is mediated by the waste produced in the fungus garden soon after the incorporation of the unsuitable leaves, as foragers can learn plant odors and cues from the damaged fungus that are both present in the recently produced waste particles. We asked whether avoidance learning of plants unsuitable for the symbiotic fungus can take place entirely at the colony dump. In order to investigate whether cues available in the waste chamber induce plant avoidance in naïve subcolonies, we exchanged the waste produced by subcolonies fed either fungicide-treated privet leaves or untreated leaves and measured the acceptance of untreated privet leaves before and after the exchange of waste. Second, we evaluated whether foragers could perceive the avoidance cues directly at the dump by quantifying the visits of labeled foragers to the waste chamber. Finally, we asked whether foragers learn to specifically avoid untreated leaves of a plant after a confinement over 3 hours in the dump of subcolonies that were previously fed fungicide-treated leaves of that species. After the exchange of the waste chambers, workers from subcolonies that had access to waste from fungicide-treated privet leaves learned to avoid that plant. One-third of the labeled foragers visited the dump. Furthermore, naïve foragers learned to avoid a specific, previously unsuitable plant if exposed solely to cues of the dump during confinement. We suggest that cues at the dump enable foragers to predict the unsuitable effects of plants even if they had never been experienced in the fungus garden.
Eclosion in flies and other insects is a circadian-gated behaviour under control of a central and a peripheral clock. It is not influenced by the motivational state of an animal, and thus presents an ideal paradigm to study the relation and signalling pathways between central and peripheral clocks, and downstream peptidergic regulatory systems. Little is known, however, about eclosion rhythmicity under natural conditions, and research into this direction is hampered by the physically closed design of current eclosion monitoring systems.
We describe a novel open eclosion monitoring system (WEclMon) that allows the puparia to come into direct contact with light, temperature and humidity. We demonstrate that the system can be used both in the laboratory and outdoors, and shows a performance similar to commercial closed funnel-type monitors. Data analysis is semi-automated based on a macro toolset for the open imaging software Fiji. Due to its open design, the WEclMon is also well suited for optogenetic experiments. A small screen to identify putative neuroendocrine signals mediating time from the central clock to initiate eclosion showed that optogenetic activation of ETH-, EH and myosuppressin neurons can induce precocious eclosion. Genetic ablation of myosuppressin-expressing neurons did, however, not affect eclosion rhythmicity.
Septins are a highly conserved family of small GTPases that form cytoskeletal filaments. Their cellular functions, especially in the nervous system, still remain largely enigmatic, but there are accumulating lines of evidence that septins play important roles in neuronal physiology and pathology. In order to further dissect septin function in the nervous system a detailed temporal resolved analysis in the genetically well tractable model vertebrate zebrafish (Danio rerio) is crucially necessary. To close this knowledge gap we here provide a reference dataset describing the expression of selected septins (sept3, sept5a and sept5b) in the zebrafish central nervous system. Strikingly, proliferation zones are devoid of expression of all three septins investigated, suggesting that they have a role in post-mitotic neural cells. Our finding that three septins are mainly expressed in non-proliferative regions was further confirmed by double-stainings with a proliferative marker. Our RNA in situ hybridization (ISH) study, detecting sept3, sept5a and sept5b mRNAs, shows that all three septins are expressed in largely overlapping regions of the developing brain. However, the expression of sept5a is much more confined compared to sept3 and sept5b. In contrast, the expression of all the three analyzed septins is largely similar in the adult brain.
Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.
A recent study by Peng and Yang in Scientific Reports using confocal-microscopy based automated quantification of anti-synapsin labeled microglomeruli in the mushroom bodies of honeybee brains reports potentially incorrect numbers of microglomerular densities. Whereas several previous studies using visually supervised or automated counts from confocal images and analyses of serial 3D electron-microscopy data reported consistent numbers of synaptic complexes per volume, Peng and Yang revealed extremely low numbers differing by a factor of 18 or more from those obtained in visually supervised counts, and by a factor 22–180 from numbers in two other studies using automated counts. This extreme discrepancy is especially disturbing as close comparison of raw confocal images of anti-synapsin labeled whole-mount brain preparations are highly similar across these studies. We conclude that these discrepancies may reside in potential misapplication of confocal imaging followed by erroneous use of automated image analysis software. Consequently, the reported microglomerular densities during maturation and after manipulation by insecticides require validation by application of appropriate confocal imaging methods and analyses tools that rely on skilled observers. We suggest several improvements towards more reliable or standardized automated or semi-automated synapse counts in whole mount preparations of insect brains.
Background:
Intrauterine exposure to gestational diabetes mellitus (GDM) confers a lifelong increased risk for metabolic and other complex disorders to the offspring. GDM-induced epigenetic modifications modulating gene regulation and persisting into later life are generally assumed to mediate these elevated disease susceptibilities. To identify candidate genes for fetal programming, we compared genome-wide methylation patterns of fetal cord bloods (FCBs) from GDM and control pregnancies.
Methods and results:
Using Illumina’s 450K methylation arrays and following correction for multiple testing, 65 CpG sites (52 associated with genes) displayed significant methylation differences between GDM and control samples. Four candidate genes, ATP5A1, MFAP4, PRKCH, and SLC17A4, from our methylation screen and one, HIF3A, from the literature were validated by bisulfite pyrosequencing. The effects remained significant after adjustment for the confounding factors maternal BMI, gestational week, and fetal sex in a multivariate regression model. In general, GDM effects on FCB methylation were more pronounced in women with insulin-dependent GDM who had a more severe metabolic phenotype than women with dietetically treated GDM.
Conclusions:
Our study supports an association between maternal GDM and the epigenetic status of the exposed offspring. Consistent with a multifactorial disease model, the observed FCB methylation changes are of small effect size but affect multiple genes/loci. The identified genes are primary candidates for transmitting GDM effects to the next generation. They also may provide useful biomarkers for the diagnosis, prognosis, and treatment of adverse prenatal exposures.
The availability of pollen in agricultural landscapes is essential for the successful growth and reproduction of honey bee colonies (Apis mellifera L.). The quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment, we rotated 16 honey bee colonies across 16 agricultural landscapes, used traps to collect samples of collected pollen and observed intra-colonial dance communication to gain information about foraging distances. DNA metabarcoding was applied to analyze mixed pollen samples. Neither the amount of collected pollen nor pollen diversity was related to landscape diversity. However, we found a strong seasonal variation in the amount and diversity of collected pollen in all sites independent of landscape diversity. The observed increase in foraging distances with decreasing landscape diversity suggests that honey bees compensated for lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. Our results underscore the importance of a diverse pollen diet for honey bee colonies. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes.
OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased number of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info.
Staphylococcus aureus (S. aureus) infections are a major clinical problem and range from mild skin and soft-tissue infections to severe and even lethal infections such as pneumonia, endocarditis, sepsis, osteomyelitis, and toxic shock syndrome. Toxins that are released from S. aureus mediate many of these effects. Here, we aimed to identify molecular mechanisms how α-toxin, a major S. aureus toxin, induces inflammation. Methods: Macrophages were isolated from the bone marrow of wildtype and acid sphingomyelinase-deficient mice, stimulated with S. aureus α-toxin and activation of the acid sphingomyelinase was quantified. The subcellular formation of ceramides was determined by confocal microscopy. Release of cathepsins from lysosomes, activation of inflammasome proteins and formation of Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) were analyzed by western blotting, confocal microscopy and ELISA. Results: We demonstrate that S. aureus α-toxin activates the acid sphingomyelinase in ex vivo macrophages and triggers a release of ceramides. Ceramides induced by S. aureus α-toxin localize to lysosomes and mediate a release of cathepsin B and D from lysosomes into the cytoplasm. Cytosolic cathepsin B forms a complex with Nlrc4. Treatment of macrophages with α-toxin induces the formation of IL-1β and TNF-α. These events are reduced or abrogated, respectively, in cells lacking the acid sphingomyelinase and upon treatment of macrophages with amitriptyline, a functional inhibitor of acid sphingomyelinase. Pharmacological inhibition of cathepsin B prevented activation of the inflammasome measured as release of IL-1β, while the formation of TNF-α was independent of cathepsin B. Conclusion: We demonstrate a novel mechanism how bacterial toxins activate the inflammasome and mediate the formation and release of cytokines: S. aureus α-toxin triggers an activation of the acid sphingomyelinase and a release of ceramides resulting in the release of lysosomal cathepsin B and formation of pro-inflammatory cytokines.
