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Olpidiopsis is a genus of obligate holocarpic endobiotic oomycetes. Most of the species classified in the genus are known only from their morphology and life cycle, and a few have been examined for their ultrastructure or molecular phylogeny. However, the taxonomic placement of all sequenced species is provisional, as no sequence data are available for the type species, O. saprolegniae, to consolidate the taxonomy of species currently placed in the genus. Thus, efforts were undertaken to isolate O. saprolegniae from its type host, Saprolegnia parasitica and to infer its phylogenetic placement based on 18S rDNA sequences. As most species of Olpidiopsis for which sequence data are available are from rhodophyte hosts, we have also isolated the type species of the rhodophyte-parasitic genus Pontisma, P. lagenidioides and obtained partial 18S rDNA sequences. Phylogenetic reconstructions in the current study revealed that O. saprolegniae from Saprolegnia parasitica forms a monophyletic group with a morphologically similar isolate from S. ferax, and a morphologically and phylogenetically more divergent species from S. terrestris. However, they were widely separated from a monophyletic, yet unsupported clade containing P. lagenidioides and red algal parasites previously classified in Olpidiopsis. Consequently, all holocarpic parasites in red algae should be considered to be members of the genus Pontisma as previously suggested by some researchers. In addition, a new species of Olpidiopsis, O. parthenogenetica is introduced to accommodate the pathogen of S. terrestris.
Zoos attract millions of visitors every year, many of whom are schoolchildren. For this reason, zoos are important institutions for the environmental education of future generations. Empirical studies on the educational impact of environmental education programs in zoos are still rare. To address this issue, we conducted two studies: In study 1, we investigated students’ interests in different biological topics, including zoos (n = 1,587). Data analysis of individual topics revealed large differences of interest, with advanced students showing less interest in zoos. In study 2, we invited school classes of this age group to visit different guided tours at the zoo and tested connection to nature before and after each educational intervention (n = 608). The results showed that the guided tours are an effective tool to raise students’ connection to nature. Add-on components have the potential to further promote connection to nature. The education programs are most effective with students with a low initial nature connection.
Brain aging is one of the major risk factors for the development of several neurodegenerative diseases. Therefore, mitochondrial dysfunction plays an important role in processes of both, brain aging and neurodegeneration. Aged mice including NMRI mice are established model organisms to study physiological and molecular mechanisms of brain aging. However, longitudinal data evaluated in one cohort are rare but are important to understand the aging process of the brain throughout life, especially since pathological changes early in life might pave the way to neurodegeneration in advanced age. To assess the longitudinal course of brain aging, we used a cohort of female NMRI mice and measured brain mitochondrial function, cognitive performance, and molecular markers every 6 months until mice reached the age of 24 months. Furthermore, we measured citrate synthase activity and respiration of isolated brain mitochondria. Mice at the age of three months served as young controls. At six months of age, mitochondria-related genes (complex IV, creb-1, β-AMPK, and Tfam) were significantly elevated. Brain ATP levels were significantly reduced at an age of 18 months while mitochondria respiration was already reduced in middle-aged mice which is in accordance with the monitored impairments in cognitive tests. mRNA expression of genes involved in mitochondrial biogenesis (cAMP response element-binding protein 1 (creb-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), nuclear respiratory factor-1 (Nrf-1), mitochondrial transcription factor A (Tfam), growth-associated protein 43 (GAP43), and synaptophysin 1 (SYP1)) and the antioxidative defense system (catalase (Cat) and superoxide dismutase 2 (SOD2)) was measured and showed significantly decreased expression patterns in the brain starting at an age of 18 months. BDNF expression reached, a maximum after 6 months. On the basis of longitudinal data, our results demonstrate a close connection between the age-related decline of cognitive performance, energy metabolism, and mitochondrial biogenesis during the physiological brain aging process.
High tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. TIFP may hamper adequate uptake of macromolecular therapeutics in tumor tissue. In addition, TIFP generates mechanical forces affecting the tumor cortex, which might influence the growth parameters of tumor cells. This seems likely as, in other tissues (namely, blood vessels or the skin), mechanical stretch is known to trigger proliferation. Therefore, we hypothesize that TIFP-induced stretch modulates proliferation-associated parameters. Solid epithelial tumors (A431 and A549) were grown in Naval Medical Research Institute nude mice, generating a TIFP of about 10 mm Hg (A431) or 5 mm Hg (A549). Tumor drainage of the central cystic area led to a rapid decline of TIFP, together with visible relaxation of the tumor cortex. It was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis that TIFP lowering yields a decreased phosphorylation of proliferation-associated p44/42 mitogen-activated protein kinase and tumor relaxation. In confirmation, immunohistochemical staining showed a decrease of tumor-associated proliferation marker Ki-67 after TIFP lowering. These data suggest that the mechanical stretch induced by TIFP is a positive modulator of tumor proliferation.
Here, we describe a new immersion-based clearing method suitable for optical clearing of thick adult human brain samples while preserving its lipids and lipophilic labels such as 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). This clearing procedure is simple, easy to implement, and allowed for clearing of 5 mm thick human brain tissue samples within 12 days. Furthermore, we show for the first time the advantageous effect of the Periodate-Lysine-Paraformaldehyde (PLP) fixation as compared to the more commonly used 4% paraformaldehyde (PFA) on clearing performance.
Tsetse flies are the transmitting vector of trypanosomes causing human sleeping sickness and animal trypanosomiasis in sub-saharan Africa. 3-alkylphenols are used as attractants in tsetse fly traps to reduce the spread of the disease. Here we present an inexpensive production method for 3-ethylphenol (3-EP) and 3-propylphenol (3-PP) by microbial fermentation of sugars. Heterologous expression in the yeast Saccharomyces cerevisiae of phosphopantetheinyltransferase-activated 6-methylsalicylic acid (6-MSA) synthase (MSAS) and 6-MSA decarboxylase converted acetyl-CoA as a priming unit via 6-MSA into 3-methylphenol (3-MP). We exploited the substrate promiscuity of MSAS to utilize propionyl-CoA and butyryl-CoA as alternative priming units and the substrate promiscuity of 6-MSA decarboxylase to produce 3-EP and 3-PP in yeast fermentations. Increasing the formation of propionyl-CoA by expression of a bacterial propionyl-CoA synthetase, feeding of propionate and blocking propionyl-CoA degradation led to the production of up to 12.5 mg/L 3-EP. Introduction of a heterologous ‘reverse ß-oxidation’ pathway provided enough butyryl-CoA for the production of 3-PP, reaching titers of up to 2.6 mg/L. As the concentrations of 3-alkylphenols are close to the range of the concentrations deployed in tsetse fly traps, the yeast broths might become promising and inexpensive sources for attractants, producible on site by rural communities in Africa.
High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease
(2019)
Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.
Data supporting the role of the non-glycosylated isoform of MIC26 in determining cristae morphology
(2015)
Membrane architecture is crucially important for mitochondrial function and integrity. The MICOS complex is located at crista junctions and determines cristae membrane morphology and the formation of crista junctions. Here we provide data of the bona fide MICOS subunit MIC26 for determining cristae morphology. MiRNA-mediated downregulation of MIC26 results in higher protein levels of MIC27 and in lower levels of Mic10. Using a miRNA-resistant form to MIC26 we show that this effect is specific to MIC26. Our data further demonstrate that depletion of MIC26 primarily affects the level of the 22 kDa mitochondrial isoform of MIC26 but not the amount of the secreted 55 kDa isoform of MIC26. Depletion of MIC27, however, increases secretion of the latter isoform. Overexpression of a myc-tagged version of MIC26 resulted in altered cristae morphology with swollen and partly vesicular cristae-structures.
Transcriptional basis for differential thermosensitivity of seedlings of various tomato genotypes
(2020)
Transcriptional reprograming after the exposure of plants to elevated temperatures is a hallmark of stress response which is required for the manifestation of thermotolerance. Central transcription factors regulate the stress survival and recovery mechanisms and many of the core responses controlled by these factors are well described. In turn, pathways and specific genes contributing to variations in the thermotolerance capacity even among closely related plant genotypes are not well defined. A seedling-based assay was developed to directly compare the growth and transcriptome response to heat stress in four tomato genotypes with contrasting thermotolerance. The conserved and the genotype-specific alterations of mRNA abundance in response to heat stress were monitored after exposure to three different temperatures. The transcripts of the majority of genes behave similarly in all genotypes, including the majority of heat stress transcription factors and heat shock proteins, but also genes involved in photosynthesis and mitochondrial ATP production. In turn, genes involved in hormone and RNA-based regulation, such as auxin- and ethylene-related genes, or transcription factors like HsfA6b, show a differential regulation that associates with the thermotolerance pattern. Our results provide an inventory of genes likely involved in core and genotype-dependent heat stress response mechanisms with putative role in thermotolerance in tomato seedlings.
Background: Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which is sometimes associated with severe central nervous system disease in children. There is currently no specific medication for EV71 infection. Quercetin, one of the most widely distributed flavonoids in plants, has been demonstrated to inhibit various viral infections. However, investigation of the anti-EV71 mechanism has not been reported to date.
Methods: The anti-EV71 activity of quercetin was evaluated by phenotype screening, determining the cytopathic effect (CPE) and EV71-induced cells apoptosis. The effects on EV71 replication were evaluated further by determining virus yield, viral RNA synthesis and protein expression, respectively. The mechanism of action against EV71 was determined from the effective stage and time-of-addition assays. The possible inhibitory functions of quercetin via viral 2Apro, 3Cpro or 3Dpol were tested. The interaction between EV71 3Cpro and quercetin was predicted and calculated by molecular docking.
Results: Quercetin inhibited EV71-mediated cytopathogenic effects, reduced EV71 progeny yields, and prevented EV71-induced apoptosis with low cytotoxicity. Investigation of the underlying mechanism of action revealed that quercetin exhibited a preventive effect against EV71 infection and inhibited viral adsorption. Moreover, quercetin mediated its powerful therapeutic effects primarily by blocking the early post-attachment stage of viral infection. Further experiments demonstrated that quercetin potently inhibited the activity of the EV71 protease, 3Cpro, blocking viral replication, but not the activity of the protease, 2Apro, or the RNA polymerase, 3Dpol. Modeling of the molecular binding of the 3Cpro-quercetin complex revealed that quercetin was predicted to insert into the substrate-binding pocket of EV71 3Cpro, blocking substrate recognition and thereby inhibiting EV71 3Cpro activity.
Conclusions: Quercetin can effectively prevent EV71-induced cell injury with low toxicity to host cells. Quercetin may act in more than one way to deter viral infection, exhibiting some preventive and a powerful therapeutic effect against EV71. Further, quercetin potently inhibits EV71 3Cpro activity, thereby blocking EV71 replication.
