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A novel chromatin configuration is described in lampbrush chromosomes of Pleurodeles waltlii oocytes which is different from transcriptionally inactive chromatin as weil as from the various forms of transcribed chromatin hitherto described. This novel type of chromatin is not arranged in Christmas tree-Iike configurations of densely packed lateral ribonucleoprotein (RNP) fibriIs but is characterized by a periodic alternating pattern of thick and thin regions which occur in clusters 01 some 10,000 repeats. Each thickened unit with an average length of 45 nm contains two c10sely spaced particles, the putative RNA polymerases, and each thickened unit is separated from the next one by a beaded chromatin spacer with a length of about 80 nm. This chromatin spacer contains on average two particles of approximately 14 nm in diameter, assumed to be nucleosomes. The thickened regions are interpreted to represent short transcriptional units containing approximately 130 base pairs of DNA which are separated from each other by nontranscribed spacers of 240-400 base pairs of DNA. The possibility is discussed that these transcriptional units represent 5S rRNA or tRNA genes.
Many plants combat herbivore and pathogen attack indirectly by attracting predators of their herbivores. Here we describe a novel type of insect-plant interaction where a carnivorous plant uses such an indirect defence to prevent nutrient loss to kleptoparasites. The ant Camponotus schmitzi is an obligate inhabitant of the carnivorous pitcher plant Nepenthes bicalcarata in Borneo. It has recently been suggested that this ant-plant interaction is a nutritional mutualism, but the detailed mechanisms and the origin of the ant-derived nutrient supply have remained unexplained. We confirm that N. bicalcarata host plant leaves naturally have an elevated \(^{15}N/^{14}N\) stable isotope abundance ratio (\(\delta ^{15}N\)) when colonised by C. schmitzi. This indicates that a higher proportion of the plants' nitrogen is insect-derived when C. schmitzi ants are present (ca. 100%, vs. 77% in uncolonised plants) and that more nitrogen is available to them. We demonstrated direct flux of nutrients from the ants to the host plant in a \(^{15}N\) pulse-chase experiment. As C. schmitzi ants only feed on nectar and pitcher contents of their host, the elevated foliar \(\delta ^{15}N\) cannot be explained by classic ant-feeding (myrmecotrophy) but must originate from a higher efficiency of the pitcher traps. We discovered that C. schmitzi ants not only increase the pitchers' capture efficiency by keeping the pitchers' trapping surfaces clean, but they also reduce nutrient loss from the pitchers by predating dipteran pitcher inhabitants (infauna). Consequently, nutrients the pitchers would have otherwise lost via emerging flies become available as ant colony waste. The plants' prey is therefore conserved by the ants. The interaction between C. schmitzi, N. bicalcarata and dipteran pitcher infauna represents a new type of mutualism where animals mitigate the damage by nutrient thieves to a plant.
A novel USP11-TCEAL1-mediated mechanism protects transcriptional elongation by RNA Polymerase II
(2024)
Deregulated expression of MYC oncoproteins is a driving event in many human cancers. Therefore, understanding and targeting MYC protein-driven mechanisms in tumor biology remain a major challenge.
Oncogenic transcription in MYCN-amplified neuroblastoma leads to the formation of the MYCN-BRCA1-USP11 complex that terminates transcription by evicting stalling RNAPII from chromatin. This reduces cellular stress and allows reinitiation of new rounds of transcription. Basically, tumors with amplified MYC genes have a high demand on well orchestration of transcriptional processes-dependent and independent from MYC proteins functions in gene regulation. To date, the cooperation between promoter-proximal termination and transcriptional elongation in cancer cells remains still incomplete in its understanding.
In this study the putative role of the dubiquitinase Ubiquitin Specific Protease 11 (USP11) in transcription regulation was further investigated. First, several USP11 interaction partners involved in transcriptional regulation in neuroblastoma cancer cells were identified. In particular, the transcription elongation factor A like 1 (TCEAL1) protein, which assists USP11 to engage protein-protein interactions in a MYCN-dependent manner, was characterized. The data clearly show that TCEAL1 acts as a pro-transcriptional factor for RNA polymerase II (RNAPII)-medi- ated transcription. In detail, TCEAL1 controls the transcription factor S-II (TFIIS), a factor that assists RNAPII to escape from paused sites. The findings claim that TCEAL1 outcompetes the transcription elongation factor TFIIS in a non-catalytic manner on chromatin of highly expressed genes. This is reasoned by the need regulating TFIIS function in transcription. TCEAL1 equili- brates excessive backtracking and premature termination of transcription caused by TFIIS.
Collectively, the work shed light on the stoichiometric control of TFIIS demand in transcriptional regulation via the USP11-TCEAL1-USP7 complex. This complex protects RNAPII from TFIIS-mediated termination helping to regulate productive transcription of highly active genes in neuroblastoma.