The Kryptolebias marmoratus is unique because it is the only selffertilizing hermaphroditic vertebrate, known to date. It primarily reproduces by internal self-fertilization in a mixed ovary/testis gonad. Here, we report on a high-quality genome assembly for the K. marmoratus South Korea (SK) strain highlighting the diversity and distribution of transposable elements (TEs). We find that K. marmoratus genome maintains number and composition of TEs. This can be an important genomic attribute promoting genome recombination in this selfing fish, while, in addition to a mixed mating strategy, it may also represent a mechanism contributing to the evolutionary adaptation to ecological pressure of the species. Future work should help clarify this point further once genomic information is gathered for other taxa of the family Rivulidae that do not self-fertilize. We provide a valuable genome resource that highlights the potential impact of TEs on the genome evolution of a fish species with an uncommon life cycle.
A system-wide understanding of cellular function requires knowledge of all functional interactions between the expressed proteins. The STRING database aims to collect and integrate this information, by consolidating known and predicted protein–protein association data for a large number of organisms. The associations in STRING include direct (physical) interactions, as well as indirect (functional) interactions, as long as both are specific and biologically meaningful. Apart from collecting and reassessing available experimental data on protein–protein interactions, and importing known pathways and protein complexes from curated databases, interaction predictions are derived from the following sources: (i) systematic co-expression analysis, (ii) detection of shared selective signals across genomes, (iii) automated text-mining of the scientific literature and (iv) computational transfer of interaction knowledge between organisms based on gene orthology. In the latest version 10.5 of STRING, the biggest changes are concerned with data dissemination: the web frontend has been completely redesigned to reduce dependency on outdated browser technologies, and the database can now also be queried from inside the popular Cytoscape software framework. Further improvements include automated background analysis of user inputs for functional enrichments, and streamlined download options. The STRING resource is available online, at http://string-db.org/.
Background:
The cardiac hormones atrial (ANP) and B-type natriuretic peptides (BNP) moderate arterial blood pressure and improve energy metabolism as well as insulin sensitivity via their shared cGMP-producing guanylyl cyclase-A (GC-A) receptor. Obesity is associated with impaired NP/GC-A/cGMP signaling, which possibly contributes to the development of type 2 diabetes and its cardiometabolic complications. In vitro, synthetic ANP, via GC-A, stimulates glucose-dependent insulin release from cultured pancreatic islets and β-cell proliferation. However, the relevance for systemic glucose homeostasis in vivo is not known. To dissect whether the endogenous cardiac hormones modulate the secretory function and/or proliferation of β-cells under (patho)physiological conditions in vivo, here we generated a novel genetic mouse model with selective disruption of the GC-A receptor in β-cells.
Methods:
Mice with a floxed GC-A gene were bred to Rip-CreTG mice, thereby deleting GC-A selectively in β-cells (β GC-A KO). Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion were monitored in normal diet (ND)- and high-fat diet (HFD)-fed mice. β-cell size and number were measured by immunofluorescence-based islet morphometry.
Results:
In vitro, the insulinotropic and proliferative actions of ANP were abolished in islets isolated from β GC-A KO mice. Concordantly, in vivo, infusion of BNP mildly enhanced baseline plasma insulin levels and glucose-induced insulin secretion in control mice. This effect of exogenous BNP was abolished in β GC-A KO mice, corroborating the efficient inactivation of the GC-A receptor in β-cells. Despite this under physiological, ND conditions, fasted and fed insulin levels, glucose-induced insulin secretion, glucose tolerance and β-cell morphology were similar in β GC-A KO mice and control littermates. However, HFD-fed β GC-A KO animals had accelerated glucose intolerance and diminished adaptative β-cell proliferation.
Conclusions:
Our studies of β GC-A KO mice demonstrate that the cardiac hormones ANP and BNP do not modulate β-cell's growth and secretory functions under physiological, normal dietary conditions. However, endogenous NP/GC-A signaling improves the initial adaptative response of β-cells to HFD-induced obesity. Impaired β-cell NP/GC-A signaling in obese individuals might contribute to the development of type 2 diabetes.
Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transposase of Tn1549 complexed with circular transposon DNA intermediates. The structures reveal individual transposition steps and explain how specific DNA distortion and cleavage mechanisms enable DNA strand exchange with an absolute minimum homology requirement. This appears to uniquely allow Tn916-like conjugative transposons to bypass DNA homology and insert into diverse genomic sites, expanding gene transfer. We further uncover a structural regulatory mechanism that prevents premature cleavage of the transposon DNA before a suitable target DNA is found and generate a peptide antagonist that interferes with the transposase-DNA structure to block transposition. Our results reveal mechanistic principles of conjugative transposition that could help control the spread of antibiotic resistance genes.
In social groups, infections have the potential to spread rapidly and cause disease outbreaks. Here, we show that in a social insect, the ant Lasius neglectus, the negative consequences of fungal infections (Metarhizium brunneum) can be mitigated by employing an efficient multicomponent behaviour, termed destructive disinfection, which prevents further spread of the disease through the colony. Ants specifically target infected pupae during the pathogens non-contagious incubation period, utilising chemical 'sickness cues' emitted by pupae. They then remove the pupal cocoon, perforate its cuticle and administer antimicrobial poison, which enters the body and prevents pathogen replication from the inside out. Like the immune system of a metazoan body that specifically targets and eliminates infected cells, ants destroy infected brood to stop the pathogen completing its lifecycle, thus protecting the rest of the colony. Hence, in an analogous fashion, the same principles of disease defence apply at different levels of biological organisation.
Background:
In previous studies, the gram-positive firmicute genus Paenibacillus was found with significant abundances in nests of wild solitary bees. Paenibacillus larvae is well-known for beekeepers as a severe pathogen causing the fatal honey bee disease American foulbrood, and other members of the genus are either secondary invaders of European foulbrood or considered a threat to honey bees. We thus investigated whether Paenibacillus is a common bacterium associated with various wild bees and hence poses a latent threat to honey bees visiting the same flowers.
Results:
We collected 202 samples from 82 individuals or nests of 13 bee species at the same location and screened each for Paenibacillus using high-throughput sequencing-based 16S metabarcoding. We then isolated the identified strain Paenibacillus MBD-MB06 from a solitary bee nest and sequenced its genome. We did find conserved toxin genes and such encoding for chitin-binding proteins, yet none specifically related to foulbrood virulence or chitinases. Phylogenomic analysis revealed a closer relationship to strains of root-associated Paenibacillus rather than strains causing foulbrood or other accompanying diseases. We found anti-microbial evidence within the genome, confirmed by experimental bioassays with strong growth inhibition of selected fungi as well as gram-positive and gram-negative bacteria.
Conclusions:
The isolated wild bee associate Paenibacillus MBD-MB06 is a common, but irregularly occurring part of wild bee microbiomes, present on adult body surfaces and guts and within nests especially in megachilids. It was phylogenetically and functionally distinct from harmful members causing honey bee colony diseases, although it shared few conserved proteins putatively toxic to insects that might indicate ancestral predisposition for the evolution of insect pathogens within the group. By contrast, our strain showed anti-microbial capabilities and the genome further indicates abilities for chitin-binding and biofilm-forming, suggesting it is likely a useful associate to avoid fungal penetration of the bee cuticula and a beneficial inhabitant of nests to repress fungal threats in humid and nutrient-rich environments of wild bee nests.