The UN's sustainable development goals (SDGs), which aim to solve important economic, social, and environmental problems of humanity, are to be supported by education for sustainable development (ESD). Empirical studies on the success of the implementation of the SDGs in the field of education are still pending. For this reason, using the loss of global biodiversity as an example, this study examined the extent to which high school students, teacher trainees in biology, and biology bachelor students can identify the causes of the global biodiversity loss. A new questioning tool was developed and tested on 889 participants. In addition, the relationship between connection to nature and the personal assessment about biodiversity threats was examined. The factor analysis of the scale used showed that 11 out of 16 items were assigned to the intended factor. The comparison between high school students, teacher trainees in biology, and biology bachelor students showed no significant difference in overall assessment of the reasons for global biodiversity loss. When comparing the three risk levels in which the risk factors for biodiversity could be divided, across the three student groups, only minor differences were found. Therefore, a specific education of prospective teachers is necessary, as they have to pass on the competence as multipliers to their students. No significant difference could be found when examining the relationship between connection to nature and the overall scores of the assessment scale for the reasons of biodiversity loss. However, it was found that people who felt more connected to nature were more capable of assessing the main causes of risk for global biodiversity, while people who felt less connected to nature achieved better scores for the medium factors
Precise knowledge on the binding sites of an RNA-binding protein (RBP) is key to understanding the complex post-transcriptional regulation of gene expression. This information can be obtained from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments. Here, we present a complete data analysis workflow to reliably detect RBP binding sites from iCLIP data. The workflow covers all steps from the initial quality control of the sequencing reads up to peak calling and quantification of RBP binding. For each tool, we explain the specific requirements for iCLIP data analysis and suggest optimised parameter settings.
Risk evaluations for agricultural chemicals are necessary to preserve healthy populations of honey bee colonies. Field studies on whole colonies are limited in behavioural research, while results from lab studies allow only restricted conclusions on whole colony impacts. Methods for automated long-term investigations of behaviours within comb cells, such as brood care, were hitherto missing. In the present study, we demonstrate an innovative video method that enables within-cell analysis in honey bee (Apis mellifera) observation hives to detect chronic sublethal neonicotinoid effects of clothianidin (1 and 10 ppb) and thiacloprid (200 ppb) on worker behaviour and development. In May and June, colonies which were fed 10 ppb clothianidin and 200 ppb thiacloprid in syrup over three weeks showed reduced feeding visits and duration throughout various larval development days (LDDs). On LDD 6 (capping day) total feeding duration did not differ between treatments. Behavioural adaptation was exhibited by nurses in the treatment groups in response to retarded larval development by increasing the overall feeding timespan. Using our machine learning algorithm, we demonstrate a novel method for detecting behaviours in an intact hive that can be applied in a versatile manner to conduct impact analyses of chemicals, pests and other stressors.
Elevated tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. Clinically, TIFP may hamper the uptake of chemotherapeutic drugs into the tumor tissue reducing their therapeutic efficacy. In this study, a means of modulating TIFP to increase the flux of macromolecules into tumor tissue is presented, which is based on the rationale that elevated plasma colloid osmotic pressure (COP) pulls water from tumor interstitium lowering the TIFP. Concentrated human serum albumin: (20% HSA), used as an agent to enhance COP, reduced the TIFP time-dependently from 8 to 2 mm Hg in human tumor xenograft models bearing A431 epidermoid vulva carcinomas. To evaluate whether this reduction facilitates the uptake of macromolecules, the intratumoral distribution of fluorescently conjugated dextrans (2.5 mg/ml) and cetuximab (2.0 mg/ml) was probed using novel time domain nearinfrared fluorescence imaging. This method permitted discrimination and semiquantification of tumor-accumulated conjugate from background and unspecific probe fluorescence. The coadministration of 20% HSA together with either dextrans or cetuximab was found to lower the TIFP significantly and increase the concentration of the substances within the tumor tissue in comparison to control tumors. Furthermore, combined administration of 20%HSA plus cetuximab reduced the tumor growth significantly in comparison to standard cetuximab treatment. These data demonstrate that increased COP lowers the TIFP within hours and increases the uptake of therapeutic macromolecules into the tumor interstitium leading to reduced tumor growth. This model represents a novel approach to facilitate the delivery of therapeutics into tumor tissue, particularly monoclonal antibodies.
Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.
Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.
The transition from local to global patterns governs the differentiation of mouse blastocysts
(2020)
During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterized by the expression of the transcription factors NANOG and GATA6, respectively. Here, we investigate the spatio-temporal distribution of NANOG and GATA6 expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single cell-based neighbourhood analyses. We define the cell neighbourhood by local features, which include the expression levels of both fate markers expressed in each cell and its neighbours, and the number of neighbouring cells. We further include the position of a cell relative to the centre of the ICM as a global positional feature. Our analyses reveal a local three-dimensional pattern that is already present in early blastocysts: 1) Cells expressing the highest NANOG levels are surrounded by approximately nine neighbours, while 2) cells expressing GATA6 cluster according to their GATA6 levels. This local pattern evolves into a global pattern in the ICM that starts to emerge in mid blastocysts. We show that FGF/MAPK signalling is involved in the three-dimensional distribution of the cells and, using a mutant background, we further show that the GATA6 neighbourhood is regulated by NANOG. Our quantitative study suggests that the three-dimensional cell neighbourhood plays a role in Epi and PrE precursor specification. Our results highlight the importance of analysing the three-dimensional cell neighbourhood while investigating cell fate decisions during early mouse embryonic development.
Wolves (Canis lupus) are currently showing a remarkable comeback in the highly frag-mented cultural landscapes of Germany. We here show that wolf numbers increasedexponentially between 2000 and 2015 with an annual increase of about 36%. Wedemonstrate that the first territories in each newly colonized region were establishedover long distances from the nearest known reproducing pack on active militarytraining areas (MTAs). We show that MTAs, rather than protected areas, served asstepping-stones for the recolonization of Germany facilitating subsequent spreadingof wolf territories in the surrounding landscape. We did not find any significant differ-ence between MTAs and protected areas with regard to habitat. One possible reasonfor the importance of MTAs may be their lower anthropogenic mortality rates com-pared to protected and other areas. To our knowledge, this is the first documented casewhere MTAs facilitate the recolonization of an endangered species across large areas.
In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon (FEP) foils. The ultra-thin FEP-foil cuvettes have a wall thickness of about 10–12 µm. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible. Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters. Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.
Several members of the genus Legionella cause Legionnaires’ disease, a potentially debilitating form of pneumonia. Studies frequently focus on the abundant number of virulence factors present in this genus. However, what is often overlooked is the role of secondary metabolites from Legionella. Following whole genome sequencing, we assembled and annotated the Legionella parisiensis DSM 19216 genome. Together with 14 other members of the Legionella, we performed comparative genomics and analysed the secondary metabolite potential of each strain. We found that Legionella contains a huge variety of biosynthetic gene clusters (BGCs) that are potentially making a significant number of novel natural products with undefined function. Surprisingly, only a single Sfp-like phosphopantetheinyl transferase is found in all Legionella strains analyzed that might be responsible for the activation of all carrier proteins in primary (fatty acid biosynthesis) and secondary metabolism (polyketide and non-ribosomal peptide synthesis). Using conserved active site motifs, we predict some novel compounds that are probably involved in cell-cell communication, differing to known communication systems. We identify several gene clusters, which may represent novel signaling mechanisms and demonstrate the natural product potential of Legionella.
The facile synthesis and detailed investigation of a class of highly potent protease inhibitors based on 1,4-naphthoquinones with a dipeptidic recognition motif (HN-l-Phe-l-Leu-OR) in the 2-position and an electron-withdrawing group (EWG) in the 3-position is presented. One of the compound representatives, namely the acid with EWG = CN and with R = H proved to be a highly potent rhodesain inhibitor with nanomolar affinity. The respective benzyl ester (R = Bn) was found to be hydrolyzed by the target enzyme itself yielding the free acid. Detailed kinetic and mass spectrometry studies revealed a reversible covalent binding mode. Theoretical calculations with different density functionals (DFT) as well as wavefunction-based approaches were performed to elucidate the mode of action.
Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ("wide" and "narrow"), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.
Tigers are indisputably in danger of extinction due to habitat loss and demand for their parts. Tigers are extirpated in the wild from every country bar one in mainland East and Southeast Asia. Although consumption of tiger products is known to be established in China, less is known about demand for tiger products in Southeast Asia. In this study, we investigate tiger product demand in Vietnam, a major illegal wildlife consumer country. There has been little research into consumption, in particular the level of use, the products being consumed, variation in use of products between areas, and the motivations of consuming tiger products. Through a quantitative survey of 1120 individuals, we show that use of tiger products could be as high as ~11% of the sample in both urban centers of Vietnam, Hanoi and Ho Chi Minh City. Tiger bone glue is the predominant product used, for medicinal purposes. In Hanoi, it is generally purchased by the individual for self-use, while in Ho Chi Minh City it is generally purchased as a gift. In both cities, individuals were generally highly satisfied with the product, indicating entrenched belief in efficacy among consumers. Ultimately, our results show that tiger product use is relatively pervasive. We suggest that conservation organizations should focus on behavior change campaigns that are informed by the results here, and that are specific to each area and to the specific use of tiger product glue for medicine. By reducing demand, beleaguered tiger populations will have a greater chance of stabilization and eventual growth.
Communication sounds are ubiquitous in the animal kingdom, where they play a role in advertising physiological states and/or socio-contextual scenarios. Human screams, for example, are typically uttered in fearful contexts and they have a distinctive feature termed as “roughness”, which depicts amplitude fluctuations at rates from 30–150 Hz. In this article, we report that the occurrence of fast acoustic periodicities in harsh sounding vocalizations is not unique to humans. A roughness-like structure is also present in vocalizations emitted by bats (species Carollia perspicillata) in distressful contexts. We report that 47.7% of distress calls produced by bats carry amplitude fluctuations at rates ~1.7 kHz (>10 times faster than temporal modulations found in human screams). In bats, rough-like vocalizations entrain brain potentials and are more effective in accelerating the bats’ heart rate than slow amplitude modulated sounds. Our results are consistent with a putative role of fast amplitude modulations (roughness in humans) for grabbing the listeners attention in situations in which the emitter is in distressful, potentially dangerous, contexts.
Ataxin-2 (human gene symbol ATXN2) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide–sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters.
Nightly selection of resting sites and group behavior reveal antipredator strategies in giraffe
(2020)
This study presents the first findings on nocturnal behavior patterns of wild Angolan giraffe. We characterized their nocturnal behavior and analyzed the influence of ecological factors such as group size, season, and habitat use. Giraffe were observed using night vision systems and thermal imaging cameras on Okapuka Ranch, Namibia. A total of 77 giraffe were observed during 24 nights over two distinct periods—July–August 2016 (dry season) and February–March 2017 (wet season). Photoperiod had a marked influence on their activity and moving behavior. At dusk, giraffe reduced the time spent moving and increasingly lay down and slept at the onset of darkness. Body postures that likely correspond to rapid eye movement (REM) sleep posture (RSP) were observed 15.8 ± 18.3 min after giraffe sat down. Season had a significant effect with longer RSP phases during the dry season (dry: 155.2 ± 191.1 s, n = 79; wet: 85.8 ± 94.9 s, n = 73). Further analyses of the influence of social behavior patterns did not show an effect of group size on RSP lengths. When a group of giraffe spent time at a specific resting site, several individuals were alert (vigilant) while other group members sat down or took up RSP. Simultaneous RSP events within a group were rarely observed. Resting sites were characterized by single trees or sparse bushes on open areas allowing for good visibility in a relatively sheltered location.