A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis
(1981)
The amplified, extrachromosomal nucleoli of Xenopus oocytes contain a meshwork of -4-nm-thick filaments, which are densely coiled into higher-order fibrils of diameter 30-40 nm and are resistant to treatment with high- and low-salt concentrations, nucleases (DNase I, pancreatic RNase, micrococcal nuclease), sulfhydryl agents, and various nonionic detergents. This filamentous "skeleton" has been prepared from manually isolated nuclear contents and nucleoli as weil as from nucleoli isolated by fluorescence-activated particle sorting. The nucleolar skeletons are observed in light and electron microscopy and are characterized by ravels of filaments that are especially densely packed in the nucleolar cortex. DNA as weil as RNA are not constituents of this structure, and precursors to ribosomal RNAs are completely removed from the extraction-resistant filaments by treatment with high-salt buffer or RN ase. Fractions of isolated nucleolar skeletons show specific enrichment of an acidic major protein of 145,000 mol wt and an apparent pi value of -6.15, accompanied in some preparations by various amounts of minor proteins. The demonstration of this skeletal structure in "free" extrachromosomal nucleoli excludes the problem of contaminations by nonnucleolar material such as perinucleolar heterochromatin normally encountered in studies of nucleoli from somatic cells. It is suggested that this insoluble protein filament complex forms a skeleton specific to the nucleolus proper that is different from other extraction-resistant components of the nucleus such as matrix and lamina and is involved in the spatial organization of the nucleolar chromatin and its transcriptional products. In studies of the organization of the interphase nucleus, considerable progress has been made in the elucidation of the arrangement of chromatin components and transcriptional products. However, relatively little is known about the composition and function of another category of nuclear structures, the nonnucleoproteinaceous architectural components that are insoluble in solutions of low and high ionic strength, despite numerous studies dedicated to this problem. Such structures include (a) the nuclear envelope and its pore complexes (I, 15, 18, 23, 37, 41), (b) a peripheral layer of insoluble protein ("lamina"; I, 15, 22, 23, 59), (e) certain skeletal proteins related to the chromosome "scaffold" described by Laemmli and coworkers (see references 2 and 3), and (d) ill-defined tangles of fibrillar structures of the nuclear interior that are collectively described as residual "matrix" (6, 21 ; for reviews, see references THE JOURNAL OF CEll BrOlOGY . VOlUME 90 AUGUST 1981 289-299 © The RockefeIler University Press · 0021 -9525/ 81 / 08/ 0289/ 11 $1 .00 4 and 12). The latter, preparatively
A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism
(2021)
Members of the protein kinase D (PKD) family (PKD1, 2, and 3) integrate hormonal and nutritional inputs to regulate complex cellular metabolism. Despite the fact that a number of functions have been annotated to particular PKDs, their molecular targets are relatively poorly explored. PKD3 promotes insulin sensitivity and suppresses lipogenesis in the liver of animals fed a high-fat diet. However, its substrates are largely unknown. Here we applied proteomic approaches to determine PKD3 targets. We identified more than 300 putative targets of PKD3. Furthermore, biochemical analysis revealed that PKD3 regulates cAMP-dependent PKA activity, a master regulator of the hepatic response to glucagon and fasting. PKA regulates glucose, lipid, and amino acid metabolism in the liver, by targeting key enzymes in the respective processes. Among them the PKA targets phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine. Consistently, we showed that PKD3 is activated by glucagon and promotes glucose and tyrosine levels in hepatocytes. Therefore, our data indicate that PKD3 might play a role in the hepatic response to glucagon.
A long-range restriction map of part of the short arm of ehromosome 11 including the WAGR region has been constructed using pulsed-field gel electrophoresis and a number of infrequently cutting restriction enzymes. A total of 15.4 Mbp has been mapped in detall, extending from proximal 11p14 to the distal part of 11p12. The map localizes 35 different DNA probes and reveals at least nine areas with features eharaeteristle of BTF islands, some of which may be candidates for the different loci underlying the phenotype of the WAGR syndrome. This map will furthermore allow screening of DNA from individuals with WAGR-related phenotypes and from Wilms tumors for associated chromosomal rearrangements.
Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.
We present a quantitative 3D analysis of the motility of the blood parasite Trypanosoma brucei. Digital in-line holographic microscopy has been used to track single cells with high temporal and spatial accuracy to obtain quantitative data on their behavior. Comparing bloodstream form and insect form trypanosomes as well as mutant and wildtype cells under varying external conditions we were able to derive a general two-state-run-and-tumble-model for trypanosome motility. Differences in the motility of distinct strains indicate that adaption of the trypanosomes to their natural environments involves a change in their mode of swimming.
For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival.
Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells.
Background
Currently established methods to identify viable and non-viable cells of cyanobacteria are either time-consuming (eg. plating) or preparation-intensive (eg. fluorescent staining). In this paper we present a new and fast viability assay for unicellular cyanobacteria, which uses red chlorophyll fluorescence and an unspecific green autofluorescence for the differentiation of viable and non-viable cells without the need of sample preparation.