Animal-microbe mutualisms are typically maintained by vertical symbiont transmission or partner choice. A third mechanism, screening of high-quality symbionts, has been predicted in theory, but empirical examples are rare. Here we demonstrate that ambrosia beetles rely on ethanol within host trees for promoting gardens of their fungal symbiont and producing offspring. Ethanol has long been known as the main attractant for many of these fungus-farming beetles as they select host trees in which they excavate tunnels and cultivate fungal gardens. More than 300 attacks by Xylosandrus germanus and other species were triggered by baiting trees with ethanol lures, but none of the foundresses established fungal gardens or produced broods unless tree tissues contained in vivo ethanol resulting from irrigation with ethanol solutions. More X. germanus brood were also produced in a rearing substrate containing ethanol. These benefits are a result of increased food supply via the positive effects of ethanol on food-fungus biomass. Selected Ambrosiella and Raffaelea fungal isolates from ethanol-responsive ambrosia beetles profited directly and indirectly by (i) a higher biomass on medium containing ethanol, (ii) strong alcohol dehydrogenase enzymatic activity, and (iii) a competitive advantage over weedy fungal garden competitors (Aspergillus, Penicillium) that are inhibited by ethanol. As ambrosia fungi both detoxify and produce ethanol, they may maintain the selectivity of their alcohol-rich habitat for their own purpose and that of other ethanol-resistant/producing microbes. This resembles biological screening of beneficial symbionts and a potentially widespread, unstudied benefit of alcohol-producing symbionts (e.g., yeasts) in other microbial symbioses.
Galectin-1 (Gal-1) has been described to promote tumour growth by inducing angiogenesis and to contribute to the tumour immune escape. We had previously identified up-regulation of Gal-1 in preclinical models of aggressive neuroblastoma (NB), the most common extracranial tumour of childhood. While Gal-1 did not confer a survival advantage in the absence of exogenous stressors, Gal-1 contributed to enhanced cell migratory and invasive properties. Here, we review these findings and extend them by analyzing Gal-1 mediated effects on immune cell regulation and radiation resistance. In line with previous results, cell autonomous effects as well as paracrine functions contribute to Gal-1 mediated pro-tumourigenic functions. Interfering with Gal-1 functions in vivo will add to a better understanding of the role of the Gal-1 axis in the complex tumour-host interaction during immune-, chemo- and radiotherapy of neuroblastoma.
Meniscal pathologies are among the most common injuries of the femorotibial joint in both human and equine patients. Pathological forces and ensuing injuries of the cranial horn of the equine medial meniscus are considered analogous to those observed in the human posterior medial horn. Biomechanical properties of human menisci are site-and depth-specific. However, the influence of equine meniscus topography and composition on its biomechanical properties is yet unknown. A better understanding of equine meniscus composition and biomechanics could advance not only veterinary therapies for meniscus degeneration or injuries, but also further substantiate the horse as suitable translational animal model for (human) meniscus tissue engineering. Therefore, the aim of this study was to investigate the composition and structure of the equine knee meniscus in a site-and age-specific manner and their relationship with potential site-specific biomechanical properties. The meniscus architecture was investigated histologically. Biomechanical testing included evaluation of the shore hardness (SH), stiffness and energy loss of the menisci. The SH was found to be subjected to both age and site-specific changes, with an overall higher SH of the tibial meniscus surface and increase in SH with age. Stiffness and energy loss showed neither site nor age related significant differences. The macroscopic and histologic similarities between equine and human menisci described in this study, support continued research in this field.
Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain
(2018)
The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsinguanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio > 1000. After light excitation the putative signaling state forms with tau = 31 ms and decays with tau = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 angstrom) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.
Background
Gut microbes influence their hosts in many ways, in particular by modulating the impact of diet. These effects have been studied most extensively in humans and mice. In this work, we used whole genome metagenomics to investigate the relationship between the gut metagenomes of dogs, humans, mice, and pigs.
Results
We present a dog gut microbiome gene catalog containing 1,247,405 genes (based on 129 metagenomes and a total of 1.9 terabasepairs of sequencing data). Based on this catalog and taxonomic abundance profiling, we show that the dog microbiome is closer to the human microbiome than the microbiome of either pigs or mice. To investigate this similarity in terms of response to dietary changes, we report on a randomized intervention with two diets (high-protein/low-carbohydrate vs. lower protein/higher carbohydrate). We show that diet has a large and reproducible effect on the dog microbiome, independent of breed or sex. Moreover, the responses were in agreement with those observed in previous human studies.
Conclusions
We conclude that findings in dogs may be predictive of human microbiome results. In particular, a novel finding is that overweight or obese dogs experience larger compositional shifts than lean dogs in response to a high-protein diet.
Teleost fish provide some of the most intriguing examples of sexually dimorphic coloration, which is often advantageous for only one of the sexes. Mapping studies demonstrated that the genetic loci underlying such color patterns are frequently in tight linkage to the sex-determining locus of a species, ensuring sex-specific expression of the corresponding trait. Several genes affecting color synthesis and pigment cell development have been previously described, but the color loci on the sex chromosomes have mostly remained elusive as yet. Here, we summarize the current knowledge about the genetics of such color loci in teleosts, mainly from studies on poeciliids and cichlids. Further studies on these color loci will certainly provide important insights into the evolution of sex chromosomes.
By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca\(^{2+}\) mobilization and T cell proliferation. NSM-deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM-deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70, and PKCζ, and functional immune synapses are organized and stabilized via MTOC polarization.
Chloroquine (CQ) treatment failure in Plasmodium falciparum parasites has been documented for decades, but the pharmacological explanation of this phenotype is not fully understood. Current concepts attribute CQ resistance to reduced accumulation of the drug at a given external CQ concentration ([CQ] ex) in resistant compared to sensitive parasites. The implication of this explanation is that the mechanisms of CQ-induced toxicity in resistant and sensitive strains are similar once lethal internal concentrations have been reached. To test this hypothesis, we investigated the mechanism of CQ-induced toxicity in CQ-sensitive (CQS) versus CQ-resistant (CQR) parasites by analyzing the time-course of cellular responses in these strains after exposure to varying [CQ] ex as determined in 72 h toxicity assays. Parasite killing was delayed in CQR parasites for up to 10 h compared to CQS parasites when exposed to equipotent [CQ] ex. In striking contrast, brief exposure (1 h) to lethal [CQ] ex in CQS but not CQR parasites caused the appearance of hitherto undescribed hemozoin (Hz)-containing compartments in the parasite cytosol. Hz-containing compartments were very rarely observed in CQR parasites even after CQ exposures sufficient to cause irreversible cell death. These findings challenge current concepts that CQ killing of malaria parasites is solely concentration-dependent, and instead suggest that CQS and CQR strains fundamentally differ in the consequences of CQ exposure.
Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2\(^{2+}\)) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca\(^{2+}\) might be desirable. Moreover, there is need for an efficient light-gated potassium (K\(^{+}\)) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca\(^{2+}\) and K\(^{+}\) in cell physiology, light-activated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca\(^{2+}\) or for K\(^{+}\), respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca\(^{2+}\)-permeant channel, and to body extension when expressing the light-sensitive K\(^{+}\) channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.
Background:
The treatment strategies for colorectal cancer located in the right side of the colon have changed dramatically during the last decade. Due to the introduction of complete mesocolic excision (CME) with central ligation of the vessels and systematic lymph node dissection, the long-term survival of affected patients has increased significantly. It has also been proposed that right-sided colon resection can be performed laparoscopically with the same extent of resection and equal long-term results.
Methods:
A retrospective evaluation of a prospectively expanded database on right-sided colorectal cancer or adenoma treated at the University Hospital of Wuerzburg between 2009 and 2016 was performed. All patients underwent CME. This data was analyzed alone and in comparison to the published data describing laparoscopic right-sided colon resection for colon cancer.