Plants, fungi and algae are important components of global biodiversity and are fundamental to all ecosystems. They are the basis for human well-being, providing food, materials and medicines. Specimens of all three groups of organisms are accommodated in herbaria, where they are commonly referred to as botanical specimens.The large number of specimens in herbaria provides an ample, permanent and continuously improving knowledge base on these organisms and an indispensable source for the analysis of the distribution of species in space and time critical for current and future research relating to global biodiversity. In order to make full use of this resource, a research infrastructure has to be built that grants comprehensive and free access to the information in herbaria and botanical collections in general. This can be achieved through digitization of the botanical objects and associated data.The botanical research community can count on a long-standing tradition of collaboration among institutions and individuals. It agreed on data standards and standard services even before the advent of computerization and information networking, an example being the Index Herbariorum as a global registry of herbaria helping towards the unique identification of specimens cited in the literature.In the spirit of this collaborative history, 51 representatives from 30 institutions advocate to start the digitization of botanical collections with the overall wall-to-wall digitization of the flat objects stored in German herbaria. Germany has 70 herbaria holding almost 23 million specimens according to a national survey carried out in 2019. 87% of these specimens are not yet digitized. Experiences from other countries like France, the Netherlands, Finland, the US and Australia show that herbaria can be comprehensively and cost-efficiently digitized in a relatively short time due to established workflows and protocols for the high-throughput digitization of flat objects.Most of the herbaria are part of a university (34), fewer belong to municipal museums (10) or state museums (8), six herbaria belong to institutions also supported by federal funds such as Leibniz institutes, and four belong to non-governmental organizations. A common data infrastructure must therefore integrate different kinds of institutions.Making full use of the data gained by digitization requires the set-up of a digital infrastructure for storage, archiving, content indexing and networking as well as standardized access for the scientific use of digital objects. A standards-based portfolio of technical components has already been developed and successfully tested by the Biodiversity Informatics Community over the last two decades, comprising among others access protocols, collection databases, portals, tools for semantic enrichment and annotation, international networking, storage and archiving in accordance with international standards. This was achieved through the funding by national and international programs and initiatives, which also paved the road for the German contribution to the Global Biodiversity Information Facility (GBIF).Herbaria constitute a large part of the German botanical collections that also comprise living collections in botanical gardens and seed banks, DNA- and tissue samples, specimens preserved in fluids or on microscope slides and more. Once the herbaria are digitized, these resources can be integrated, adding to the value of the overall research infrastructure. The community has agreed on tasks that are shared between the herbaria, as the German GBIF model already successfully demonstrates.We have compiled nine scientific use cases of immediate societal relevance for an integrated infrastructure of botanical collections. They address accelerated biodiversity discovery and research, biomonitoring and conservation planning, biodiversity modelling, the generation of trait information, automated image recognition by artificial intelligence, automated pathogen detection, contextualization by interlinking objects, enabling provenance research, as well as education, outreach and citizen science.We propose to start this initiative now in order to valorize German botanical collections as a vital part of a worldwide biodiversity data pool.
The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tcey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.
Petroleum products including crude oils and refined distillates are unique environmental pollutants consisting of thousands of compounds with varying physical-chemical properties and resulting toxicity for aquatic biota. Hence, for a reliable risk assessment individual petroleum product toxicity profiles are needed. Furthermore, the influence of oil spill response strategies like the application of chemical dispersants has to be implemented. The present study addressed the toxicity of water-accommodated fractions (WAFs) of two different oil types on fish early life stages on different biological organization levels in the laboratory model species Danio rerio. Experiments with a 3rd generation dispersant used in loading rated resembling the exposure in experiments with chemically dispersed oils were included, enabling a direct comparability of results. This approach is of high importance as especially the investigation of dispersant toxicity in relevant exposure concentrations is rather scarce. Zebrafish embryos were exposed to different WAFs shortly after and up to 120 hour post fertilization (hpf). Besides phenotypic effects including edema and spine deformations, reduced responses to dark stimuli, increased CYP1A activity and marginal AChE inhibition were observed in sublethal effect concentrations. Both oil types had varying strength of toxicity, which did not correlate with corresponding chemical analysis of target PAHs. Chemically dispersed oils induced stronger acute toxicity in zebrafish embryos compared to native (initial) oil exposure, which was further reflected by very low exposure concentrations for biomarker endpoints. Based on a comparison to the dispersant alone, a higher toxicity of dispersed oils was related to a combination of dispersant toxicity and an elevated crude oil compound bioavailability, due to dispersion-related partitioning kinetics. In contrast to LEWAF and CEWAF neither typical morphological effects nor mechanism-specific toxicity were observed for the dispersant alone, indicating narcosis as the responsible cause of effects.
Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein.
Correction to: Scientific Reports https://doi.org/10.1038/srep21848, published online 22 February 2016
This Article contains an error. Among the studied species, the orangutan was erroneously specified as Bornean orangutan Pongo pygmaeus. In fact, the studied individual was a Sumatran orangutan Pongo abelii.
Most mammals rely on the extraction of acoustic information from the environment in order to survive. However, the mechanisms that support sound representation in auditory neural networks involving sensory and association brain areas remain underexplored. In this study, we address the functional connectivity between an auditory region in frontal cortex (the frontal auditory field, FAF) and the auditory cortex (AC) in the bat Carollia perspicillata. The AC is a classic sensory area central for the processing of acoustic information. On the other hand, the FAF belongs to the frontal lobe, a brain region involved in the integration of sensory inputs, modulation of cognitive states, and in the coordination of behavioral outputs. The FAF-AC network was examined in terms of oscillatory coherence (local-field potentials, LFPs), and within an information theoretical framework linking FAF and AC spiking activity. We show that in the absence of acoustic stimulation, simultaneously recorded LFPs from FAF and AC are coherent in low frequencies (1–12 Hz). This “default” coupling was strongest in deep AC layers and was unaltered by acoustic stimulation. However, presenting auditory stimuli did trigger the emergence of coherent auditory-evoked gamma-band activity (>25 Hz) between the FAF and AC. In terms of spiking, our results suggest that FAF and AC engage in distinct coding strategies for representing artificial and natural sounds. Taken together, our findings shed light onto the neuronal coding strategies and functional coupling mechanisms that enable sound representation at the network level in the mammalian brain.
Interspecies hydrogen transfer in anoxic ecosystems is essential for the complete microbial breakdown of organic matter to methane. Acetogenic bacteria are key players in anaerobic food webs and have been considered as prime candidates for hydrogen cycling. We have tested this hypothesis by mutational analysis of the hydrogenase in the model acetogen Acetobacterium woodii. Hydrogenase-deletion mutants no longer grew on H2 + CO2 or organic substrates such as fructose, lactate, or ethanol. Heterotrophic growth could be restored by addition of molecular hydrogen to the culture, indicating that hydrogen is an intermediate in heterotrophic growth. Indeed, hydrogen production from fructose was detected in a stirred-tank reactor. The mutant grew well on organic substrates plus caffeate, an alternative electron acceptor that does not require molecular hydrogen but NADH as reductant. These data are consistent with the notion that molecular hydrogen is produced from organic substrates and then used as reductant for CO2 reduction. Surprisingly, hydrogen cycling in A. woodii is different from the known modes of interspecies or intraspecies hydrogen cycling. Our data are consistent with a novel type of hydrogen cycling that connects an oxidative and reductive metabolic module in one bacterial cell, "intracellular syntrophy."
Gene families evolve by the processes of speciation (creating orthologs), gene duplication (paralogs), and horizontal gene transfer (xenologs), in addition to sequence divergence and gene loss. Orthologs in particular play an essential role in comparative genomics and phylogenomic analyses. With the continued sequencing of organisms across the tree of life, the data are available to reconstruct the unique evolutionary histories of tens of thousands of gene families. Accurate reconstruction of these histories, however, is a challenging computational problem, and the focus of the Quest for Orthologs Consortium. We review the recent advances and outstanding challenges in this field, as revealed at a symposium and meeting held at the University of Southern California in 2017. Key advances have been made both at the level of orthology algorithm development and with respect to coordination across the community of algorithm developers and orthology end-users. Applications spanned a broad range, including gene function prediction, phylostratigraphy, genome evolution, and phylogenomics. The meetings highlighted the increasing use of meta-analyses integrating results from multiple different algorithms, and discussed ongoing challenges in orthology inference as well as the next steps toward improvement and integration of orthology resources.
The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.
The ability to vocalize is ubiquitous in vertebrates, but neural networks underlying vocal control remain poorly understood. Here, we performed simultaneous neuronal recordings in the frontal cortex and dorsal striatum (caudate nucleus, CN) during the production of echolocation pulses and communication calls in bats. This approach allowed us to assess the general aspects underlying vocal production in mammals and the unique evolutionary adaptations of bat echolocation. Our data indicate that before vocalization, a distinctive change in high-gamma and beta oscillations (50–80 Hz and 12–30 Hz, respectively) takes place in the bat frontal cortex and dorsal striatum. Such precise fine-tuning of neural oscillations could allow animals to selectively activate motor programs required for the production of either echolocation or communication vocalizations. Moreover, the functional coupling between frontal and striatal areas, occurring in the theta oscillatory band (4–8 Hz), differs markedly at the millisecond level, depending on whether the animals are in a navigational mode (that is, emitting echolocation pulses) or in a social communication mode (emitting communication calls). Overall, this study indicates that fronto-striatal oscillations could provide a neural correlate for vocal control in bats.
In recent environmental research, relational values (RVs) have emerged as a new group of values to explain environmental behavior. Although this new concept is attracting attention, empirical studies on the subject are still rare. On this basis, we have conducted three studies to analyze an existing measurement tool for RVs and compared the construct with the concept of connection to nature. In study 1, we confirmed convergent and discriminant validity of the RV scale by comparing it with the Two Factor Model of Environmental Values (2-MEV) model using a sample of n = 350 university students. Additionally, study 1 verified reliability using test–retest reliability on three different groups of students (n1 = 53; n2 = 37; n3 = 48). In study 2, principal component analyses were performed to examine the structure of RVs and to compare it to the concept of connection to nature by reusing the sample 350 university students from study 1. The results show that RVs and connection to nature are not fundamentally distinct constructs, but overlap. However, if the structure of the RV measurement is forced to a single factor, no perfect fit is found, making a multidimensional solution more likely. A third study was conducted to review the results from study 2 using confirmatory factor analysis on a new sample of 878 university and high school students. Study 3 confirmed RVs as a multidimensional construct with three factors: care, community, and connection. It also proved the overlap of the connection to nature and RV concepts to some extent.