Results
The viability assay for unicellular cyanobacteria using red and green autofluorescence was established and validated for the model organism Synechocystis sp. PCC 6803. Both autofluorescence signals could be observed simultaneously allowing a direct classification of viable and non-viable cells. The results were confirmed by plating/colony count, absorption spectra and chlorophyll measurements. The use of an automated fluorescence microscope and a novel ImageJ based image analysis plugin allow a semi-automated analysis.
Conclusions
The new method simplifies the process of viability analysis and allows a quick and accurate analysis. Furthermore results indicate that a combination of the new assay with absorption spectra or chlorophyll concentration measurements allows the estimation of the vitality of cells.
Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. Here, we dissect the function of the key regulator of serotonin signaling—the serotonin transporter–in innate olfactory attraction to ethanol in Drosophila melanogaster. We generated a mutated version of the serotonin transporter that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. We identified four serotonergic neurons that inhibit the olfactory attraction to ethanol and two additional neurons that counteract this inhibition by strengthening olfactory information. Our results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior.
Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV derivative HIV(SNAP), which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIV(SNAP) represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy.
Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.
Background
Bees (Hymenoptera: Apoidea: Anthophila) are the most important group of pollinators with about 20,507 known species worldwide. Despite the critical role of bees in providing pollination services, studies aiming at understanding which species are present across disturbance gradients are scarce. Limited taxononomic information for the existing and unidentified bee species in Tanzania make their conservation haphazard. Here, we present a dataset of bee species records obtained from a survey in nothern Tanzania i.e. Kilimanjaro, Arusha and Manyara regions. Our findings serve as baseline data necessary for understanding the diversity and distribution of bees in the northern parts of the country, which is a critical step in devising robust conservation and monitoring strategies for their populations.
New information
In this paper, we present information on 45 bee species belonging to 20 genera and four families sampled using a combination of sweep-netting and pan trap methods. Most species (27, ~ 60%) belong to the family Halictidae followed by 16 species (35.5%) from the family Apidae. Megachilidae and Andrenidae were the least represented, each with only one species (2.2%). Additional species of Apidae and Megachilidae sampled during this survey are not yet published on Global Biodiversity Information Facility (GBIF), once they will be available on GBIF, they will be published in a subsequent paper. From a total of 953 occurrences, highest numbers were recorded in Kilimanjaro Region (n = 511), followed by Arusha (n = 410) and Manyara (n = 32), but this pattern reflects the sampling efforts of the research project rather than real bias in the distributions of bee species in northern Tanzania.
The identification of biomarker signatures is important for cancer diagnosis and prognosis. However, the detection of clinical reliable signatures is influenced by limited data availability, which may restrict statistical power. Moreover, methods for integration of large sample cohorts and signature identification are limited. We present a step-by-step computational protocol for functional gene expression analysis and the identification of diagnostic and prognostic signatures by combining meta-analysis with machine learning and survival analysis. The novelty of the toolbox lies in its all-in-one functionality, generic design, and modularity. It is exemplified for lung cancer, including a comprehensive evaluation using different validation strategies. However, the protocol is not restricted to specific disease types and can therefore be used by a broad community. The accompanying R package vignette runs in ~1 h and describes the workflow in detail for use by researchers with limited bioinformatics training.
Sex-specific markers are a prerequisite for understanding reproductive biology, genetic factors involved in sex differences, mechanisms of sex determination, and ultimately the evolution of sex chromosomes. The Western mosquitofish, Gambusia affinis, may be considered a model species for sex-chromosome evolution, as it displays female heterogamety (ZW/ZZ), and is also ecologically interesting as a worldwide invasive species. Here, de novo RNA-sequencing on the gonads of sexually mature G. affinis was used to identify contigs that were highly transcribed in females but not in males (i.e., transcripts with ovary-specific expression). Subsequently, 129 primer pairs spanning 79 contigs were tested by PCR to identify sex-specific transcripts. Of those primer pairs, one female-specific DNA marker was identified, Sanger sequenced and subsequently validated in 115 fish. Sequence analyses revealed a high similarity between the identified sex-specific marker and the 3' UTR of the aminomethyl transferase (amt) gene of the closely related platyfish (Xiphophorus maculatus). This is the first time that RNA-seq has been used to successfully characterize a sex-specific marker in a fish species in the absence of a genome map. Additionally, the identified sex-specific marker represents one of only a handful of such markers in fishes.
During recent years a number of severe clinical syndromes, collectively termed laminopathies, turned out to be caused by various, distinct mutations in the human LMNA gene. Arising from this, remarkable progress has been made to unravel the molecular pathophysiology underlying these disorders. A great benefit in this context was the generation of an A-type lamin deficient mouse line (Lmna\(^{−/−}\)) by Sullivan and others,1 which has become one of the most frequently used models in the field and provided profound insights to many different aspects of A-type lamin function. Here, we report the unexpected finding that these mice express a truncated Lmna gene product on both transcriptional and protein level. Combining different approaches including mass spectrometry, we precisely define this product as a C-terminally truncated lamin A mutant that lacks domains important for protein interactions and post-translational processing. Based on our findings we discuss implications for the interpretation of previous studies using Lmna\(^{−/−}\) mice and the concept of human laminopathies.