Results:
The database contains 279 patients, who underwent right-sided colon resection due to colorectal cancer or colorectal adenoma (255 open; 24 laparoscopic). Operation data (time, length of stay, time on ICU) was equal or superior to laparoscopy, which is comparable to the published results. Surprisingly, the surrogate parameter for correct CME (the number of removed lymph nodes) was significantly higher in the open group. In a subgroup analysis only including patients who were feasible for laparoscopic resection and had been operated with an open procedure by an experienced surgeon, operation time was significantly shorter and the number of removed lymph nodes is significantly higher in the open group.
Conclusion:
So far, several studies demonstrate that laparoscopic right-sided colon resection is comparable to open resection. Our data suggests that a consequent CME during an open operation leads to significantly more removed lymph nodes than in laparoscopically resected patients and in several so far published data of open control groups from Europe. Further prospective randomized trials comparing the long-term outcome are urgently needed before laparoscopy for right-sided colon resection can be recommended ubiquitously.
Background:
The compound eyes of insects allow them to catch photons and convert the energy into electric signals. All compound eyes consist of numerous ommatidia, each comprising a fixed number of photoreceptors. Different ommatidial types are characterized by a specific set of photoreceptors differing in spectral sensitivity. In honey bees, males and females possess different ommatidial types forming distinct retinal mosaics. However, data are lacking on retinal ontogeny and the mechanisms by which the eyes are patterned. In this study, we investigated the intrinsic temporal and circadian expression patterns of the opsins that give rise to the ultraviolet, blue and green sensitive photoreceptors, as well as the morphological maturation of the retina during pupal development of honey bees.
Results:
qPCR and histological labeling revealed that temporal opsin mRNA expression differs between sexes and correlates with rhabdom elongation during photoreceptor development. In the first half of the pupal stage, when the rhabdoms of the photoreceptors are still short, worker and (dorsal) drone retinae exhibit similar expression patterns with relatively high levels of UV (UVop) and only marginal levels of blue (BLop) and green (Lop1) opsin mRNA. In the second half of pupation, when photoreceptors and rhabdoms elongate, opsin expression in workers becomes dominated by Lop1 mRNA. In contrast, the dorsal drone eye shows high expression levels of UVop and BLop mRNA, whereas Lop1 mRNA level decreases. Interestingly, opsin expression levels increase up to 22-fold during early adult life. We also found evidence that opsin expression in adult bees is under the control of the endogenous clock.
Conclusions:
Our data indicate that the formation of the sex-specific retinal composition of photoreceptors takes place during the second half of the pupal development, and that opsin mRNA expression levels continue to increase in young bees, which stands in contrast to Drosophila, where the highest expression levels are found during the late pupal stage and remain constant in adults. From an evolutionary perspective, we hypothesize that the delayed retinal maturation during the early adult phase is linked to the delayed transition from indoor to outdoor activities in bees, when vision becomes important.
Crop diversification has been proposed as farm management tool that could mitigate the externalities of conventional farming while reducing productivity-biodiversity trade-offs. Yet evidence for the acclaimed biodiversity benefits of landscape-level crop diversity is ambiguous. Effects may strongly depend on spatial scale and the level of landscape heterogeneity (e.g. overall habitat diversity). At the same time, contrasting within-taxon responses obscure benefits to specific functional groups (i.e. species with shared characteristics or requirements) if studied at the community level. The objectives of this study were to 1) disentangle the relative effects of crop diversity and landscape heterogeneity on avian species richness across five spatial scales ranging from 250 to 3000 m radii around focal winter wheat fields; and 2) assess whether functional groups (feeding guild, conservation status, habitat preference, nesting behaviour) determine the strength and direction of responses to crop diversity and landscape heterogeneity. In central Germany, 14 landscapes were selected along independent gradients of crop diversity (annual arable crops) and landscape heterogeneity. Bird species richness in each landscape was estimated using four point counts throughout the breeding season. We found no effects of landscape-level crop diversity on bird richness and functional groups. Instead, landscape heterogeneity was strongly associated with increased total bird richness across all spatial scales. In particular, insect-feeding and non-farmland birds were favoured in heterogeneous landscapes, as were species not classified as endangered or vulnerable on the regional Red List. Crop-nesting farmland birds, however, were less species-rich in these landscapes. Accordingly, crop diversification may be less suitable for conserving avian diversity and associated ecosystem services (e.g. biological pest control), although confounding interactions with management intensity need yet to be confirmed. In contrast, enhancement of landscape heterogeneity by increasing perennial habitat diversity, reducing field sizes and the amount of cropland has the potential to benefit overall bird richness. Specialist farmland birds, however, may require more targeted management approaches.
Bees receive nectar and pollen as reward for pollinating plants. Pollen of different plant species varies widely in nutritional composition. In order to select pollen of appropriate nutritional quality, bees would benefit if they could distinguish different pollen types. Whether they rely on visual, olfactory and/or chemotactile cues to distinguish between different pollen types, has however been little studied. In this study, we examined whether and how Apis mellifera workers differentiate between almond and apple pollen. We used differential proboscis extension response conditioning with olfactory and chemotactile stimulation, in light and darkness, and in summer and winter bees. We found that honeybees were only able to differentiate between different pollen types, when they could use both chemotactile and olfactory cues. Visual cues further improved learning performance. Summer bees learned faster than winter bees. Our results thus highlight the importance of multisensory information for pollen discrimination.
The honeybee olfactory pathway comprises an intriguing pattern of convergence and divergence: ~60.000 olfactory sensory neurons (OSN) convey olfactory information on ~900 projection neurons (PN) in the antennal lobe (AL). To transmit this information reliably, PNs employ relatively high spiking frequencies with complex patterns. PNs project via a dual olfactory pathway to the mushroom bodies (MB). This pathway comprises the medial (m-ALT) and the lateral antennal lobe tract (l-ALT). PNs from both tracts transmit information from a wide range of similar odors, but with distinct differences in coding properties. In the MBs, PNs form synapses with many Kenyon cells (KC) that encode odors in a spatially and temporally sparse way. The transformation from complex information coding to sparse coding is a well-known phenomenon in insect olfactory coding. Intrinsic neuronal properties as well as GABAergic inhibition are thought to contribute to this change in odor representation. In the present study, we identified intrinsic neuronal properties promoting coding differences between PNs and KCs using in-situ patch-clamp recordings in the intact brain. We found very prominent K+ currents in KCs clearly differing from the PN currents. This suggests that odor coding differences between PNs and KCs may be caused by differences in their specific ion channel properties. Comparison of ionic currents of m- and l-ALT PNs did not reveal any differences at a qualitative level.
Due to intensive agriculture honeybees are threatened by various pesticides. The use of one group of them, the neonicotinoids, was recently restricted by the European Union. These chemicals bind to the nicotinic acetylcholine receptor (nAchR) in the honeybee brain. Recently, Bayer AG released a new pesticide by the name of “Sivanto” against sucking insects. It is assumed to be harmless for honeybees, although its active ingredient, flupyradifurone, binds nAchR similar to the neonicotinoids. We investigated if this pesticide affects the taste for sugar and cognitive performance in honeybee foragers. These bees are directly exposed to the pesticide while foraging for pollen or nectar. Our results demonstrate that flupyradifurone can reduce taste and appetitive learning performance in honeybees foraging for pollen and nectar, although only the highest concentration had significant effects. Most likely, honeybee foragers will not be exposed to these high concentrations. Therefore, the appropriate use of this pesticide is considered safe for honeybees, at least with respect to the behaviors studied here.
After a large outbreak in Brazil, novel drugs against Zika virus became extremely necessary. Evaluation of virus-based pharmacological strategies concerning essential host factors brought us to the idea that targeting the Axl receptor by blocking its dimerization function could be critical for virus entry. Starting from experimentally validated compounds, such as RU-301, RU-302, warfarin, and R428, we identified a novel compound 2′ (R428 derivative) to be the most potent for this task amongst a number of alternative compounds and leads. The improved affinity of compound 2′ was confirmed by molecular docking as well as molecular dynamics simulation techniques using implicit solvation models. The current study summarizes a new possibility for inhibition of the Axl function as a potential target for future antiviral therapies.
