Temperature drives variation in flying insect biomass across a German malaise trap network O_LIAmong the many concerns for biodiversity in the Anthropocene, recent reports of flying insect loss are particularly alarming, given their importance as pollinators and as a food source for many predators. Few insect monitoring programs cover large spatial scales required to provide more generalizable estimates of insect responses to global change drivers.
C_LIO_LIWe ask how climate and surrounding habitat affect flying insect biomass and day of peak biomass using data from the first year of a new standardized distributed monitoring network at 84 locations across Germany comprising spatial gradient of land-cover types from protected to urban areas.
C_LIO_LIFlying insect biomass increased linearly with monthly temperature across Germany. However, the effect of temperature on flying insect biomass flipped to negative in the hot months of June and July when local temperatures most exceeded long-term averages.
C_LIO_LILand-cover explained little variation in insect biomass, but biomass was lowest in forested sites. Grasslands, pastures and orchards harbored the highest insect biomass. The date of peak biomass was primarily driven by surrounding land-cover type, with grasslands especially having earlier insect biomass phenologies.
C_LIO_LIStandardized, large-scale monitoring is pivotal to uncover underlying processes of insect decline and to develop climate-adapted strategies to promote insect diversity. In a temperate climate region, we find that the benefits of temperature on flying insect biomass diminish in a German summer at locations where temperatures most exceeded long-term averages. These results highlighting the importance of local adaptation in climate change-driven impacts on insect communities.
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The spatial configuration of biotic interactions shapes coexistence-area relation-ships in an annual plant community The increase of species richness with area is a universal phenomenon on Earth. How-ever, this observation contrasts with our poor understanding of how these species-area relationships (SARs) emerge from the collective effects of area, spatial heterogeneity, and local interactions. By combining a structuralist approach with five years of empirical observations in a highly-diverse Mediterranean grassland, we show that spatial hetero-geneity plays a little role in the accumulation of species richness with area in our system. Instead, as we increase the sampled area more species combinations are realized, and they coexist mainly due to direct pairwise interactions rather than by changes in single-species dominance or by indirect interactions. We also identify a small set of transient species with small population sizes that are consistently found across spatial scales. These findings empirically support the importance of the architecture of species interactions together with stochastic events for driving coexistence- and species-area relationships.
SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein Histidines Inteins are auto-processing domains that implement a multi-step biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis. Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wild-type precursor protein. Structural analysis and molecular dynamics simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing. Host evolutionary history and ecological context modulate the adaptive potential of the microbiome Can the microbiome serve as a reservoir of adaptive potential for hosts? To address this question, we leveraged [~]150 generations of experimental evolution in Drosophila melanogaster in a stressful high sugar (HS) diet. We performed a fully reciprocal transplant experiment using the dominant control and HS bacteria and measured life-history and metabolic traits. If the microbiome can indeed confer benefits to hosts, then transplant recipients should gain fitness benefits compared to controls. Interestingly, we found that these benefits do exist, but are non-linear. The benefits depend on specific interactions between host genotype and the microbiome, and these interactions are modulated by the environment. Mismatches between fly evolution and microbiome exerted fitness costs by slowing development and reducing fecundity, especially in the stressful HS diet. The dominant HS bacteria (Acetobacter pasteurianus) uniquely encoded several genes to enable uric acid degradation, mediated the toxic effects of uric acid accumulation due to the HS diet for flies. Our study demonstrates that host genotype x microbiome x environment interactions have substantial effects on host phenotype, highlighting how host evolution and ecological context together shape the adaptive potential of the microbiome. Citation needed? Wikipedia and the COVID-19 pandemic BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Mechanical bistability of the mesoderm epithelium facilitates mesoderm invagination during Drosophila gastrulation Apical constriction driven by non-muscle myosin II ("myosin") provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of myosin, we find that during Drosophila mesoderm invagination, myosin contractility is critical to prevent tissue relaxation during the early, "priming" stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. The binary response suggests that the mesoderm is mechanically bistable during gastrulation. Combined modeling analysis and experimental measurements suggest that the observed mechanical bistability may arise from apicobasal shrinkage of the surrounding ectoderm, which promotes mesoderm invagination by facilitating a buckling transition. Our results suggest that Drosophila mesoderm invagination requires a joint action of local myosin contractility and mechanical bistability of the epithelium to trigger epithelial buckling. Microbial Communities Performing Hydrogen Solventogenic Metabolism of Volatile Fatty Acids 1This work evaluates four different physicochemical pretreatments (acidic, thermal, acidic-thermal and thermal-acidic) on an anaerobic inoculum used for alcohol production from acetate and butyrate. All experiments were conducted in single batches using acetate and butyrate as substrates at 30 {degrees}C and with a pressurized headspace of pure H2 at 2.15 atm (218.2 MPa). Thermal and acidic-thermal pretreatments lead to higher production of both ethanol and butanol. Mathematical modelling shows that the highest attainable concentrations of ethanol and butanol produced were 122 mg L-1 and 97 mg L-1 for the thermal pretreatment (after 17.5 days) and 87 mg L-1 and 143 mg L-1 for the acidic-thermal pretreatment (after 18.9 days). Acetate was produced in all assays. Thermodynamic data indicated that a high H2 partial pressure favoured solventogenic metabolic pathways. Finally, sequencing data showed that both thermal and acidic-thermal pretreatments selected mainly the bacterial genera Pseudomonas, Brevundimonas and Clostridium. The acidic-thermal pretreatment selected a bacterial community more adapted to the conversion of acetate and butyrate into ethanol and butanol, respectively. Thermal-acidic pretreatment was unstable, showing significant variability between replicates. Acidic pretreatment showed the lowest alcohol production. TAK1 blockade as a therapy for retinal neovascularization Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-{beta}-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-{beta}1 and other pro-inflammatory cytokines. TAK1 is also a key mediator of inflammation, innate immune responses, apoptosis and tissue homeostasis and plays an important role in physiological angiogenesis. Its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. We investigated the regulatory role of TAK1 in pathological angiogenesis in the retina. Transcriptome analysis of human retina featuring retinal neovascularization revealed enrichment of known TAK1-mediated signaling pathways. Selective inhibition of TAK1 activation by 5Z-7-oxozeaenol attenuated aberrant retinal angiogenesis in rats following oxygen-induced retinopathy. Transcriptome profiling revealed that TAK1 activation in human microvascular endothelial cells under TNF stimulation led to increase the gene expression related to cytokines and leukocyte-endothelial interaction, mainly through nuclear factor kappa B (NF{kappa}B) signaling pathways. These results reveal that inhibition of TAK1 signaling may have therapeutic value for the treatment of pathological angiogenesis in the retina. Multi-omics investigation of tacrolimus nephrotoxicity BackgroundTacrolimus, an immunosuppressive drug prescribed to a majority of transplanted patients is nephrotoxic, through still unclear mechanisms. This study aims to evaluate the impact of tacrolimus on a lineage of proximal tubular cells using a multi-omics approach.