Zeit ist einer jener Begriffe, für die man die Augustinische Charakterisierung gelten lassen wollte, es sei klar, was sie bedeuten, solange nicht danach gefragt werde (Augustinus Confessiones Lib. XI, 17). Die Frage aber nach dem, was "Zeit" eigentlich ist, erscheint umso berechtigter, als es insbesondere die Naturwissenschaften sind, die für sich in Anspruch nehmen, hier Antworten geben zu können. Die zu erwartenden Antworten wären danach wesentlich empirischer Natur – also direkt oder indirekt experimentell gestützt und mithin Ergebnis dieser Forschung. ...
Communication with the hematopoietic system is a vital component of regulating brain function in health and disease. Traditionally, the major routes considered for this neuroimmune communication are by individual molecules such as cytokines carried by blood, by neural transmission, or, in more severe pathologies, by the entry of peripheral immune cells into the brain. In addition, functional mRNA from peripheral blood can be directly transferred to neurons via extracellular vesicles (EVs), but the parameters that determine their uptake are unknown. Using varied animal models that stimulate neuronal activity by peripheral inflammation, optogenetics, and selective proteasome inhibition of dopaminergic (DA) neurons, we show that the transfer of EVs from blood is triggered by neuronal activity in vivo. Importantly, this transfer occurs not only in pathological stimulation but also by neuronal activation caused by the physiological stimulus of novel object placement. This discovery suggests a continuous role of EVs under pathological conditions as well as during routine cognitive tasks in the healthy brain.
The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. In spite of the rich knowledge about yeast FAS in structure and function, its assembly remained elusive until recently, when co-translational interaction of the β-subunit with the nascent α-subunit was found to initiate assembly. Here, we characterize the co-translational assembly of yeast FAS at a molecular level. We show that the co-translationally formed interface is sensitive to subtle perturbations, so that the exchange of two amino acids located in the emerging interface can prevent assembly. On the other hand, assembly can also be initiated via the co-translational interaction of the subunits at other sites, which implies that this process is not strictly site or sequence specific. We further highlight additional steps in the biogenesis of yeast FAS, as the formation of a dimeric subunit that orchestrates complex formation and acts as platform for post-translational phosphopantetheinylation. The presented data supports the understanding of the recently discovered prevalence of eukaryotic complexes for co-translational assembly, and is valuable for further harnessing FAS in the biotechnological production of aliphatic compounds.
Sorting nexins are a conserved protein family involved in vesicle transport, membrane trafficking and protein sorting. The sorting nexin ATG24/SNX4 has been demonstrated to be involved in different autophagy pathways and in endosomal trafficking. However, its impact on cellular quality control and on aging and development is still elusive. Here we report studies analyzing the function of PaATG24 in the aging model Podospora anserina. Ablation of PaATG24 leads to a reduced growth rate, infertility, and to a pronounced lifespan reduction. These characteristics are accompanied by alterations of the morphology and size distribution of vacuoles and severe impairments in non-selective and selective autophagy of peroxisomes (pexophagy) and mitochondria (mitophagy). While general autophagy and pexophagy are almost completely blocked, a PaATG24-independent form of mitophagy is induced during aging. In the ΔPaAtg24 mutant a strong accumulation of peroxisomes occurs while mitochondrial abundance is only slightly increased. These mitochondria are partially affected in function. Most strikingly, although some PaATG24-independent mitophagy exists, it appears that this is not sufficient to remove dysfunctional mitochondria efficiently enough to prevent premature aging. Overall our data emphasize the key role of mitochondria in aging and of mitophagy in quality control to keep a population of “healthy” mitochondria during aging.
The insertion of membrane proteins requires proteinaceous complexes in the cytoplasm, the membrane, and the lumen of organelles. Most of the required complexes have been described, while the components for insertion of β‐barrel‐type proteins into the outer membrane of chloroplasts remain unknown. The same holds true for the signals required for the insertion of β‐barrel‐type proteins. At present, only the processing of Toc75‐III, the β‐barrel‐type protein of the central chloroplast translocon with an atypical signal, has been explored in detail. However, it has been debated whether Toc75‐V/ outer envelope protein 80 (OEP80), a second protein of the same family, contains a signal and undergoes processing. To substantiate the hypothesis that Toc75‐V/OEP80 is processed as well, we reinvestigated the processing in a protoplast‐based assay as well as in native membranes. Our results confirm the existence of a cleavable segment. By protease protection and pegylation, we observed intermembrane space localization of the soluble N‐terminal domain. Thus, Toc75‐V contains a cleavable N‐terminal signal and exposes its polypeptide transport‐associated domains to the intermembrane space of plastids, where it likely interacts with its substrates.
Makorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3’ UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells we show that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. We observe increased association of the translational repressor Bru1 with osk mRNA upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3’ UTR.
The hydrogen-dependent carbon dioxide reductase is a soluble enzyme complex that directly utilizes hydrogen (H2) for the reduction of carbon dioxide (CO2) to formate in the first step of the acetyl-coenzyme A- or Wood-Ljungdahl pathway (WLP). HDCR consists of 2 catalytic subunits, a hydrogenase and a formate dehydrogenase (FDH) and two small subunits carrying iron-sulfur clusters. The enzyme complex has been purified and characterized from two acetogenic bacteria, from the mesophile Acetobacterium woodii and, recently, from the thermophile Thermoanaerobacter kivui. Physiological studies toward the importance of the HDCR for growth and formate metabolism in acetogens have not been carried out yet, due to the lack of genetic tools. Here, we deleted the genes encoding HDCR in T. kivui taking advantage of the recently developed genetic system. As expected, the deletion mutant (strain TKV_MB013) did not grow with formate as single substrate or under autotrophic conditions with H2 + CO2. Surprisingly, the strain did also not grow on any other substrate (sugars, mannitol or pyruvate), except for when formate was added. Concentrated cell suspensions quickly consumed formate in the presence of glucose only. In conclusion, HDCR provides formate which was essential for growth of the T. kivui mutant. Alternatively, extracellularly added formate served as terminal electron acceptor in addition to CO2, complementing the growth deficiency. The results show a tight coupling of multi-carbon substrate oxidation to the WLP. The metabolism in the mutant can be viewed as a coupled formate + CO2 respiration, which may be an ancient metabolic trait.
Brain metastases are the most common intracranial tumor in adults and are associated with poor patient prognosis and median survival of only a few months. Treatment options for brain metastasis patients remain limited and largely depend on surgical resection, radio- and/or chemotherapy. The development and pre-clinical testing of novel therapeutic strategies require reliable experimental models and diagnostic tools that closely mimic technologies that are used in the clinic and reflect histopathological and biochemical changes that distinguish tumor progression from therapeutic response. In this study, we sought to test the applicability of magnetic resonance (MR) spectroscopy in combination with MR imaging to closely monitor therapeutic efficacy in a breast-to-brain metastasis model. Given the importance of radiotherapy as the standard of care for the majority of brain metastases patients, we chose to monitor the post-irradiation response by magnetic resonance spectroscopy (MRS) in combination with MR imaging (MRI) using a 7 Tesla small animal scanner. Radiation was applied as whole brain radiotherapy (WBRT) using the image-guided Small Animal Radiation Research Platform (SARRP). Here we describe alterations in different metabolites, including creatine and N-acetylaspartate, that are characteristic for brain metastases progression and lactate, which indicates hypoxia, while choline levels remained stable. Radiotherapy resulted in normalization of metabolite levels indicating tumor stasis or regression in response to treatment. Our data indicate that the use of MR spectroscopy in addition to MRI represents a valuable tool to closely monitor not only volumetrical but also metabolic changes during tumor progression and to evaluate therapeutic efficacy of intervention strategies. Adapting the analytical technology in brain metastasis models to those used in clinical settings will increase the translational significance of experimental evaluation and thus contribute to the advancement of pre-clinical assessment of novel therapeutic strategies to improve treatment options for brain metastases patients.
Impact of F1Fo-ATP-synthase dimer assembly factors on mitochondrial function and organismic aging
(2018)
In aerobic organisms, mitochondrial F1Fo-ATP-synthase is the major site of ATP production. Beside this fundamental role, the protein complex is involved in shaping and maintenance of cristae. Previous electron microscopic studies identified the dissociation of F1Fo-ATP-synthase dimers and oligomers during organismic aging correlating with a massive remodeling of the mitochondrial inner membrane. Here we report results aimed to experimentally proof this impact and to obtain further insights into the control of these processes. We focused on the role of the two dimer assembly factors PaATPE and PaATPG of the aging model Podospora anserina. Ablation of either protein strongly affects mitochondrial function and leads to an accumulation of senescence markers demonstrating that the inhibition of dimer formation negatively influences vital functions and accelerates organismic aging. Our data validate a model that links mitochondrial membrane remodeling to aging and identify specific molecular components triggering this process.
Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.
Wie herzig!
(2019)
Doch Vorsicht – dies ist kein Einblick in die Hirnwindungen eines verliebten Teenagers. Vielmehr handelt es sich hier um einen wissenschaftlichen Blick in die Großhirnrinde einer Maus. Die Forscherinnen und Forscher um Prof. Amparo Acker-Palmer vom Buchmann Institut für Molekulare Lebenswissenschaften und dem Institut für Zellbiologie und Neurowissenschaften der Goethe-Universität haben 2018 in der Zeitschrift "Science" darüber berichtet, dass Blutgefäße bei der Entwicklung neuronaler Zellnetzwerke im Gehirn eine bislang unbekannte Rolle spielen ...
Acetylcholine (ACh) is the major excitatory neurotransmitter in the insect central nervous system (CNS). However, besides the neuronal expression of ACh receptors (AChR), the existence of non-neuronal AChR in honeybees is plausible. The cholinergic system is a popular target of insecticides because the pharmacology of insect nicotinic acetylcholine receptors (nAChRs) differs substantially from their vertebrate counterparts. Neonicotinoids are agonists of the nAChR and are largely used in crop protection. In contrast to their relatively high safety for humans and livestock, neonicotinoids pose a threat to pollinating insects such as bees. In addition to its effects on behavior, it becomes increasingly evident that neonicotinoids affect developmental processes in bees that appear to be independent of neuronal AChRs. Brood food (royal jelly, worker jelly, or drone jelly) produced in the hypopharyngeal glands of nurse bees contains millimolar concentrations of ACh, which is required for proper larval development. Neonicotinoids reduce the secreted ACh-content in brood food, reduce hypopharyngeal gland size, and lead to developmental impairments within the colony. We assume that potential hazards of neonicotinoids on pollinating bees occur neuronally causing behavioral impairments on adult individuals, and non-neuronally causing developmental disturbances as well as destroying gland functioning.