Background:
Sex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination.
Results:
We now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal.
Conclusions:
Our work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.
Ras genes are among the most commonly mutated genes in human cancer; yet our understanding of their oncogenic activity at the molecular mechanistic level is incomplete. To identify downstream events that mediate ras-induced cellular transformation in vivo, we analyzed global microRNA expression in three different models of Ras-induction and tumor formation in zebrafish. Six microRNAs were found increased in Ras-induced melanoma, glioma and in an inducible model of ubiquitous Ras expression. The upregulation of the microRNAs depended on the activation of the ERK and AKT pathways and to a lesser extent, on mTOR signaling. Two Ras-induced microRNAs (miR-146a and 193a) target Jmjd6, inducing downregulation of its mRNA and protein levels at the onset of Ras expression during melanoma development. However, at later stages of melanoma progression, jmjd6 levels were found elevated. The dynamic of Jmjd6 levels during progression of melanoma in the zebrafish model suggests that upregulation of the microRNAs targeting Jmjd6 may be part of an anti-cancer response. Indeed, triple transgenic fish engineered to express a microRNA-resistant Jmjd6 from the onset of melanoma have increased tumor burden, higher infiltration of leukocytes and shorter melanoma-free survival. Increased JMJD6 expression is found in several human cancers, including melanoma, suggesting that the up-regulation of Jmjd6 is a critical event in tumor progression.
The following link has been created to allow review of record GSE37015: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jjcrbiuicyyqgpc&acc=GSE37015.
The cell wall synthesis pathway producing peptidoglycan is a highly coordinated and tightly regulated process. Although the major components of bacterial cell walls have been known for decades, the complex regulatory network controlling peptidoglycan synthesis and many details of the cell division machinery are not well understood. The eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp play an important role in cell wall biosynthesis and drug resistance in S. aureus. We show that stp deletion has a pronounced impact on cell wall synthesis. Deletion of stp leads to a thicker cell wall and decreases susceptibility to lysostaphin. Stationary phase Δstp cells accumulate peptidoglycan precursors and incorporate higher amounts of incomplete muropeptides with non-glycine, monoglycine and monoalanine interpeptide bridges into the cell wall. In line with this cell wall phenotype, we demonstrate that the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. Mass spectrometric analyses identify Thr32, Thr36 and Ser415 as phosphoacceptors. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Moreover, Stk interacts with FemA and FemB, but is unable to phosphorylate them. Our data indicate that Stk and Stp modulate cell wall synthesis and cell division at several levels.
1. Global warming can disrupt mutualistic interactions between solitary bees and plants when increasing temperature differentially changes the timing of interacting partners. One possible scenario is for insect phenology to advance more rapidly than plant phenology.
2. However, empirical evidence for fitness consequences due to temporal mismatches is lacking for pollinators and it remains unknown if bees have developed strategies to mitigate fitness losses following temporal mismatches.
3. We tested the effect of temporal mismatches on the fitness of three spring-emerging solitary bee species, including one pollen specialist. Using flight cages, we simulated (i) a perfect synchronization (from a bee perspective): bees and flowers occur simultaneously, (ii) a mismatch of 3days and (iii) a mismatch of 6days, with bees occurring earlier than flowers in the latter two cases.
4. A mismatch of 6days caused severe fitness losses in all three bee species, as few bees survived without flowers. Females showed strongly reduced activity and reproductive output compared to synchronized bees. Fitness consequences of a 3-day mismatch were species-specific. Both the early-spring species Osmia cornuta and the mid-spring species Osmia bicornis produced the same number of brood cells after a mismatch of 3days as under perfect synchronization. However, O.cornuta decreased the number of female offspring, whereas O.bicornis spread the brood cells over fewer nests, which may increase offspring mortality, e.g. due to parasitoids. The late-spring specialist Osmia brevicornis produced fewer brood cells even after a mismatch of 3days. Additionally, our results suggest that fitness losses after temporal mismatches are higher during warm than cold springs, as the naturally occurring temperature variability revealed that warm temperatures during starvation decreased the survival rate of O.bicornis.
5. We conclude that short temporal mismatches can cause clear fitness losses in solitary bees. Although our results suggest that bees have evolved species-specific strategies to mitigate fitness losses after temporal mismatches, the bees were not able to completely compensate for impacts on their fitness after temporal mismatches with their food resources.
Background and main text: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex and controversial clinical condition without having established causative factors. Increasing numbers of cases during past decade have created awareness among patients as well as healthcare professionals. Chronic viral infection as a cause of ME/CFS has long been debated. However, lack of large studies involving well-designed patient groups and validated experimental set ups have hindered our knowledge about this disease. Moreover, recent developments regarding molecular mechanism of pathogenesis of various infectious agents cast doubts over validity of several of the past studies.
Conclusions: This review aims to compile all the studies done so far to investigate various viral agents that could be associated with ME/CFS. Furthermore, we suggest strategies to better design future studies on the role of viral infections in ME/CFS.
Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.
Species' functional traits set the blueprint for pair-wise interactions in ecological networks. Yet, it is unknown to what extent the functional diversity of plant and animal communities controls network assembly along environmental gradients in real-world ecosystems. Here we address this question with a unique dataset of mutualistic bird-fruit, bird-flower and insect-flower interaction networks and associated functional traits of 200 plant and 282 animal species sampled along broad climate and land-use gradients on Mt. Kilimanjaro. We show that plant functional diversity is mainly limited by precipitation, while animal functional diversity is primarily limited by temperature. Furthermore, shifts in plant and animal functional diversity along the elevational gradient control the niche breadth and partitioning of the respective other trophic level. These findings reveal that climatic constraints on the functional diversity of either plants or animals determine the relative importance of bottom-up and top-down control in plant-animal interaction networks.
It’s a matter of design - how pitfall trap design affects trap samples and possible predictions
(2018)
Background:
Pitfall traps are commonly used to assess ground dwelling arthropod communities. The effects of different pitfall trap designs on the trapping outcome are poorly investigated however they might affect conclusions drawn from pitfall trap data greatly.
Methods:
We tested four pitfall trap types which have been used in previous studies for their effectiveness: a simple type, a faster exchangeable type with an extended plastic rim plate and two types with guidance barriers (V- and X-shaped). About 20 traps were active for 10 weeks and emptied biweekly resulting in 100 trap samples.
Results:
Pitfall traps with guidance barriers were up to five times more effective than simple pitfall traps and trap samples resulted in more similar assemblage approximations. Pitfall traps with extended plastic rim plates did not only perform poorly but also resulted in distinct carabid assemblages with less individuals of small species and a larger variation.
Discussion:
Due to the obvious trait filtering and resulting altered assemblages, we suggest not to use pitfall traps with extended plastic rim plates. In comprehensive biodiversity inventories, a smaller number of pitfall traps with guidance barriers and a larger number of spatial replicates is of advantage, while due to comparability reasons, the use of simple pitfall traps will be recommended in most other cases.
The fruit fly Drosophila melanogaster possesses approximately 150 brain clock neurons that control circadian behavioral rhythms. Even though individual clock neurons have self-sustaining oscillators, they interact and synchronize with each other through a network. However, little is known regarding the factors responsible for these network interactions. In this study, we investigated the role of CCHamide1 (CCHa1), a neuropeptide expressed in the anterior dorsal neuron 1 (DN1a), in intercellular communication of the clock neurons. We observed that CCHa1 connects the DN1a clock neurons to the ventral lateral clock neurons (LNv) via the CCHa1 receptor, which is a homolog of the gastrin-releasing peptide receptor playing a role in circadian intercellular communications in mammals. CCHa1 knockout or knockdown flies have a generally low activity level with a special reduction of morning activity. In addition, they exhibit advanced morning activity under light-dark cycles and delayed activity under constant dark conditions, which correlates with an advance/delay of PAR domain Protein 1 (PDP1) oscillations in the small-LNv (s-LNv) neurons that control morning activity. The terminals of the s-LNv neurons show rather high levels of Pigment-dispersing factor (PDF) in the evening, when PDF is low in control flies, suggesting that the knockdown of CCHa1 leads to increased PDF release; PDF signals the other clock neurons and evidently increases the amplitude of their PDP1 cycling. A previous study showed that high-amplitude PDP1 cycling increases the siesta of the flies, and indeed, CCHa1 knockout or knockdown flies exhibit a longer siesta than control flies. The DN1a neurons are known to be receptive to PDF signaling from the s-LNv neurons; thus, our results suggest that the DN1a and s-LNv clock neurons are reciprocally coupled via the neuropeptides CCHa1 and PDF, and this interaction fine-tunes the timing of activity and sleep.