Developmental delay or mental retardation is a frequent component of multi-system anomaly syndromes associated with chromosomal deletions. Isolation of genes involved in the mental dysfunction in these disorders should define loci important in brain formation or function. We have identified a highly conserved locus in the distal part of 11 p 13 that is prominently expressed in fetal brain. Minimal expression is observed in a number of other fetal tissues. The gene maps distal to PAX-6 but proximal to the loci for brain-derived neurotrophic factor (BDNF) and the beta subunit of follicle stimulating hormone (FSHB), within a region previously implicated in the mental retardation component of some WAGR syndrome patients. Within fetal brain, the corresponding transcript is prominent in frontal, motor and primary visual cortex as weil as in the caudate-putamen. The characteristics of this gene, including the striking evolutionary conservation at the locus, suggest that the encoded protein may function in brain development.
Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identifed the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efciently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
The variable regions (V1–V9) of the 18S rDNA are routinely used in barcoding and phylogenetics. In handling these data for trypanosomes, we have noticed a misunderstanding that has apparently taken a life of its own in the literature over the years. In particular, in recent years, when studying the phylogenetic relationship of trypanosomes, the use of V7/V8 was systematically established. However, considering the current numbering system for all other organisms (including other Euglenozoa), V7/V8 was never used. In Maia da Silva et al. [Parasitology 2004, 129, 549–561], V7/V8 was promoted for the first time for trypanosome phylogenetics, and since then, more than 70 publications have replicated this nomenclature and even discussed the benefits of the use of this region in comparison to V4. However, the primers used to amplify the variable region of trypanosomes have actually amplified V4 (concerning the current 18S rDNA numbering system).
The ultrastructure of twO kinds of transcription ally active chromatin, the lampbrush chromosome loops and the nucleoli from amphibian oocytes and primary nuclei of the green alga Acetabularia, has been examined after manual isolation and dispersion in low salt media of slightly alkaline pH using various electron microscopic staining techniques (positive staining, metal shadowing, negative staining, preparation on positively charged films, etc.) and compared with the appearance of chromatin from various somatic cells (hen erythrocytes, rat hepatocytes, ClIltured murine sarcoma cells) prepared in parallel. While typical nucleosomes were revealed with all the techniques for chromatin from the latter three cell system, no nucleosomes were identified in either the lampbrush chromosome structures or the nucleolar chromatin. Nucleosomal arrays were absent not only in maximally fibril-covered matrix units but also in fibril-free regions between transcriptional complexes, including the apparent spacer intercepts between different transcriptional units. Moreover, comparisons of the length of the repeating units of rDNA in the transcribed state with those determined in the isolated rDNA and with the lengths of the first stable product of rDNA transcription, the pre-rRNA, demonstrated that the transcribed rDNA was not significantly shortened and/or condensed but rather extended in the transcriptional units. Distinct granules of about nucleosomal size which were sometimes found in apparent spacer regions as well as within matrix units of reduced fibril density were shown not to represent nucleosomes since their number per spacer unit was not inversely correlated with the length of the specific unit and also on the basis of their resistance to treatment with the detergent Sarkosyl NL-30. It is possible to structurally distinguish between transcriptionally active chromatin in which the DNA is extended in a non-nucleosomal form of chromatin and condensed, inactive chromatin within the typical nucleosomal package. The characteristic extended structure of transcriptionally active chromatin is found not only in the transcribed genes but also in non-transcribed regions within or between ("spacer") transcriptional units as well as in transcriptional units that are untranscribed amidst transcribed ones and/or have been inactivated for relatively short time. It is hypothesized that activation of transcription involves a transition from a nucleosomal to an extended chromatin organisation and that this structural transition is not specific for single "activated" genes but may involve larger chromatin regions, including adjacent untranscribed intercepts.
Available data on the occurrence and expression of somatic histone HI during oogenesis and early embryogenesis of Xenopus laevis are contradictory. In particular the reported presence of a large storage pool of histone HIA in oocytes is difficult to reconcile with the high transcriptional activity of all gene classes in this specific cell type. In the present study we have used polyclonal antibodies raised against somatic Xenopus histone HI (HIA and HIA/B) for combined immunoblotting experiments to quantitate HI pools and immunolocalization studies to visualize chromosome- bound HI. Both approaches failed to detect soluble or chromosomal histone HI in vitellogenic oocytes, eggs, and cleavage-stage embryos up to early blastula. In addition, chromatin assembled in Xenopus egg extract was also negative for histone HI as revealed by immunofluorescence microscopy. Lampbrush chromosomes not only lacked histone HI but also the previously identified histone HI-like B4 protein (Smith et al., 1988, Genes Dev. 2,1284-1295). In contrast, chromosomes of eggs and early embryos fluoresced brightly with anti-B4 antibodies. Our results lend further support to the view that histone HI expression is developmentally regulated during Xenopus oogenesis and embryogenesis similar to what is known from other species.