Pyridine 2,4-dicarboxylic acid is a structural analog of 2-oxoglutarate and is known to inhibit 2-oxoglutare-dependent dioxygenases. The effect of this inhibitor in tomato seedlings grown in MS media supplied with various concentrations of PDCA was investigated, resulting in shorter roots and hypocotyls in a dose-dependent manner. The partial inhibition of growth in roots was more drastic compared to hypocotyls and was attributed to a decrease in the elongation of root and hypocotyl cells. Concentrations of 100 and 250 μM of PDCA decreased hydroxyproline content in roots while only the 250 μM treatment reduced the hydroxyproline content in shoots. Seedlings treated with 100 μM PDCA exhibited enhanced growth of hypocotyl and cotyledon cells and higher hydroxyproline content resulting in cotyledons with greater surface area. However, no alterations in hypocotyl length were observed. Prolyl 4 hydroxylases (P4Hs) are involved in the O-glycosylation of AGPs and were also highly expressed during seedling growth. Moreover PDCA induced a decrease in the accumulation of HRGPs and particularly in AGPs-bound epitopes in a dose dependent-manner while more drastic reduction were observed in roots compared to shoots. In addition, bulged root epidermal cells were observed at the high concentration of 250 μM which is characteristic of root tissues with glycosylation defects. These results indicate that PDCA induced pleiotropic effects during seedling growth while further studies are required to better investigate the physiological significance of this 2-oxoglutarate analog. This pharmacological approach might be used as a tool to better understand the physiological significance of HRGPs and probably P4Hs in various growth and developmental programs in plants.
Background: Developmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes. In this context, transparent model organisms such as the zebrafish Danio rerio in which developing tissues and organs can be easily observed and imaged using fluorescent proteins have become very popular. One limiting factor however is the acquisition of a sufficient amount of data, in standardized and reproducible conditions, to allow robust quantitative analysis. One way to improve this is by developing mounting methods to increase the number of embryos that can be imaged simultaneously in near-to-identical orientation.
Results: Here we present an improved mounting method allowing semi-automated and high-content imaging of zebrafish embryos. It is based on a 3D-printed stamp which is used to create a 2D coordinate system of multiple μ-wells in an agarose cast. Each μ-well models a negative of the average zebrafish embryo morphology between 22 and 96 h-post-fertilization. Due to this standardized and reproducible arrangement, it is possible to define a custom well plate in the respective imaging software that allows for a semi-automated imaging process. Furthermore, the improvement in Z-orientation significantly reduces post-processing and improves comparability of volumetric data while reducing light exposure and thus photo-bleaching and photo-toxicity, and improving signal-to-noise ratio (SNR).
Conclusions: We present here a new method that allows to standardize and improve mounting and imaging of embryos. The 3D-printed stamp creates a 2D coordinate system of μ-wells in an agarose cast thus standardizing specimen mounting and allowing high-content imaging of up to 44 live or mounted zebrafish embryos simultaneously in a semi-automated, well-plate like manner on inverted confocal microscopes. In summary, image data quality and acquisition efficiency (amount of data per time) are significantly improved. The latter might also be crucial when using the services of a microscopy facility.
Aims: The examination of histological sections is still the gold standard in diagnostic pathology. Important histopathological diagnostic criteria are nuclear shapes and chromatin distribution as well as nucleus-cytoplasm relation and immunohistochemical properties of surface and intracellular proteins. The aim of this investigation was to evaluate the benefits and drawbacks of three-dimensional imaging of CD30+ cells in classical Hodgkin Lymphoma (cHL) in comparison to CD30+ lymphoid cells in reactive lymphoid tissues.
Materials and results: Using immunoflourescence confocal microscopy and computer-based analysis, we compared CD30+ neoplastic cells in Nodular Sclerosis cHL (NScCHL), Mixed Cellularity cHL (MCcHL), with reactive CD30+ cells in Adenoids (AD) and Lymphadenitis (LAD). We confirmed that the percentage of CD30+ cell volume can be calculated. The amount in lymphadenitis was approx. 1.5%, in adenoids around 2%, in MCcHL up to 4,5% whereas the values for NScHL rose to more than 8% of the total cell cytoplasm. In addition, CD30+ tumour cells (HRS-cells) in cHL had larger volumes, and more protrusions compared to CD30+ reactive cells. Furthermore, the formation of large cell networks turned out to be a typical characteristic of NScHL.
Conclusion: In contrast to 2D histology, 3D laser scanning offers a visualisation of complete cells, their network interaction and spatial distribution in the tissue. The possibility to differentiate cells in regards to volume, surface, shape, and cluster formation enables a new view on further diagnostic and biological questions. 3D includes an increased amount of information as a basis of bioinformatical calculations.
Visual aesthetic experiences unfold over time, yet most of our understanding of such experiences comes from experiments using static visual stimuli and measuring static responses. Here, we investigated the temporal dynamics of subjective aesthetic experience using temporally extended stimuli (movie clips) in combination with continuous behavioral ratings. Two groups of participants, a rate group (n = 25) and a view group (n = 25), watched 30-second video clips of landscapes and dance performances in test and retest blocks. The rate group reported continuous ratings while watching the videos, with an overall aesthetic judgment at the end of each video, in both test and retest blocks. The view group, however, passively watched the videos in the test block, reporting only an overall aesthetic judgment at the end of each clip. In the retest block, the view group reported both continuous and overall judgments. When comparing the two groups, we found that the task of making continuous ratings did not influence overall ratings or agreement across participants. In addition, the degree of temporal variation in continuous ratings over time differed substantially by observer (from slower "integrators" to "fast responders"), but less so by video. Reliability of continuous ratings across repeated exposures was in general high, but also showed notable variance across participants. Together, these results show that temporally extended stimuli produce aesthetic experiences that are not the same from person to person, and that continuous behavioral ratings provide a reliable window into the temporal dynamics of such aesthetic experiences while not materially altering the experiences themselves.
Divergent selection between ecologically dissimilar habitats promotes local adaptation, which can lead to reproductive isolation (RI). Populations in the Poecilia mexicana species complex have independently adapted to toxic hydrogen sulfide and show varying degrees of RI. Here, we examined the variation in the mate choice component of prezygotic RI. Mate choice tests across drainages (with stimulus males from another drainage) suggest that specific features of the males coupled with a general female preference for yellow color patterns explain the observed variation. Analyses of male body coloration identified the intensity of yellow fin coloration as a strong candidate to explain this pattern, and common-garden rearing suggested heritable population differences. Male sexual ornamentation apparently evolved differently across sulfide-adapted populations, for example because of differences in natural counterselection via predation. The ubiquitous preference for yellow color ornaments in poeciliid females likely undermines the emergence of strong RI, as female discrimination in favor of own males becomes weaker when yellow fin coloration in the respective sulfide ecotype increases. Our study illustrates the complexity of the (partly non-parallel) pathways to divergence among replicated ecological gradients. We suggest that future work should identify the genomic loci involved in the pattern reported here, making use of the increasing genomic and transcriptomic datasets available for our study system.
Cell–matrix adhesion and cell migration are physiologically important processes that also play a major role in cancer spreading. In cultured cells, matrix adhesion depends on integrin-containing contacts such as focal adhesions. Flotillin-1 and flotillin-2 are frequently overexpressed in cancers and are associated with poor survival. Our previous studies have revealed a role for flotillin-2 in cell–matrix adhesion and in the regulation of the actin cytoskeleton. We here show that flotillins are important for cell migration in a wound healing assay and influence the morphology and dynamics of focal adhesions. Furthermore, anchorage-independent growth in soft agar is enhanced by flotillins. In the absence of flotillins, especially flotillin-2, phosphorylation of focal adhesion kinase and extracellularly regulated kinase is diminished. Flotillins interact with α-actinin, a major regulator of focal adhesion dynamics. These findings are important for understanding the molecular mechanisms of how flotillin overexpression in cancers may affect cell migration and, especially, enhance metastasis formation.
Bone remodeling can be mimicked in vitro by co-culture models. Based on bone cells, such co-cultures help to study synergistic morphological changes and the impact of materials and applied substances. Hence, we examined the formation of osteoclasts on bovine bone materials to prove the bone resorption functionality of the osteoclasts in three different co-culture set-ups using human monocytes (hMCs) and (I) human mesenchymal stem cells (hMSCs), (II) osteogenic differentiated hMSCs (hOBs), and (III) hOBs in addition of soluble monocyte-colony stimulating factor (M-CSF) and cytokine receptor activator of NFκB ligand (RANKL). We detected osteoclast-specific actin morphology, as well as the expression of cathepsin K and CD51/61 in single cells in set-up II and in numerous cells in set-up III. Resorption pits on bone material as characteristic proof of functional osteoclasts were not found in set-up I and II, but we detected such resorption pits in set–up III. We conclude in co-culture models without M-CSF and RANKL that monocytes can differentiate into osteoclasts that show the characteristic actin structures and protein expression. However, to receive functional bone resorbing osteoclasts in vitro, the addition of M-CSF and RANKL is needed. Moreover, we suggest the use of bone or bone-like materials for future studies evaluating osteoclastogenesis.
In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity.
Volatile organic compounds are secondary metabolites emitted by all organisms, especially by plants and microbes. Their role as aboveground signals has been established for decades. Recent evidence suggests that they might have a non-negligible role belowground and might be involved in root–root and root–microbial/pest interactions. Our aim here was to make a comprehensive review of belowground volatile diversity using a meta-analysis approach. At first we synthesized current literature knowledge on plant root volatiles and classified them in terms of chemical diversity. In a second step, relying on the mVOC database of microbial volatiles, we classified volatiles based on their emitters (bacteria vs. fungi) and their specific ecological niche (i.e., rhizosphere, soil). Our results highlight similarities and differences among root and microbial volatiles and also suggest that some might be niche specific. We further explored the possibility that volatiles might be involved in intra- and inter-specific root–root communication and discuss the ecological implications of such scenario. Overall this work synthesizes current knowledge on the belowground volatilome and the potential signaling role of its constituents. It also highlights that the total diversity of belowground volatiles might be orders of magnitude larger that the few hundreds of compounds described to date.
Symbiotic nitrogen fixation (SNF) in root nodules of grain legumes such as chickpea is a highly complex process that drastically affects the gene expression patterns of both the prokaryotic as well as eukaryotic interacting cells. A successfully established symbiotic relationship requires mutual signaling mechanisms and a continuous adaptation of the metabolism of the involved cells to varying environmental conditions. Although some of these processes are well understood today many of the molecular mechanisms underlying SNF, especially in chickpea, remain unclear. Here, we reannotated our previously published transcriptome data generated by deepSuperSAGE (Serial Analysis of Gene Expression) to the recently published draft genome of chickpea to assess the root- and nodule-specific transcriptomes of the eukaryotic host cells. The identified gene expression patterns comprise up to 71 significantly differentially expressed genes and the expression of twenty of these was validated by quantitative real-time PCR with the tissues from five independent biological replicates. Many of the differentially expressed transcripts were found to encode proteins implicated in sugar metabolism, antioxidant defense as well as biotic and abiotic stress responses of the host cells, and some of them were already known to contribute to SNF in other legumes. The differentially expressed genes identified in this study represent candidates that can be used for further characterization of the complex molecular mechanisms underlying SNF in chickpea.