The human body is constantly attacked by pathogens. Various lines of defence have evolved, among which the immune system is principal. In contrast to most pathogens, the African trypanosomes thrive freely in the blood circulation, where they escape immune destruction by antigenic variation and incessant motility. These unicellular parasites are flagellate microswimmers that also withstand the harsh mechanical forces prevailing in the bloodstream. They undergo complex developmental cycles in the bloodstream and organs of the mammalian host, as well as the disease-transmitting tsetse fly. Each life cycle stage has been shaped by evolution for manoeuvring in distinct microenvironments. Here, we introduce trypanosomes as blueprints for nature-inspired design of trypanobots, micromachines that, in the future, could explore the human body without affecting its physiology. We review cell biological and biophysical aspects of trypanosome motion. While this could provide a basis for the engineering of microbots, their actuation and control still appear more like fiction than science. Here, we discuss potentials and challenges of trypanosome-inspired microswimmer robots.
Cryptochromes (CRYs) are a class of flavoproteins that sense blue light. In animals, CRYs are expressed in the eyes and in the clock neurons that control sleep/wake cycles and are implied in the generation and/or entrainment of circadian rhythmicity. Moreover, CRYs are sensing magnetic fields in insects as well as in humans. Here, we show that in the fruit fly Drosophila melanogaster CRY plays a light-independent role as “assembling” protein in the rhabdomeres of the compound eyes. CRY interacts with actin and appears to increase light sensitivity of the eyes by keeping the “signalplex” of the phototransduction cascade close to the membrane. By this way, CRY also enhances light-responses of the circadian clock.
Eric Davidson once wrote about Theodor Boveri: "From his own researches, and perhaps most important, his generalized interpretations, derive the paradigms that underlie modern inquiries into the genomic basis of embryogenesis" (Davidson, 1985). As luck would have it, the "primary data" of Boveri's experimental work, namely the microscope slides prepared by him and his wife Marcella during several stays at the Zoological Station in Naples (1901/02, 1911/12 and 1914), have survived at the University of Wurzburg. More than 600 slides exist and despite their age they are in a surprisingly good condition. The slides are labelled and dated in Boveri's handwriting and thus can be assigned to his published experimental work on sea urchin development. The results allowed Boveri to unravel the role of the cell nucleus and its chromosomes in development and inheritance. Here, I present an overview of the slides in the context of Boveri's work along with photographic images of selected specimens taken from the original slides. It is planned to examine the slides in more detail, take high-resolution focal image series of significant specimens and make them online available.
Synaptic vesicles (SVs) are a key component of neuronal signaling and fulfil different roles depending on their composition. In electron micrograms of neurites, two types of vesicles can be distinguished by morphological criteria, the classical “clear core” vesicles (CCV) and the typically larger “dense core” vesicles (DCV), with differences in electron density due to their diverse cargos. Compared to CCVs, the precise function of DCVs is less defined. DCVs are known to store neuropeptides, which function as neuronal messengers and modulators [1]. In C. elegans, they play a role in locomotion, dauer formation, egg-laying, and mechano- and chemosensation [2]. Another type of DCVs, also referred to as granulated vesicles, are known to transport Bassoon, Piccolo and further constituents of the presynaptic density in the center of the active zone (AZ), and therefore are important for synaptogenesis [3].
To better understand the role of different types of SVs, we present here a new automated approach to classify vesicles. We combine machine learning with an extension of our previously developed vesicle segmentation workflow, the ImageJ macro 3D ART VeSElecT. With that we reliably distinguish CCVs and DCVs in electron tomograms of C. elegans NMJs using image-based features. Analysis of the underlying ground truth data shows an increased fraction of DCVs as well as a higher mean distance between DCVs and AZs in dauer larvae compared to young adult hermaphrodites. Our machine learning based tools are adaptable and can be applied to study properties of different synaptic vesicle pools in electron tomograms of diverse model organisms.
Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.
A comparative view on sex differentiation and gametogenesis genes in lungfish and coelacanths
(2018)
Gonadal sex differentiation and reproduction are the keys to the perpetuation of favorable gene combinations and positively selected traits. In vertebrates, several gonad development features that differentiate tetrapods and fishes are likely to be, at least in part, related to the water-to-land transition. The collection of information from basal sarcopterygians, coelacanths, and lungfishes, is crucial to improve our understanding of the molecular evolution of pathways involved in reproductive functions, since these organisms are generally regarded as “living fossils” and as the direct ancestors of tetrapods. Here, we report for the first time the characterization of >50 genes related to sex differentiation and gametogenesis in Latimeria menadoensis and Protopterus annectens. Although the expression profiles of most genes is consistent with the intermediate position of basal sarcopterygians between actinopterygian fish and tetrapods, their phylogenetic placement and presence/absence patterns often reveal a closer affinity to the tetrapod orthologs. On the other hand, particular genes, for example, the male gonad factor gsdf (Gonadal Soma-Derived Factor), provide examples of ancestral traits shared with actinopterygians, which disappeared in the tetrapod lineage.
Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.
TelAP1 links telomere complexes with developmental expression site silencing in African trypanosomes
(2018)
During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host, immune evasion of T. brucei bloodstream form (BSF) cells relies on antigenic variation, which includes monoallelic expression and periodic switching of variant surface glycoprotein (VSG) genes. The active VSG is transcribed from only 1 of the 15 subtelomeric expression sites (ESs). During differentiation from BSF to the insect-resident procyclic form (PCF), the active ES is transcriptionally silenced. We used mass spectrometry-based interactomics to determine the composition of telomere protein complexes in T. brucei BSF and PCF stages to learn more about the structure and functions of telomeres in trypanosomes. Our data suggest a different telomere complex composition in the two forms of the parasite. One of the novel telomere-associated proteins, TelAP1, forms a complex with telomeric proteins TbTRF, TbRAP1 and TbTIF2 and influences ES silencing kinetics during developmental differentiation.
Overwintering temperature and body condition shift emergence dates of spring-emerging solitary bees
(2018)
Solitary bees in seasonal environments must align their life-cycles with favorable environmental conditions and resources; the timing of their emergence is highly fitness relevant. In several bee species, overwintering temperature influences both emergence date and body weight at emergence. High variability in emergence dates among specimens overwintering at the same temperatures suggests that the timing of emergence also depends on individual body conditions. However, possible causes for this variability, such as individual differences in body size or weight, have been rarely studied. In a climate chamber experiment using two spring-emerging mason bees (Osmia cornuta and O. bicornis), we investigated the relationship between temperature, emergence date, body weight, and body size, the last of which is not affected by overwintering temperature. Our study showed that body weight declined during hibernation more strongly in warm than in cold overwintering temperatures. Although bees emerged earlier in warm than in cold overwintering temperatures, at the time of emergence, bees in warm overwintering temperatures had lower body weights than bees in cold overwintering temperatures (exception of male O. cornuta). Among specimens that experienced the same overwintering temperatures, small and light bees emerged later than their larger and heavier conspecifics. Using a simple mechanistic model we demonstrated that spring-emerging solitary bees use a strategic approach and emerge at a date that is most promising for their individual fitness expectations. Our results suggest that warmer overwintering temperatures reduce bee fitness by causing a decrease in body weight at emergence. We showed furthermore that in order to adjust their emergence dates, bees use not only temperature but also their individual body condition as triggers. This may explain differing responses to climate warming within and among bee populations and may have consequences for bee-plant interactions as well as for the persistence of bee populations under climate change.