MethodsLLC-PK1 cells were exposed to 5 M of tacrolimus for 24h. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, FBP1 and FBP2 was measured using RT-qPCR.

ResultsIn our cell model, tacrolimus impacted different metabolic pathways including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p<0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis. Apart from glucose synthesis, gluconeogenesis is an important process in kidney mediated acid-base balance control.

ConclusionThe variations found using this multi-omics approach clearly point towards a dysregulation of energy production in epithelial cells of the renal tubule, and potentially of their functions, that may be implicated in tacrolimus nephrotoxicity in the clinics. A novel two-generation approach to the population dynamics of gregarious parasitoids Parasitoids, as important natural enemies, help maintain balance in natural ecosystems. Their population dynamics is generally predicted from the number of individuals. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the mothers manipulation of offspring fertility due to the clutch size-body size- fertility correlation. As a result of this correlation, when females deliberately adjust the number of offspring laid in a host, they determine not only the number but also the body sizes and reproductive potentials of those offspring. For the first time in the model species Anaphes flavipes, we determined the parasitoids offspring fertility from clutch size. Using this, we experimentally clarified the advantage of specific clutch size combinations and we show that identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can cause higher fertility in the F2 generation. Based on these results, we propose a novel two-generation approach which includes the clutch size-body size-fertility correlation. Our novel approach provides a new perspective for determining the individual fitness levels of gregarious parasitoids with new options for the modelling of parasitoid population dynamics. Onset and kinetics of B cell polarization depend on substrate mechanics and antigen concentration The immune synapse is the tight contact zone between a lymphocyte and a cell presenting its cognate antigen. This structure serves as a signaling platform and entails a polarization of intra-cellular components, necessary to the immunological function of the cell. While the surface properties of the presenting cell are known to control the formation of the synapse, their impact on polarization has not yet been studied.

Using functional lipid droplets as tunable artificial presenting cells combined with a microfluidic pairing device, we simultaneously observe synchronized synapses and dynamically quantify polarization patterns of individual B cells. By assessing how the ligand concentration, the surface fluidity and the substrate rigidity impact this polarization, we show that its onset depends on the antigen concentration at the synapse, and that the substrate rigidity controls both its onset and its kinetics. Our experimental system enables a fine phenotyping of monoclonal cell populations based on their synaptic readout.

Significance statementTo fulfill their immune function, B cells acquire antigens presented on the surface of antigen-presenting cells and organize their cytoskeleton and lysosomal traffic towards the contact. The polarization mechanism is evolutionary conserved, making the system interesting both from the immunology and the cell biology point of view. We developed and finely characterized antigen-coated droplets as surrogate presenting cells with a physiological-flow microfluidic cell-pairing chip to explore the B cell lysosome polarization in response to changes in droplet mechanics and antigen concentration. We observed that polarization is sensitive to mechanical properties (responding more strongly but less fast on stiffer substrates) and to the local antigen concentration at the contact. Hence, surprisingly, a late cell response is sensitive to the mechanics of the contact. Protein degradation sets the fraction of active ribosomes at vanishing growth Growing cells adopt common basic strategies to achieve optimal resource allocation under limited resource availability. Our current understanding of such "growth laws" neglects degradation, assuming that it occurs slowly compared to the cell cycle duration. Here we argue that this assumption cannot hold at slow growth, leading to important consequences. We propose a simple framework showing that at slow growth protein degradation is balanced by a fraction of "maintenance" ribosomes. Consequently, active ribosomes do not drop to zero at vanishing growth, but as growth rate diminishes, an increasing fraction of active ribosomes performs maintenance. Through a detailed analysis of compiled data, we show that the predictions of this model agree with data from E. coli and S. cerevisiae. Intriguingly, we also find that protein degradation increases at slow growth, which we interpret as a consequence of active waste management and/or recycling. Our results highlight protein turnover as an underrated factor for our understanding of growth laws across kingdoms.