Along with barley and rice, maize provides staple food for more than half of the world population. Maize ears are regularly infected with fungal pathogens of the Fusarium genus, which, besides reducing yield, also taint grains with toxic metabolites. In an earlier work, we have shown that maize ears infection with single Fusarium strains was detectable through volatile sensing. In nature, infection most commonly occurs with more than a single fungal strain; hence we tested how the interactions of two strains would modulate volatile emission from infected ears. For this purpose, ears of a hybrid and a dwarf maize variety were simultaneously infected with different strains of Fusarium graminearum and F. verticillioides and, the resulting volatile profiles were compared to the ones of ears infected with single strains. Disease severity, fungal biomass, and the concentration of the oxylipin 9-hydroxy octadecadienoic acid, a signaling molecule involved in plant defense, were monitored and correlated to volatile profiles. Our results demonstrate that in simultaneous infections of hybrid and dwarf maize, the most competitive fungal strains had the largest influence on the volatile profile of infected ears. In both concurrent and single inoculations, volatile profiles reflected disease severity. Additionally, the data further indicate that dwarf maize and hybrid maize might emit common (i.e., sesquiterpenoids) and specific markers upon fungal infection. Overall this suggests that volatile profiles might be a good proxy for disease severity regardless of the fungal competition taking place in maize ears. With the appropriate sensitivity and reliability, volatile sensing thus appears as a promising tool for detecting fungal infection of maize ears under field conditions.
Circular RNAs (circRNAs), an important class of regulatory RNAs, have been shown to be the most prevalent in the brain compared with other tissues. However the processes governing their biogenesis in neurons are still elusive. Moreover, little is known about whether and how different biogenesis factors work in synchrony to generate neuronal circRNAs. To address this question, we pharmacologically inhibited the spliceosome and profiled rat neuronal circRNAs using RNA sequencing. We identified over 100 circRNAs that were up-regulated and a few circRNAs that were down-regulated upon spliceosome inhibition. Bioinformatic analysis revealed that up-regulated circRNAs possess significantly longer flanking introns compared with the un-changed circRNA population. Moreover, the flanking introns of up-regulated circRNAs harbor a higher number of distinct repeat sequences and more reverse complementary motifs compared with the unchanged circRNAs. Taken together, our data demonstrate that the biogenesis of circRNAs containing distinct intronic features becomes favored under conditions of limited spliceosome activity.
The identification of heat stress (HS)-resilient germplasm is important to ensure food security under less favorable environmental conditions. For that, germplasm with an altered activity of factors regulating the HS response is an important genetic tool for crop improvement. Heat shock binding protein (HSBP) is one of the main negative regulators of HS response, acting as a repressor of the activity of HS transcription factors. We identified a TILLING allele of Solanum lycopersicum (tomato) HSBP1. We examined the effects of the mutation on the functionality of the protein in tomato protoplasts, and compared the thermotolerance capacity of lines carrying the wild-type and mutant alleles of HSBP1. The methionine-to-isoleucine mutation in the central heptad repeats of HSBP1 leads to a partial loss of protein function, thereby reducing the inhibitory effect on Hsf activity. Mutant seedlings show enhanced basal thermotolerance, while mature plants exhibit increased resilience in repeated HS treatments, as shown by several physiological parameters. Importantly, plants that are homozygous for the wild-type or mutant HSBP1 alleles showed no significant differences under non-stressed conditions. Altogether, these results indicate that the identified mutant HSBP1 allele can be used as a genetic tool in breeding, aiming to improve the thermotolerance of tomato varieties.
Truffle fungi are well known for their enticing aromas partially emitted by microbes colonizing truffle fruiting bodies. The identity and diversity of these microbes remain poorly investigated, because few studies have determined truffle-associated bacterial communities while considering only a small number of fruiting bodies. Hence, the factors driving the assembly of truffle microbiomes are yet to be elucidated. Here we investigated the bacterial community structure of more than 50 fruiting bodies of the black truffle Tuber aestivum in one French and one Swiss orchard using 16S rRNA gene amplicon high-throughput sequencing. Bacterial communities from truffles collected in both orchards shared their main dominant taxa: while 60% of fruiting bodies were dominated by α-Proteobacteria, in some cases the β-Proteobacteria or the Sphingobacteriia classes were the most abundant, suggesting that specific factors (i.e., truffle maturation and soil properties) shape differently truffle-associated microbiomes. We further attempted to assess the influence in truffle microbiome variation of factors related to collection season, truffle mating type, degree of maturation, and location within the truffle orchards. These factors had differential effects between the two truffle orchards, with season being the strongest predictor of community variation in the French orchard, and spatial location in the Swiss one. Surprisingly, genotype and fruiting body maturation did not have a significant effect on microbial community composition. In summary, our results show, regardless of the geographical location considered, the existence of heterogeneous bacterial communities within T. aestivum fruiting bodies that are dominated by three bacterial classes. They also indicate that factors shaping microbial communities within truffle fruiting bodies differ across local conditions.
This review summarizes studies of protection against singlet oxygen and radical damage by carotenoids. The main focus is on how substitutions of the carotenoid molecules determine high antioxidant activities such as singlet oxygen quenching and radical scavenging. Applied assays were carried out either in vitro in solvents or with liposomes, and in a few cases with living organisms. In the latter, protection by carotenoids especially of photosynthesis against light- and UV-stress is of major importance, but also heterotrophic organisms suffer from high light and UV exposure which can be alleviated by carotenoids. Carotenoids to be compared include C30, C40 and C50 molecules either acyclic, monocyclic or bicyclic with different substitutions including sugar and fatty acid moieties. Although some studies are difficult to compare, there is a tendency towards mono and bicyclic carotenoids with keto groups at C-4/C-4’ and the longest possible polyene structure functions to act best in singlet oxygen quenching and radical scavenging. Size of the carotenoid and lipophilic substituents such as fatty acids seem to be of minor importance for their activity but hydroxyl groups at an acyclic end and especially glycosylation of these hydroxyl groups enhance carotenoid activity.
Hereditary Parkinson’s disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCAA53T in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As an exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension, and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was also detected for other microtubule and microfilament factors, namely MAP2S1801, MARK1S394, MAP1AT1794, KIF1AS1537, 4.1NS541, 4.1GS86, and ADD2S528. While the MAP1B heavy chain supports regeneration and growth cones, its light chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2S299, VPS13DS2429, and VPS13CS2480 in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in Drosophila melanogaster that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly, ANK2 gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.
The haloarchaeon Haloferax volcanii contains nearly 2800 small non-coding RNAs (sRNAs). One intergenic sRNA, sRNA132, was chosen for a detailed characterization. A deletion mutant had a growth defect and thus underscored the importance of sRNA132. A microarray analysis identified the transcript of an operon for a phosphate-specific ABC transporter as a putative target of sRNA132. Both the sRNA132 and the operon transcript accumulated under low phosphate concentrations, indicating a positive regulatory role of sRNA132. A kinetic analysis revealed that sRNA132 is essential shortly after the onset of phosphate starvation, while other regulatory processes take over after several hours. Comparison of the transcriptomes of wild-type and the sRNA132 gene deletion mutant 30 min after the onset of phosphate starvation revealed that sRNA132 controls a regulon of about 40 genes. Remarkably, the regulon included a second operon for a phosphate-specific ABC transporter, which also depended on sRNA132 for rapid induction in the absence of phosphate. Competitive growth experiments of the wild-type and ABC transporter operon deletion mutants underscored the importance of both transporters for growth at low phosphate concentrations. Northern blot analyses of four additional members of the sRNA132 regulon verified that all four transcripts depended on sRNA132 for rapid regulation after the onset of phosphate starvation. Importantly, this is the first example for the transient importance of a sRNA for any archaeal and bacterial species. In addition, this study unraveled the first sRNA regulon for haloarchaea.
As a flavor and platform chemical, m-cresol (3-methylphenol) is a valuable industrial compound that currently is mainly synthesized by chemical methods from fossil resources. In this study, we present the first biotechnological de novo production of m-cresol from sugar in complex yeast extract-peptone medium with the yeast Saccharomyces cerevisiae. A heterologous pathway based on the decarboxylation of the polyketide 6-methylsalicylic acid (6-MSA) was introduced into a CEN.PK yeast strain. For synthesis of 6-MSA, expression of different variants of 6-MSA synthases (MSASs) were compared. Overexpression of codon-optimized MSAS from Penicillium patulum together with activating phosphopantetheinyl transferase npgA from Aspergillus nidulans resulted in up to 367 mg/L 6-MSA production. Additional genomic integration of the genes had a strongly promoting effect and 6-MSA titers reached more than 2 g/L. Simultaneous expression of 6-MSA decarboxylase patG from A. clavatus led to the complete conversion of 6-MSA and production of up to 589 mg/L m-cresol. As addition of 450–750 mg/L m-cresol to yeast cultures nearly completely inhibited growth our data suggest that the toxicity of m-cresol might be the limiting factor for higher production titers.
Mapping biodiversity is the marathon of the 21st Century as an answer to the present extinction crisis. A century in which science is also characterised by large scientific datasets collected through new technologies aiming to fill gaps in our knowledge of species distributions. However, most species records rely on observations that are not linked to specimens, which does not allow verification of species hypotheses by other scientists. Natural history museums form a verifiable source of biodiversity records which were made by taxonomists. Nonetheless, these museums seem to be forgotten by biologists in scientific fields other than taxonomy or systematics. Naturalis Biodiversity Center (NBC) in Leiden is care keeper of large collections of marine organisms, which were sampled in the Northeast Atlantic during the CANCAP and Tyro Mauritania II expeditions (1976–1988). Many octocorals were sampled and deposited in the NBC collection, where they became available for study and were partially identified by the senior author (M.G.) in the 1980s. Nonetheless, no checklist or taxonomic revision was published so far with the complete results. In 2016 the first author visited NBC to examine NE Atlantic Plexauridae octocorals. Plexauridae octocoral-vouchered records were listed and mapped to reveal high standard primary biodiversity records unreported so far for the NE Atlantic Ocean. Twenty-four Plexauridae species with ~ six putative new species to science were discovered and eleven new biogeographical records were made from distinct Macaronesian archipelagos. Finally, new depth range records were found for three species at sea basin level and for eight species at a regional scale.
In homeostatic scaling at central synapses, the depth and breadth of cellular mechanisms that detect the offset from the set-point, detect the duration of the offset and implement a cellular response are not well understood. To understand the time-dependent scaling dynamics we treated cultured rat hippocampal cells with either TTX or bicucculline for 2 hr to induce the process of up- or down-scaling, respectively. During the activity manipulation we metabolically labeled newly synthesized proteins using BONCAT. We identified 168 newly synthesized proteins that exhibited significant changes in expression. To obtain a temporal trajectory of the response, we compared the proteins synthesized within 2 hr or 24 hr of the activity manipulation. Surprisingly, there was little overlap in the significantly regulated newly synthesized proteins identified in the early- and integrated late response datasets. There was, however, overlap in the functional categories that are modulated early and late. These data indicate that within protein function groups, different proteomic choices can be made to effect early and late homeostatic responses that detect the duration and polarity of the activity manipulation.