Most parasites and parasitoids are adapted to overcome defense mechanisms of their specific hosts and hence colonize a narrow range of host species. Accordingly, an increase in host functional or phylogenetic dissimilarity is expected to increase the species diversity of parasitoids. However, the local diversity of parasitoids may be driven by the accessibility and detectability of hosts, both increasing with increasing host abundance. Yet, the relative importance of these two mechanisms remains unclear. We parallelly reared communities of saproxylic beetle as potential hosts and associated parasitoid Hymenoptera from experimentally felled trees. The dissimilarity of beetle communities was inferred from distances in seven functional traits and from their evolutionary ancestry. We tested the effect of host abundance, species richness, functional, and phylogenetic dissimilarities on the abundance, species richness, and Shannon diversity of parasitoids. Our results showed an increase of abundance, species richness, and Shannon diversity of parasitoids with increasing beetle abundance. Additionally, abundance of parasitoids increased with increasing species richness of beetles. However, functional and phylogenetic dissimilarity showed no effect on the diversity of parasitoids. Our results suggest that the local diversity of parasitoids, of ephemeral and hidden resources like saproxylic beetles, is highest when resources are abundant and thereby detectable and accessible. Hence, in some cases, resources do not need to be diverse to promote parasitoid diversity.
Background
Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA.
Hypothesis
Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing.
Methods
To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression.
Results
Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry.
Conclusions
PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression.
Background: Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA. Hypothesis: Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing. Methods: To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression. Results: Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry. Conclusions: PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression.
Unisexual reproduction, which generates clonal offspring, is an alternative strategy to sexual breeding and occurs even in vertebrates. A wide range of non-sexual reproductive modes have been described, and one of the least understood questions is how such pathways emerged and how they mechanistically proceed. The Amazon molly, Poecilia formosa, needs sperm from males of related species to trigger the parthenogenetic development of diploid eggs. However, the mechanism, of how the unreduced female gametes are produced, remains unclear. Cytological analyses revealed that the chromosomes of primary oocytes initiate pachytene but do not proceed to bivalent formation and meiotic crossovers. Comparing ovary transcriptomes of P. formosa and its sexual parental species revealed expression levels of meiosis-specific genes deviating from P. mexicana but not from P. latipinna. Furthermore, several meiosis genes show biased expression towards one of the two alleles from the parental genomes. We infer from our data that in the Amazon molly diploid oocytes are generated by apomixis due to a failure in the synapsis of homologous chromosomes. The fact that this failure is not reflected in the differential expression of known meiosis genes suggests the underlying molecular mechanism may be dysregulation on the protein level or misexpression of a so far unknown meiosis gene, and/or hybrid dysgenesis because of compromised interaction of proteins from diverged genomes.
Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.
Thin section electron microscopy of Actinomycin D treated Tetrahymena cells and amphibian oocytes (Xenopus laevis, Triturus aZpestris) reveal no reduction in the central granules in the nuclear pore complexes. Possible reasons for the diversity between these results and earlier observations using negatively stained isolated nuclear envelopes from the same objects are discussed. The results clearly show that the presence of central granules within the nuclear pores does neither depend on nuclear RNA synthesis nor does indicate nucleocytoplasmic RNA transport. This conclusion leads to a reconsideration of the nature of the central granule. The functioning of the central granule of the nuclear pore complexes is further discussed in connection with recent studies on the ultrastructure of various types of cisternal pores.
Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia
(2021)
The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD+AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD+AML. Garitano-Trojaola et al. used a combination of human acute myeloid leukemia (AML) cell lines and primary samples to show that RAC1-dependent actin cytoskeleton remodeling through BCL2 family plays a key role in resistance to the FLT3 inhibitor, Midostaurin in AML. They showed that by targeting RAC1 and BCL2, Midostaurin resistance was diminished, which potentially paves the way for an innovate treatment approach for FLT3 mutant AML.
Background
Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network.
Methods and Results
In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response.
Conclusion and Significance
Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine.
Activation of the pp60\(^{c-src}\) kinase during differentiation of monomyelocytic cells in vitro
(1986)
Tbe proto-oncogene c-src, the cellular homolog of the Rous sarcoma virus (RSV) transforming gene v-src, is expressed in a tissue-specific and age-dependent manner. Its physiological function, although still unknown, appears to be more closely related to differentiation processes than to proliferation processes. To obtain more information about the physiological role of the c-src gene in cells, we have studied differentiation-dependent alterations using the human HL-60 leukaemia cell line as a model system. Induction of monocytic and granulocytic differentiation of HL-60 cells by 12-0-tetradecanoylphorbol-13-acetate (TPA) and dimethylsulfoxide (DMSO) is associated with an activation of the pp60<sup>c-src</sup> tyrosine kinase, but not with increased c-src gene expression. Control experiments exclude an interaction of TPA and DMSO themselves with the pp60<sup>c-src</sup> kinase.
Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently (p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD.
Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.
Activity of Tracheal Cytotoxin of Bordetella pertussis in a Human Tracheobronchial 3D Tissue Model
(2021)
Bordetella pertussis is a highly contagious pathogen which causes whooping cough in humans. A major pathophysiology of infection is the extrusion of ciliated cells and subsequent disruption of the respiratory mucosa. Tracheal cytotoxin (TCT) is the only virulence factor produced by B. pertussis that has been able to recapitulate this pathology in animal models. This pathophysiology is well characterized in a hamster tracheal model, but human data are lacking due to scarcity of donor material. We assessed the impact of TCT and lipopolysaccharide (LPS) on the functional integrity of the human airway mucosa by using in vitro airway mucosa models developed by co-culturing human tracheobronchial epithelial cells and human tracheobronchial fibroblasts on porcine small intestinal submucosa scaffold under airlift conditions. TCT and LPS either alone and in combination induced blebbing and necrosis of the ciliated epithelia. TCT and LPS induced loss of ciliated epithelial cells and hyper-mucus production which interfered with mucociliary clearance. In addition, the toxins had a disruptive effect on the tight junction organization, significantly reduced transepithelial electrical resistance and increased FITC-Dextran permeability after toxin incubation. In summary, the results indicate that TCT collaborates with LPS to induce the disruption of the human airway mucosa as reported for the hamster tracheal model.
Ofall amphibians living in arid habitats, reed frogs (belonging to the super species Hyperolius viridiflavus) are the most peculiar. Froglets are able to tolerate dry periods of up to 35 days or longer immediately after metamorphosis, in climatically exposed positions. They face similar problems to estivating juveniles, i.e. enduranee of long periods of high temperature and low RH with rather limited energy and water reserves. In addition, they must have had to develop meehanisms to prevent poisoning by nitrogenous wastes that rapidly accumulate during dry periods as a metabolie consequenee of maintaining a non-torpid state. During dry periods, plasma osmolarity of H. v. taeniatus froglets strongly increased, mainly through urea accumulation. Urea accumulation was also observed during metamorphic climax. During postmetamorphic growth, chromatophores develop with the density and morphology typical of the adult pigmentary pattern. The dermal iridophore layer, which is still incomplete at this time, is fully developed within 4-8 days after metamorphosis, irrespective of maintenance conditions. These iridophores mainly contain the purines guanine and hypoxanthine. The ability of these purines to reflect light provides an excellent basis for the role of iridophores in temperature regulation. In individuals experiencing dehydration stress, the initial rate of purine synthesis is doubled in eomparison to specimens continuously maintained under wet season conditions. This increase in synthesis rate leads to a rapid increase in the thiekness of the iridophore layer, thereby effectively reducing radiation absorption. Thus, the danger of overheating is diminished during periods of water shortage when evaporative cooling must be avoided. After the development of an iridophore layer of sufficient thickness for effective radiation reflectance, synthesis of iridophore pigments does not cease. Rather, this pathway is further used during the remaining dry season for solving osmotic problems eaused by accumulation of nitrogenous wastes. During prolonged water deprivation, in spite of reduced metabolic rates, purine pigments are produced at the same rate as in wet season conditions. This leads to a higher relative proportion of nitrogen end products being stored in skin pigments under dry season conditions. At the end of an experimental dry season lasting 35 days, up to 38% of the accrued nitrogen is stored in the form of osmotically inactive purines in thc skin. Thus the osmotic problems caused by evaporative water loss and urea production are greatly reduced.