Apoptosis is a physiological cell death process essential for development, tissue homeostasis, and for immune defense of multicellular animals. Inhibitors of apoptosis proteins (IAPs) regulate apoptosis in response to various cellular assaults. Using both genetic and pharmacological approaches we demonstrate here that the IAPs not only support opportunistic survival of intracellular human pathogens like Chlamydia pneumoniae but also control plasticity of iNOS+ M1 macrophage during the course of infection and render them refractory for immune stimulation. Treatment of Th1 primed macrophages with birinapant (IAP-specific antagonist) inhibited NO generation and relevant proteins involved in innate immune signaling. Accordingly, birinapant promoted hypoxia, angiogenesis, and tumor-induced M2 polarization of iNOS+ M1 macrophages. Interestingly, birinapant-driven changes in immune signaling were accompanied with changes in the expression of various proteins involved in the metabolism, and thus revealing the new role of IAPs in immune metabolic reprogramming in committed macrophages. Taken together, our study reveals the significance of IAP targeting approaches (Smac mimetic compounds) for the management of infectious and inflammatory diseases relying on macrophage plasticity.
Aspergillus fumigatus is a saprophytic, cosmopolitan fungus that attacks patients with a weak immune system. A rational solution against fungal infection aims to manipulate fungal metabolism or to block enzymes essential for Aspergillus survival. Here we discuss and compare different bioinformatics approaches to analyze possible targeting strategies on fungal-unique pathways. For instance, phylogenetic analysis reveals fungal targets, while domain analysis allows us to spot minor differences in protein composition between the host and fungi. Moreover, protein networks between host and fungi can be systematically compared by looking at orthologs and exploiting information from host–pathogen interaction databases. Further data—such as knowledge of a three-dimensional structure, gene expression data, or information from calculated metabolic fluxes—refine the search and rapidly put a focus on the best targets for antimycotics. We analyzed several of the best targets for application to structure-based drug design. Finally, we discuss general advantages and limitations in identification of unique fungal pathways and protein targets when applying bioinformatics tools.
Obligate intracellular pathogenic Chlamydia trachomatis express several serine proteases whose roles in chlamydial development and pathogenicity are not completely understood. The chlamydial protease CPAF is expressed during the replicative phase of the chlamydial developmental cycle and is secreted into the lumen of the Chlamydia-containing vacuole called inclusion. How the secreted protease is activated in the inclusion lumen is currently not fully understood. We have identified human serine peptidase inhibitor PI15 as a potential host factor involved in the regulation of CPAF activation. Silencing expression as well as over expression of PI15 affected normal development of Chlamydia. PI15 was transported into the chlamydial inclusion lumen where it co-localized with CPAF aggregates. We show that PI15 binds to the CPAF zymogen and potentially induces CPAF protease activity at low concentrations. However, at high concentrations PI15 inhibits CPAF activity possibly by blocking its protease domain. Our findings shed light on a new aspect of chlamydial host co-evolution which involves the recruitment of host cell proteins into the inclusion to control the activation of bacterial proteases like CPAF that are important for the normal development of Chlamydia.
Meiotic chromosomes undergo rapid prophase movements, which are thought to facilitate the formation of inter-homologue recombination intermediates that underlie synapsis, crossing over and segregation. The meiotic telomere complex (MAJIN, TERB1, TERB2) tethers telomere ends to the nuclear envelope and transmits cytoskeletal forces via the LINC complex to drive these rapid movements. Here, we report the molecular architecture of the meiotic telomere complex through the crystal structure of MAJIN-TERB2, together with light and X-ray scattering studies of wider complexes. The MAJIN-TERB2 2:2 hetero-tetramer binds strongly to DNA and is tethered through long flexible linkers to the inner nuclear membrane and two TRF1-binding 1:1 TERB2-TERB1 complexes. Our complementary structured illumination microscopy studies and biochemical findings reveal a telomere attachment mechanism in which MAJIN-TERB2-TERB1 recruits telomere-bound TRF1, which is then displaced during pachytene, allowing MAJIN-TERB2-TERB1 to bind telomeric DNA and form a mature attachment plate.
Background
Cuticular hydrocarbons (CHC) have been documented to play crucial roles as species- and sex-specific cues in the chemical communication systems of a wide variety of insects. However, whether they are sufficient by themselves as the sole cue triggering sexual behavior as well as preference of con- over heterospecific mating partners is rarely assessed. We conducted behavioral assays in three representative species of parasitoid wasps (Hymenoptera: Pteromalidae) to determine their reliance on CHC as species-specific sexual signaling cues.
Results
We found a surprising degree of either unspecific or insufficient sexual signaling when CHC are singled out as recognition cues. Most strikingly, the cosmopolitan species Nasonia vitripennis, expected to experience enhanced selection pressure to discriminate against other co-occurring parasitoids, did not discriminate against CHC of a partially sympatric species from another genus, Trichomalopsis sarcophagae. Focusing on the latter species, in turn, it became apparent that CHC are even insufficient as the sole cue triggering conspecific sexual behavior, hinting at the requirement of additional, synergistic sexual cues particularly important in this species. Finally, in the phylogenetically and chemically most divergent species Muscidifurax uniraptor, we intriguingly found both CHC-based sexual signaling as well as species discrimination behavior intact although this species is naturally parthenogenetic with sexual reproduction only occurring under laboratory conditions.
Conclusions
Our findings implicate a discrepancy in the reliance on and specificity of CHC as sexual cues in our tested parasitioid wasps. CHC profiles were not sufficient for unambiguous discrimination and preference behavior, as demonstrated by clear cross-attraction between some of our tested wasp genera. Moreover, we could show that only in T. sarcophagae, additional behavioral cues need to be present for triggering natural mating behavior, hinting at an interesting shift in signaling hierarchy in this particular species. This demonstrates the importance of integrating multiple, potentially complementary signaling modalities in future studies for a better understanding of their individual contributions to natural sexual communication behavior.
Background:
Ketogenic diets (KDs) or short-term fasting are popular trends amongst supportive approaches for cancer patients. Beta-hydroxybutyrate (3-OHB) is the main physiological ketone body, whose concentration can reach plasma levels of 2–6 mM during KDs or fasting. The impact of 3-OHB on the biology of tumor cells described so far is contradictory. Therefore, we investigated the effect of a physiological concentration of 3 mM 3-OHB on metabolism, proliferation, and viability of breast cancer (BC) cells in vitro.
Methods:
Seven different human BC cell lines (BT20, BT474, HBL100, MCF-7, MDA-MB 231, MDA-MB 468, and T47D) were cultured in medium with 5 mM glucose in the presence of 3 mM 3-OHB at mild hypoxia (5% oxygen) or normoxia (21% oxygen). Metabolic profiling was performed by quantification of the turnover of glucose, lactate, and 3-OHB and by Seahorse metabolic flux analysis. Expression of key enzymes of ketolysis as well as the main monocarboxylic acid transporter MCT2 and the glucose-transporter GLUT1 was analyzed by RT-qPCR and Western blotting. The effect of 3-OHB on short- and long-term cell proliferation as well as chemo- and radiosensitivity were also analyzed.
Results:
3-OHB significantly changed the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in BT20 cells resulting in a more oxidative energetic phenotype. MCF-7 and MDA-MB 468 cells had increased ECAR only in response to 3-OHB, while the other three cell types remained uninfluenced. All cells expressed MCT2 and GLUT1, thus being able to uptake the metabolites. The consumption of 3-OHB was not strongly linked to mRNA overexpression of key enzymes of ketolysis and did not correlate with lactate production and glucose consumption. Neither 3-OHB nor acetoacetate did interfere with proliferation. Further, 3-OHB incubation did not modify the response of the tested BC cell lines to chemotherapy or radiation.