The nuclear exosome and its essential co-factor, the RNA helicase MTR4, play crucial roles in several RNA degradation pathways. Besides unwinding RNA substrates for exosome-mediated degradation, MTR4 associates with RNA-binding proteins that function as adaptors in different RNA processing and decay pathways. Here, we identify and characterize the interactions of human MTR4 with a ribosome processing adaptor, NVL, and with ZCCHC8, an adaptor involved in the decay of small nuclear RNAs. We show that the unstructured regions of NVL and ZCCHC8 contain short linear motifs that bind the MTR4 arch domain in a mutually exclusive manner. These short sequences diverged from the arch-interacting motif (AIM) of yeast rRNA processing factors. Our results suggest that nuclear exosome adaptors have evolved canonical and non-canonical AIM sequences to target human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA-binding proteins.
Nanoplastics (NP) and microplastics (MP) accumulate in our environment as a consequence of the massive consumption of plastics. Huge knowledge-gaps exist regarding uptake and fate of plastic particles in micro- and nano-dimensions in humans as well as on their impact on human health.
This study investigated the transport and effects of 50 nm and 0.5 μm COOH-modified polystyrene (PS) particles, as representatives for NP and MP, in different biological models in vitro. Acute toxicity and potential translocation of the particles were studied at the human intestinal and placental barrier using advanced in vitro co-culture models. Furthermore, embryotoxicity and genotoxicity were investigated as highly sensitive endpoints.
Polystyrene was not acutely toxic in both sizes (nano- and microparticles). No transport across the intestinal and placental barrier but a cellular uptake and intracellular accumulation of PS nano- and microparticles were determined. The particles were identified as weak embryotoxic and non-genotoxic.
In contrast to single-organ studies, this multi-endpoint study is providing a data-set with the exact same type of particles to compare organ-specific outcomes. Our study clearly shows the need to investigate other types of plastics as well as towards long-term or chronic effects of plastic particles in different biological models in vitro.
The website Sci-Hub enables users to download PDF versions of scholarly articles, including many articles that are paywalled at their journal’s site. Sci-Hub has grown rapidly since its creation in 2011, but the extent of its coverage was unclear. Here we report that, as of March 2017, Sci-Hub’s database contains 68.9% of the 81.6 million scholarly articles registered with Crossref and 85.2% of articles published in toll access journals. We find that coverage varies by discipline and publisher and that Sci-Hub preferentially covers popular, paywalled content. For toll access articles, green open access via licit services is quite limited, while Sci-Hub provides greater coverage than a major research university. Our interactive browser at https://greenelab.github.io/scihub allows users to explore these findings in more detail. For the first time, nearly all scholarly literature is available gratis to anyone with an Internet connection, suggesting the toll access business model will become unsustainable.
The website Sci-Hub provides access to scholarly literature via full text PDF downloads. The site enables users to access articles that would otherwise be paywalled. Since its creation in 2011, Sci-Hub has grown rapidly in popularity. However, until now, the extent of Sci-Hub's coverage was unclear. As of March 2017, we find that Sci-Hub's database contains 68.9% of all 81.6 million scholarly articles, which rises to 85.2% for those published in closed access journals. Furthermore, Sci-Hub contains 77.0% of the 5.2 million articles published by inactive journals. Coverage varies by discipline, with 92.8% coverage of articles in chemistry journals compared to 76.3% for computer science. Coverage also varies by publisher, with the coverage of the largest publisher, Elsevier, at 97.3%. Our interactive browser at https://greenelab.github.io/scihub allows users to explore these findings in more detail. Finally, we estimate that over a six-month period in 2015–2016, Sci-Hub provided access for 99.3% of valid incoming requests. Hence, the scope of this resource suggests the subscription publishing model is becoming unsustainable. For the first time, the overwhelming majority of scholarly literature is available gratis to anyone with an Internet connection.
The website Sci-Hub enables users to download PDF versions of scholarly articles, including many articles that are paywalled at their journal’s site. Sci-Hub has grown rapidly since its creation in 2011, but the extent of its coverage has been unclear. Here we report that, as of March 2017, Sci-Hub’s database contains 68.9% of the 81.6 million scholarly articles registered with Crossref and 85.1% of articles published in toll access journals. We find that coverage varies by discipline and publisher, and that Sci-Hub preferentially covers popular, paywalled content. For toll access articles, we find that Sci-Hub provides greater coverage than the University of Pennsylvania, a major research university in the United States. Green open access to toll access articles via licit services, on the other hand, remains quite limited. Our interactive browser at https://greenelab.github.io/scihub allows users to explore these findings in more detail. For the first time, nearly all scholarly literature is available gratis to anyone with an Internet connection, suggesting the toll access business model may become unsustainable.
As adapter molecules to convert the nucleic acid information into the amino acid sequence, tRNAs play a central role in protein synthesis. To fulfill this function in a reliable way, tRNAs exhibit highly conserved structural features common in all organisms and in all cellular compartments active in translation. However, in mitochondria of metazoans, certain dramatic deviations from the consensus tRNA structure are described, where some tRNAs lack the D- or T-arm without losing their function. In Enoplea, this miniaturization comes to an extreme, and functional mitochondrial tRNAs can lack both arms, leading to a considerable size reduction. Here, we investigate the secondary and tertiary structure of two such armless tRNAs from Romanomermis culicivorax. Despite their high AU content, the transcripts fold into a single and surprisingly stable hairpin structure, deviating from standard tRNAs. The three-dimensional form is boomerang-like and diverges from the standard L-shape. These results indicate that such unconventional miniaturized tRNAs can still fold into a tRNA-like shape, although their length and secondary structure are very unusual. They highlight the remarkable flexibility of the protein synthesis apparatus and suggest that the translational machinery of Enoplea mitochondria may show compensatory adaptations to accommodate these armless tRNAs for efficient translation.
Microenvironmental regulation of tumor progression and therapeutic response in brain metastasis
(2019)
Cellular and non-cellular components of the tumor microenvironment (TME) are emerging as key regulators of primary tumor progression, organ-specific metastasis, and therapeutic response. In the era of TME-targeted- and immunotherapies, cancer-associated inflammation has gained increasing attention. In this regard, the brain represents a unique and highly specialized organ. It has long been regarded as an immunological sanctuary site where the presence of the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCB) restricts the entry of immune cells from the periphery. Consequently, tumor cells that metastasize to the brain were thought to be shielded from systemic immune surveillance and destruction. However, the detailed characterization of the immune landscape within border-associated areas of the central nervous system (CNS), such as the meninges and the choroid plexus, as well as the discovery of lymphatics and channels that connect the CNS with the periphery, have recently challenged the dogma of the immune privileged status of the brain. Moreover, the presence of brain metastases (BrM) disrupts the integrity of the BBB and BCB. Indeed, BrM induce the recruitment of different immune cells from the myeloid and lymphoid lineage to the CNS. Blood-borne immune cells together with brain-resident cell-types, such as astrocytes, microglia, and neurons, form a highly complex and dynamic TME that affects tumor cell survival and modulates the mode of immune responses that are elicited by brain metastatic tumor cells. In this review, we will summarize recent findings on heterotypic interactions within the brain metastatic TME and highlight specific functions of brain-resident and recruited cells at different rate-limiting steps of the metastatic cascade. Based on the insight from recent studies, we will discuss new opportunities and challenges for TME-targeted and immunotherapies for BrM.
The neomycin sensing riboswitch is the smallest biologically functional RNA riboswitch, forming a hairpin capped with a U-turn loop—a well-known RNA motif containing a conserved uracil. It was shown previously that a U→C substitution of the eponymous conserved uracil does not alter the riboswitch structure due to C protonation at N3. Furthermore, cytosine is evolutionary permitted to replace uracil in other U-turns. Here, we use molecular dynamics simulations to study the molecular basis of this substitution in the neomycin sensing riboswitch and show that a structure-stabilizing monovalent cation-binding site in the wild-type RNA is the main reason for its negligible structural effect. We then use NMR spectroscopy to confirm the existence of this cation-binding site and to demonstrate its effects on RNA stability. Lastly, using quantum chemical calculations, we show that the cation-binding site is altering the electronic environment of the wild-type U-turn so that it is more similar to the cytosine mutant. The study reveals an amazingly complex and delicate interplay between various energy contributions shaping up the 3D structure and evolution of nucleic acids.
Altered microRNA (miRNA) expression is a hallmark of many cancer types. The combined analysis of miRNA and messenger RNA (mRNA) expression profiles is crucial to identifying links between deregulated miRNAs and oncogenic pathways. Therefore, we investigated the small non-coding (snc) transcriptomes of nine clear cell renal cell carcinomas (ccRCCs) and adjacent normal tissues for alterations in miRNA expression using a publicly available small RNA-Sequencing (sRNA-Seq) raw-dataset. We constructed a network of deregulated miRNAs and a set of differentially expressed genes publicly available from an independent study to in silico determine miRNAs that contribute to clear cell renal cell carcinogenesis. From a total of 1,672 sncRNAs, 61 were differentially expressed across all ccRCC tissue samples. Several with known implications in ccRCC development, like the upregulated miR-21-5p, miR-142-5p, as well as the downregulated miR-106a-5p, miR-135a-5p, or miR-206. Additionally, novel promising candidates like miR-3065, which i.a. targets NRP2 and FLT1, were detected in this study. Interaction network analysis revealed pivotal roles for miR-106a-5p, whose loss might contribute to the upregulation of 49 target mRNAs, miR-135a-5p (32 targets), miR-206 (28 targets), miR-363-3p (22 targets), and miR-216b (13 targets). Among these targets are the angiogenesis, metastasis, and motility promoting oncogenes c-MET, VEGFA, NRP2, and FLT1, the latter two coding for VEGFA receptors.
The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers) and the remaining part by caldarchaeol lipids (tetraethers). The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.
A large number of staphylinid beetles are closely associated with ants and termites (for review see Wilson 1971, Kistner 1979). Those species living with ants are commonly called myremcophiles. At least a few (Atemeles, Lomechusa) have "broken" the communication code of their host species and are thereby able to become completely integrated in the social system of the ants (Hölldolber 1967, 1970, 1971). In an attempt to understand the evolutionary pathways of this highly specialized social parasitic behavior, we studied closely related staphylinid species that do not live within the ant society but instead occupy the foraging trails and garbage dumps of an ant nest. ...
Background: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily.
Results: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic.
Conclusions: The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.
Unique features of a global human ectoparasite identified through sequencing of the bed bug genome
(2016)
The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.
Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility.
Zinc finger domains are highly structured and can mediate interactions to DNA, RNA, proteins, lipids, and small molecules. Accordingly, zinc finger proteins are very versatile and involved in many biological functions. Eukaryotes contain a wealth of zinc finger proteins, but zinc finger proteins have also been found in archaea and bacteria. Large zinc finger proteins have been well studied, however, in stark contrast, single domain zinc finger µ-proteins of less than 70 amino acids have not been studied at all, with one single exception. Therefore, 16 zinc finger µ-proteins of the haloarchaeon Haloferax volcanii were chosen and in frame deletion mutants of the cognate genes were generated. The phenotypes of mutants and wild-type were compared under eight different conditions, which were chosen to represent various pathways and involve many genes. None of the mutants differed from the wild-type under optimal or near-optimal conditions. However, 12 of the 16 mutants exhibited a phenotypic difference under at least one of the four following conditions: Growth in synthetic medium with glycerol, growth in the presence of bile acids, biofilm formation, and swarming. In total, 16 loss of function and 11 gain of function phenotypes were observed. Five mutants indicated counter-regulation of a sessile versus a motile life style in H. volcanii. In conclusion, the generation and analysis of a set of deletion mutants demonstrated the high importance of zinc finger µ-proteins for various biological functions, and it will be the basis for future mechanistic insight.