Reed frogs of the superspecies Hyperolius viridiflavus occur throughout the seasonally very dry and hot African savannas. Despite their small size (300-700 mg), estivating reed frogs do not avoid stressful conditions above ground by burrowing into the soil, but endure the inhospitable climate relatively unprotected, clinging to mostly dry grass sterns. They must have emcient mechanisms to enable them to survive e.g. very high temperatures, low relative hurnidities, and high solar radiation loads. Mechanisms must also have developed to prevent poisoning by the nitrogenous wastes that inevitably result from protein and nucleotide turnover. In contrast to fossorial amphibians, estivating reed frogs do not become torpid. Reduction in metabolism is therefore rather Iimited so that nitrogenous wastes accumulate faster in these frogs than in fossorial amphibians. This severely aggravates the osmotic problems caused by dehydration. During dry periods total plasma osmolarity greatly increases, mainly due to urea accumulation. Of the total urea accumulated over 42 days of experimental water deprivation, 30% was produced during the first 7 days. In the next 7 days rise in plasma urea content was negligible. This strong initial increase of urea is seen as a byproduct of elevated amino acid catabolism following the onset of dry conditions. Tbe rise in total plasma osmolarity due to urea accumulation, however, is not totally disadvantageous, but enables fast rehydration when water is available for very short periods only. Voiding of urine and feces eeases once evaporative water loss exceeds 10% of body weight. Tberefore, during continuous water deprivation, nitrogenous end products are not excreted. After 42 days of water deprivation, bladder fluid was substantially depleted, and urea coneentration in the remaining urine (up to 447 mM) was never greater than in plasma fluid. Feces voided at the end of the dry period after water uptake contained only small amounts of nitrogenous end products. DSF (dry season frogs) seemed not to be uricotelic. Instead, up to 35% of the total nitrogenous wastes produced over 42 days of water deprivation were deposited in an osmotically inert and nontoxic form in iridophore crystals. The increase in skin purine content averaged 150 µg/mg dry weight. If urea had been the only nitrogenous waste product during an estivation period of 42 days, lethal limits of total osmolarity (about 700 mOsm) would have been reached 10-14 days earlier. Thus iridophores are not only involved in colour change and in reducing heat load by radiation remission, but are also important in osmoregulation during dry periods. The seIective advantages of deposition of guanine rather than uric acid are discussed.
Hyperolius viridiflavus nitidulus inhabits parts of the seasonally very hot and dry West African savanna. During the long lasting dry season, the small frog is sitting unhidden on mostly dry plants and has to deal with high solar radiation load (SRL), evaporative water loss (EWL) and small energy reserves. It seems to be very badly equipped to survive such harsh climatic conditions (unfavorable surface to volume ratio, very limited capacity to störe energy and water). Therefore, it must have developed extraordinary efficient mechanisms to solve the mentioned Problems. Some of these mechanisms are to be looked for within the skin of the animal (e.g. protection against fast desiccation, deleterious effects of UV radiation and over-heating). The morphology of the wet season skin is, in most aspects, that of a "normal" anuran skin. It differs in the Organization of the processes of the melanophores and in the arrangement of the chromatophores in the Stratum spongiosum, forming no "Dermal Chromatophore Unit". During the adaptation to dry season conditions the number of iridophores in dorsal and ventral skin is increased 4-6 times compared to wet season skin. This increase is accompanied by a very conspicuous change of the wet season color pattern. Now, at air temperatures below 35° C the color becomes brownish white or grey and changes to a brilliant white at air temperatures near and over 40° C. Thus, in dry season State the frog retains its ability for rapid color change. In wet season State the platelets of the iridophores are irregularly distributed. In dry season State many platelets become arranged almost parallel to the surface. These purine crystals probably act as quarter-wave-length interference reflectors, reducing SRL by reflecting a considerable amount of the radiated energy input. EWL is as low as that of much larger xeric reptilians. The impermeability of the skin seems to be the result of several mechanisms (ground substance, iridophores, lipids, mucus) supplementing each other. The light red skin at the pelvic region and inner sides of the limbs is specialized for rapid uptake of water allowing the frog to replenish the unavoidable EWL by using single drops of dew or rain, available for only very short periods.
Background: According to the classical model of Macevicz and Oster, annual eusocial insects should show a clear dichotomous "bang-bang" strategy of resource allocation; colony fitness is maximised when a period of pure colony growth (exclusive production of workers) is followed by a single reproductive period characterised by the exclusive production of sexuals. However, in several species graded investment strategies with a simultaneous production of workers and sexuals have been observed. Such deviations from the "bang-bang" strategy are usually interpreted as an adaptive (bet-hedging) response to environmental fluctuations such as variation in season length or food availability. To generate predictions about the optimal investment pattern of insect colonies in fluctuating environments, we slightly modified Macevicz and Oster's classical model of annual colony dynamics and used a dynamic programming approach nested into a recurrence procedure for the solution of the stochastic optimal control problem. Results: 1) The optimal switching time between pure colony growth and the exclusive production of sexuals decreases with increasing environmental variance. 2) Yet, for reasonable levels of environmental fluctuations no deviation from the typical bang-bang strategy is predicted. 3) Model calculations for the halictid bee Lasioglossum malachurum reveal that bet-hedging is not likely to be the reason for the graded allocation into sexuals versus workers observed in this species. 4) When environmental variance reaches a critical level our model predicts an abrupt change from dichotomous behaviour to graded allocation strategies, but the transition between colony growth and production of sexuals is not necessarily monotonic. Both, the critical level of environmental variance as well as the characteristic pattern of resource allocation strongly depend on the type of function used to describe environmental fluctuations. Conclusion: Up to now bet-hedging as an evolutionary response to variation in season length has been the main argument to explain field observations of graded resource allocation in annual eusocial insect species. However, our model shows that the effect of moderate fluctuations of environmental conditions does not select for deviation from the classical bang-bang strategy and that the evolution of graded allocation strategies can be triggered only by extreme fluctuations. Detailed quantitative observations on resource allocation in eusocial insects are needed to analyse the relevance of alternative explanations, e.g. logistic colony growth or reproductive conflict between queen and workers, for the evolution of graded allocation strategies.