Conclusions:
We found that a physiological level of 3-OHB can change the energetic profile of some BC cell lines. However, 3-OHB failed to influence different biologic processes in these cells, e.g., cell proliferation and the response to common breast cancer chemotherapy and radiotherapy. Thus, we have no evidence that 3-OHB generally influences the biology of breast cancer cells in vitro.
Endogenous molecular circadian clocks drive daily rhythmic changes at the cellular, physiological, and behavioral level for adaptation to and anticipation of environmental signals. The core molecular system consists of autoregulatory feedback loops, where clock proteins inhibit their own transcription. A complex and not fully understood interplay of regulatory proteins influences activity, localization and stability of clock proteins to set the pace of the clock. This study focuses on the molecular function of Ribosomal S6 Kinase (RSK) in the Drosophila melanogaster circadian clock. Mutations in the human rsk2 gene cause Coffin–Lowry syndrome, which is associated with severe mental disabilities. Knock-out studies with Drosophila ortholog rsk uncovered functions in synaptic processes, axonal transport and adult behavior including associative learning and circadian activity. However, the molecular targets of RSK remain elusive. Our experiments provide evidence that RSK acts in the key pace maker neurons as a negative regulator of Shaggy (SGG) kinase activity, which in turn determines timely nuclear entry of the clock proteins Period and Timeless to close the negative feedback loop. Phosphorylation of serine 9 in SGG is mediated by the C-terminal kinase domain of RSK, which is in agreement with previous genetic studies of RSK in the circadian clock but argues against the prevailing view that only the N-terminal kinase domain of RSK proteins carries the effector function. Our data provide a mechanistic explanation how RSK influences the molecular clock and imply SGG S9 phosphorylation by RSK and other kinases as a convergence point for diverse cellular and external stimuli.
Epichloë endophytes associated with cool-season grass species can protect their hosts from herbivory and can suppress mycorrhizal colonization of the hosts’ roots. However, little is known about whether or not Epichloë endophyte infection can also change the foliar fungal assemblages of the host. We tested 52 grassland study sites along a land-use intensity gradient in three study regions over two seasons (spring vs. summer) to determine whether Epichloë infection of the host grass Lolium perenne changes the fungal community structure in leaves. Foliar fungal communities were assessed by Next Generation Sequencing of the ITS rRNA gene region. Fungal community structure was strongly affected by study region and season in our study, while land-use intensity and infection with Epichloë endophytes had no significant effects. We conclude that effects on non-systemic endophytes resulting from land use practices and Epichloë infection reported in other studies were masked by local and seasonal variability in this study’s grassland sites.
Background:
Genetic heterogeneity and consanguineous marriages make recessive inherited hearing loss in Iran the second most common genetic disorder. Only two reported pathogenic variants (c.323G>C, p.Arg108Pro and c.419A>G, p.Tyr140Cys) in the S1PR2 gene have previously been linked to autosomal recessive hearing loss (DFNB68) in two Pakistani families. We describe a segregating novel homozygous c.323G>A, p.Arg108Gln pathogenic variant in S1PR2 that was identified in four affected individuals from a consanguineous five generation Iranian family.
Methods:
Whole exome sequencing and bioinformatics analysis of 116 hearing loss-associated genes was performed in an affected individual from a five generation Iranian family. Segregation analysis and 3D protein modeling of the p.Arg108 exchange was performed.
Results:
The two Pakistani families previously identified with S1PR2 pathogenic variants presented profound hearing loss that is also observed in the affected Iranian individuals described in the current study. Interestingly, we confirmed mixed hearing loss in one affected individual. 3D protein modeling suggests that the p.Arg108 position plays a key role in ligand receptor interaction, which is disturbed by the p.Arg108Gln change.
Conclusion:
In summary, we report the third overall mutation in S1PR2 and the first report outside the Pakistani population. Furthermore, we describe a novel variant that causes an amino acid exchange (p.Arg108Gln) in the same amino acid residue as one of the previously reported Pakistani families (p.Arg108Pro). This finding emphasizes the importance of the p.Arg108 amino acid in normal hearing and confirms and consolidates the role of S1PR2 in autosomal recessive hearing loss.
Approximately half of all melanoma patients harbour activating mutations in the serine/threonine kinase BRAF. This is the basis for one of the main treatment strategies for this tumor type, the targeted therapy with BRAF and MEK inhibitors. While the initial responsiveness to these drugs is high, resistance develops after several months, frequently at sites of the previously responding tumor. This indicates that tumor response is incomplete and that a certain tumor fraction survives even in drug-sensitive patients, e.g., in a therapy-induced senescence-like state. Here, we show in several melanoma cell lines that BRAF inhibition induces a secretome with stimulating effect on fibroblasts and naive melanoma cells. Several senescence-associated factors were found to be transcribed and secreted in response to BRAF or MEK inhibition, among them members of the fibroblast growth factor family. We identified the growth factor FGF1 as mediator of resilience towards BRAF inhibition, which limits the pro-apoptotic effects of the drug and activates fibroblasts to secrete HGF. FGF1 regulation was mediated by the PI3K pathway and by FRA1, a direct target gene of the MAPK pathway. When FGFR inhibitors were applied in parallel to BRAF inhibitors, resilience was broken, thus providing a rationale for combined therapeutical application.
Forest biodiversity conservation requires precise, area-wide information on the abundance and distribution of key habitat structures at multiple spatial scales. We combined airborne laser scanning (ALS) data with color-infrared (CIR) aerial imagery for identifying individual tree characteristics and quantifying multi-scale habitat requirements using the example of the three-toed woodpecker (Picoides tridactylus) (TTW) in the Bavarian Forest National Park (Germany). This bird, a keystone species of boreal and mountainous forests, is highly reliant on bark beetles dwelling in dead or dying trees. While previous studies showed a positive relationship between the TTW presence and the amount of deadwood as a limiting resource, we hypothesized a unimodal response with a negative effect of very high deadwood amounts and tested for effects of substrate quality. Based on 104 woodpecker presence or absence locations, habitat selection was modelled at four spatial scales reflecting different woodpecker home range sizes. The abundance of standing dead trees was the most important predictor, with an increase in the probability of TTW occurrence up to a threshold of 44–50 dead trees per hectare, followed by a decrease in the probability of occurrence. A positive relationship with the deadwood crown size indicated the importance of fresh deadwood. Remote sensing data allowed both an area-wide prediction of species occurrence and the derivation of ecological threshold values for deadwood quality and quantity for more informed conservation management.
It is a common belief that feral honey bee colonies (Apis mellifera L.) were eradicated in Europe through the loss of habitats, domestication by man and spread of pathogens and parasites. Interestingly, no scientific data are available, neither about the past nor the present status of naturally nesting honeybee colonies. We expected near-natural beech (Fagus sylvatica L.) forests to provide enough suitable nest sites to be a home for feral honey bee colonies in Europe. Here, we made a first assessment of their occurrence and density in two German woodland areas based on two methods, the tracing of nest sites based on forager flight routes (beelining technique), and the direct inspection of potential cavity trees. Further, we established experimental swarms at forest edges and decoded dances for nest sites performed by scout bees in order to study how far swarms from beekeeper-managed hives would potentially move into a forest. We found that feral honey bee colonies regularly inhabit tree cavities in near-natural beech forests at densities of at least 0.11-0.14 colonies/km\(^{2}\). Colonies were not confined to the forest edges; they were also living deep inside the forests. We estimated a median distance of 2,600 m from the bee trees to the next apiaries, while scout bees in experimental swarms communicated nest sites in close distances (median: 470 m). We extrapolate that there are several thousand feral honey bee colonies in German woodlands. These have to be taken in account when assessing the role of forest areas in providing pollination services to the surrounding land, and their occurrence has implications for the species' perception among researchers, beekeepers and conservationists. This study provides a starting point for investigating the life-histories and the ecological interactions of honey bees in temperate European forest environments.