Tuberaceae is one of the most diverse lineages of symbiotic truffle-forming fungi. To understand the molecular underpinning of the ectomycorrhizal truffle lifestyle, we compared the genomes of Piedmont white truffle (Tuber magnatum), Périgord black truffle (Tuber melanosporum), Burgundy truffle (Tuber aestivum), pig truffle (Choiromyces venosus) and desert truffle (Terfezia boudieri) to saprotrophic Pezizomycetes. Reconstructed gene duplication/loss histories along a time-calibrated phylogeny of Ascomycetes revealed that Tuberaceae-specific traits may be related to a higher gene diversification rate. Genomic features in Tuber species appear to be very similar, with high transposon content, few genes coding lignocellulose-degrading enzymes, a substantial set of lineage-specific fruiting-body-upregulated genes and high expression of genes involved in volatile organic compound metabolism. Developmental and metabolic pathways expressed in ectomycorrhizae and fruiting bodies of T. magnatum and T. melanosporum are unexpectedly very similar, owing to the fact that they diverged ~100 Ma. Volatile organic compounds from pungent truffle odours are not the products of Tuber-specific gene innovations, but rely on the differential expression of an existing gene repertoire. These genomic resources will help to address fundamental questions in the evolution of the truffle lifestyle and the ecology of fungi that have been praised as food delicacies for centuries.
Drebrin (DBN) regulates cytoskeletal functions during neuronal development, and is thought to contribute to structural and functional synaptic changes associated with aging and Alzheimer’s disease. Here we show that DBN coordinates stress signalling with cytoskeletal dynamics, via a mechanism involving kinase ataxia-telangiectasia mutated (ATM). An excess of reactive oxygen species (ROS) stimulates ATM-dependent phosphorylation of DBN at serine-647, which enhances protein stability and accounts for improved stress resilience in dendritic spines. We generated a humanized DBN Caenorhabditis elegans model and show that a phospho-DBN mutant disrupts the protective ATM effect on lifespan under sustained oxidative stress. Our data indicate a master regulatory function of ATM-DBN in integrating cytosolic stress-induced signalling with the dynamics of actin remodelling to provide protection from synapse dysfunction and ROS-triggered reduced lifespan. They further suggest that DBN protein abundance governs actin filament stability to contribute to the consequences of oxidative stress in physiological and pathological conditions.
Cyanobacteria are photoautotrophic microorganisms present in almost all ecologically niches on Earth. They exist as single-cell or filamentous forms and the latter often contain specialized cells for N2 fixation known as heterocysts. Heterocysts arise from photosynthetic active vegetative cells by multiple morphological and physiological rearrangements including the absence of O2 evolution and CO2 fixation. The key function of this cell type is carried out by the metalloprotein complex known as nitrogenase. Additionally, many other important processes in heterocysts also depend on metalloproteins. This leads to a high metal demand exceeding the one of other bacteria in content and concentration during heterocyst development and in mature heterocysts. This review provides an overview on the current knowledge of the transition metals and metalloproteins required by heterocysts in heterocyst-forming cyanobacteria. It discusses the molecular, physiological, and physicochemical properties of metalloproteins involved in N2 fixation, H2 metabolism, electron transport chains, oxidative stress management, storage, energy metabolism, and metabolic networks in the diazotrophic filament. This provides a detailed and comprehensive picture on the heterocyst demands for Fe, Cu, Mo, Ni, Mn, V, and Zn as cofactors for metalloproteins and highlights the importance of such metalloproteins for the biology of cyanobacterial heterocysts.
Anaerobic ammonium oxidation (anammox) is a major process in the biogeochemical nitrogen cycle in which nitrite and ammonium are converted to dinitrogen gas and water through the highly reactive intermediate hydrazine. So far, it is unknown how anammox organisms convert the toxic hydrazine into nitrogen and harvest the extremely low potential electrons (−750 mV) released in this process. We report the crystal structure and cryo electron microscopy structures of the responsible enzyme, hydrazine dehydrogenase, which is a 1.7 MDa multiprotein complex containing an extended electron transfer network of 192 heme groups spanning the entire complex. This unique molecular arrangement suggests a way in which the protein stores and releases the electrons obtained from hydrazine conversion, the final step in the globally important anammox process.
Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5’-UTRs and with very long 3’-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3’-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome.
Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.
The mechanistic target of rapamycin (mTOR) is elevated in prostate cancer, making this protein attractive for tumor treatment. Unfortunately, resistance towards mTOR inhibitors develops and the tumor becomes reactivated. We determined whether epigenetic modulation by the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), may counteract non-responsiveness to the mTOR inhibitor, temsirolimus, in prostate cancer (PCa) cells. Prostate cancer cells, sensitive (parental) and resistant to temsirolimus, were exposed to VPA, and tumor cell growth behavior compared. Temsirolimus resistance enhanced the number of tumor cells in the G2/M-phase, correlating with elevated cell proliferation and clonal growth. The cell cycling proteins cdk1 and cyclin B, along with Akt-mTOR signaling increased, whereas p19, p21 and p27 decreased, compared to the parental cells. VPA significantly reduced cell growth and up-regulated the acetylated histones H3 and H4. Cdk1 and cyclin B decreased, as did phosphorylated mTOR and the mTOR sub-complex Raptor. The mTOR sub-member Rictor and phosphorylated Akt increased under VPA. Knockdown of cdk1, cyclin B, or Raptor led to significant cell growth reduction. HDAC inhibition through VPA counteracts temsirolimus resistance, probably by down-regulating cdk1, cyclin B and Raptor. Enhanced Rictor and Akt, however, may represent an undesired feedback loop, which should be considered when designing future therapeutic regimens.
Orthologs document the evolution of genes and metabolic capacities encoded in extant and ancient genomes. However, the similarity between orthologs decays with time, and ultimately it becomes insufficient to infer common ancestry. This leaves ancient gene set reconstructions incomplete and distorted to an unknown extent. Here we introduce the "evolutionary traceability" as a measure that quantifies, for each protein, the evolutionary distance beyond which the sensitivity of the ortholog search becomes limiting. Using yeast, we show that genes that were thought to date back to the last universal common ancestor are of high traceability. Their functions mostly involve catalysis, ion transport, and ribonucleoprotein complex assembly. In turn, the fraction of yeast genes whose traceability is not sufficient to infer their presence in last universal common ancestor is enriched for regulatory functions. Computing the traceabilities of genes that have been experimentally characterized as being essential for a self-replicating cell reveals that many of the genes that lack orthologs outside bacteria have low traceability. This leaves open whether their orthologs in the eukaryotic and archaeal domains have been overlooked. Looking at the example of REC8, a protein essential for chromosome cohesion, we demonstrate how a traceability-informed adjustment of the search sensitivity identifies hitherto missed orthologs in the fast-evolving microsporidia. Taken together, the evolutionary traceability helps to differentiate between true absence and nondetection of orthologs, and thus improves our understanding about the evolutionary conservation of functional protein networks. "protTrace," a software tool for computing evolutionary traceability, is freely available at https://github.com/BIONF/protTrace.git; last accessed February 10, 2019.
Synthesis and SAR of the antistaphylococcal natural product nematophin from Xenorhabdus nematophila
(2019)
The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead structures are needed. Here, the synthesis and an expanded structure–activity relationship of the simple and antistaphylococcal amide nematophin from Xenorhabdus nematophila and synthetic derivatives are described. Moreover, the synthesis of intrinsic fluorescent derivatives, incorporating azaindole moieties was achieved for the first time.
Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies.
Numerous cell–cell and cell–matrix interactions within the bone marrow microenvironment enable the controlled lifelong self-renewal and progeny of hematopoietic stem and progenitor cells (HSPCs). On the cellular level, this highly mutual interaction is granted by cell adhesion molecules (CAMs) integrating differentiation, proliferation, and pro-survival signals from the surrounding microenvironment to the inner cell. However, cell–cell and cell–matrix interactions are also critically involved during malignant transformation of hematopoietic stem/progenitor cells. It has become increasingly apparent that leukemia-associated gene products, such as activated tyrosine kinases and fusion proteins resulting from chromosomal translocations, directly regulate the activation status of adhesion molecules, thereby directing the leukemic phenotype. These observations imply that interference with adhesion molecule function represents a promising treatment strategy to target pre-leukemic and leukemic lesions within the bone marrow niche. Focusing on myeloid leukemia, we provide a current overview of the mechanisms by which leukemogenic gene products hijack control of cellular adhesion to subsequently disturb normal hematopoiesis and promote leukemia development.
In the mouse, most mature olfactory sensory neurons (OSNs) express one allele of one gene from the repertoire of ~1100 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express a given OR coalesce into homogeneous glomeruli, which reside at conserved positions in the olfactory bulb. ORs are intimately involved in ensuring the expression of one OR per OSN and the coalescence of OSN axons into glomeruli. But the mechanisms whereby ORs accomplish these diverse functions remain poorly understood. An experimental approach that has been informative is to substitute an OR genetically with another GPCR that is normally not expressed in OSNs, in order to determine in which aspects this GPCR can serve as surrogate OR in mouse OSNs. Thus far only the β2-adrenergic receptor (β2AR, Ardb2) has been shown to be able to serve as surrogate OR in OSNs; the β2AR could substitute for the M71 OR in all aspects examined. Can other non-olfactory GPCRs function equally well as surrogate ORs in OSNs? Here, we have generated and characterized two novel gene-targeted mouse strains in which the mouse melanocortin 4 receptor (Mc4r) or the mouse dopamine receptor D1 (Drd1a) is coexpressed with tauGFP in OSNs that express the OR locus M71. These alleles and strains are abbreviated as Mc4r→M71-GFP and Drd1a→M71-GFP. We detected strong Mc4r or Drd1a immunoreactivity in axons and dendritic knobs and cilia of OSNs that express Mc4r or Drd1a from the M71 locus. These OSNs responded physiologically to cognate agonists for Mc4r (Ro27-3225) or Drd1a (SKF81297), and not to the M71 ligand acetophenone. Axons of OSNs expressing Mc4r→M71-GFP coalesced into glomeruli. Axons of OSNs expressing Drd1a→M71-GFP converged onto restricted areas of the olfactory bulb but did not coalesce into glomeruli. Thus, OR functions in OSNs can be substituted by Mc4r or Drd1a, but not as well as by β2AR. We attribute the weak performance of Drd1a as surrogate OR to poor OSN maturation.
Gene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell-derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26-Cre males. This cross produces males that are sterile due to a complete cell-autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell-derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene-targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non-ES cell-derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326-333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.