Adding amino acids to a sucrose diet is not sufficient to support longevity of adult bumble bees
(2020)
Dietary macro-nutrients (i.e., carbohydrates, protein, and fat) are important for bee larval development and, thus, colony health and fitness. To which extent different diets (varying in macro-nutrient composition) affect adult bees and whether they can thrive on nectar as the sole amino acid source has, however, been little investigated. We investigated how diets varying in protein concentration and overall nutrient composition affected consumption, longevity, and breeding behavior of the buff-tailed bumble bee, Bombus terrestris (Hymenoptera: Apidae). Queenless micro-colonies were fed either natural nutrient sources (pollen), nearly pure protein (i.e., the milk protein casein), or sucrose solutions with low and with high essential amino acid content in concentrations as can be found in nectar. We observed micro-colonies for 110 days. We found that longevity was highest for pure pollen and lowest for pure sucrose solution and sucrose solution supplemented with amino acids in concentrations as found in the nectar of several plant species. Adding higher concentrations of amino acids to sucrose solution did only slightly increase longevity compared to sucrose alone. Consequently, sucrose solution with the applied concentrations and proportions of amino acids or other protein sources (e.g., casein) alone did not meet the nutritional needs of healthy adult bumble bees. In fact, longevity was highest and reproduction only successful in micro-colonies fed pollen. These results indicate that, in addition to carbohydrates and protein, adult bumble bees, like larvae, need further nutrients (e.g., lipids and micro-nutrients) for their well-being. An appropriate nutritional composition seemed to be best provided by floral pollen, suggesting that pollen is an essential dietary component not only for larvae but also for adult bees.
Background
Adrenalectomies are rare procedures especially in childhood. So far, no large cohort study on this topic has been published with data on to age distribution, operative procedures, hospital volume and operative outcome.
Methods
This is a retrospective analysis of anonymized nationwide hospital billing data (DRG data, 2009-2017). All adrenal surgeries (defined by OPS codes) of patients between the age 0 and 21 years in Germany were included.
Results
A total of 523 patient records were identified. The mean age was 8.6 ± 7.7 years and 262 patients were female (50.1%). The majority of patients were between 0 and 5 years old (52% overall), while 11.1% were between 6 and 11 and 38.8% older than 12 years. The most common diagnoses were malignant neoplasms of the adrenal gland (56%, mostly neuroblastoma) with the majority being younger than 5 years. Benign neoplasms in the adrenal gland (D350) account for 29% of all cases with the majority of affected patients being 12 years or older. 15% were not defined regarding tumor behavior. Overall complication rate was 27% with a clear higher complication rate in resection for malignant neoplasia of the adrenal gland. Bleeding occurrence and transfusions are the main complications, followed by the necessary of relaparotomy. There was an uneven patient distribution between hospital tertiles (low volume, medium and high volume tertile). While 164 patients received surgery in 85 different “low volume” hospitals (0.2 cases per hospital per year), 205 patients received surgery in 8 different “high volume” hospitals (2.8 cases per hospital per year; p<0.001). Patients in high volume centers were significant younger, had more extended resections and more often malignant neoplasia. In multivariable analysis younger age, extended resections and open procedures were independent predictors for occurrence of postoperative complications.
Conclusion
Overall complication rate of adrenalectomies in the pediatric population in Germany is low, demonstrating good therapeutic quality. Our analysis revealed a very uneven distribution of patient volume among hospitals.
Adrenocortical tumors are rare in children. This systematic review summarizes the published evidence on pediatric adrenocortical carcinoma (ACC) to provide a basis for a better understanding of the disease, investigate new molecular biomarkers and therapeutic targets, and define which patients may benefit from a more aggressive therapeutic approach. We included 137 studies with 3680 ACC patients (~65% female) in our analysis. We found no randomized controlled trials, so this review mainly reflects retrospective data. Due to a specific mutation in the TP53 gene in ~80% of Brazilian patients, that cohort was analyzed separately from series from other countries. Hormone analysis was described in 2569 of the 2874 patients (89%). Most patients were diagnosed with localized disease, whereas 23% had metastasis at primary diagnosis. Only 72% of the patients achieved complete resection. In 334 children (23%), recurrent disease was reported: 81% — local recurrence, 19% (n = 65) — distant metastases at relapse. Patients < 4 years old had a different distribution of tumor stages and hormone activity and better overall survival (p < 0.001). Although therapeutic approaches are typically multimodal, no consensus is available on effective standard treatments for advanced ACC. Thus, knowledge regarding pediatric ACC is still scarce and international prospective studies are needed to implement standardized clinical stratifications and risk-adapted therapeutic strategies.