Author summaryThe idea that simple quantitative relationships relate cell physiology to cellular composition dates back to the 1950s, but the recent years saw a leap in our understanding of such "growth laws", with relevant implications regarding the interdependence between growth, metabolism and biochemical networks. However, recent works on nutrient-limited growth mainly focused on laboratory conditions that are favourable to growth. Thus, our current mathematical understanding of the growth laws neglects protein degradation, under the argument that it occurs slowly compared to the timescale of the cell cycle. Instead, at slow growth the timescales of mass loss from protein degradation and dilution become comparable. In this work, we propose that protein degradation shapes the quantitative relationships between ribosome allocation and growth rate, and determines a fraction of ribosomes that do not contribute to growth and need to remain active to balance degradation. Dihydroceramide desaturase promotes the formation of intraluminal vesicles and inhibits autophagy to increase exosome production Exosomes are important for cell-cell communication. Deficiencies in the human dihydroceramide desaturase gene, DEGS1, increase the dihydroceramide-to-ceramide ratio and causes hypomyelinating leukodystrophy. However, the disease mechanism remains unknown. Here, we developed an in vivo assay with spatially controlled expression of exosome markers in Drosophila eye imaginal discs and showed that the level and activity of the DEGS1 ortholog, ifc, correlated with exosome production. Knocking out ifc decreased the density of the exosome precursor intraluminal vesicles (ILVs) in the multivesicular endosomes and reduced the number of exosomes released. While ifc overexpression and autophagy inhibition both enhanced exosome production, combining the two had no additive effect. Moreover, DEGS1 activity was sufficient to drive ILV formation in vitro. Together, DEGS1/Ifc controls the dihydroceramide-to-ceramide ration and enhances exosome secretion by promoting ILV formation and preventing the autophagic degradation of MVEs.

These findings provide a potential cause for the neuropathy associated with DEGS1-deficient mutations.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/376046v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@c7479corg.highwire.dtl.DTLVardef@12e7f46org.highwire.dtl.DTLVardef@1a5e7f0org.highwire.dtl.DTLVardef@11e7f08_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIAn in vivo system was developed for observing exosome production in Drosophila.
C_LIO_LIDihydroceramide desaturase (DEGS1/Ifc) promotes exosome production by two means.
C_LIO_LIIfc drives membrane invagination to promote the formation of intraluminal vesicles.
C_LIO_LIIfc inhibits autophagic degradation of MVEs and increases exosome release.
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eTOC BlurbThe level and activity of dihydroceramide desaturase (human DEGS1 and Drosophila Ifc) correlate with exosome production. Wu et al. show that DEGS1 drives the formation of intraluminal vesicles in vivo and in vitro. Overexpressing Ifc inhibits autophagy and reduces the degradation of multivesicular endosomes, thus increases exosome release. Kinesin-3 mediated axonal delivery of presynaptic neurexin stabilizes dendritic spines and postsynaptic components The functional properties of neural circuits are defined by the patterns of synaptic connections between their partnering neurons, but the mechanisms that stabilize circuit connectivity are poorly understood. We systemically examined this question at synapses onto newly characterized dendritic spines of C. elegans GABAergic motor neurons. We show that the presynaptic adhesion protein, neurexin/NRX-1, is required for stabilization of postsynaptic structure. We find that early postsynaptic developmental events proceed without a strict requirement for synaptic activity and are not disrupted by deletion of neurexin/nrx-1. However, in the absence of presynaptic NRX-1, dendritic spines and receptor clusters become destabilized and collapse prior to adulthood. We demonstrate that kinesin-3/UNC-104 delivers NRX-1 to presynaptic terminals and show that ongoing delivery is required for postsynaptic maintenance in mature animals. By defining the dynamics and temporal order of synapse formation events in vivo, we describe a mechanism for stabilizing mature circuit connectivity through neurexin-based adhesion. Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study Recently, there have been great advances in cardiovascular channelopathy modeling and drug safety pharmacology using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). The automated patch-clamp (APC) technique overcomes the disadvantages of the manual patch-clamp (MPC) technique, which is labor intensive and gives low output. However, the application of the APC platform is still limited in iPSC-CM based research, due to the difficulty in maintaining the high quality of single iPSC-CMs during dissociation and recording. In this study, we improved the method for single iPSC-CM preparation by applying 2.5 M blebbistatin (BB, an excitation-contraction coupling uncoupler) throughout APC procedures (dissociation, filtration, storage, and recording). Under non-BB buffered condition, iPSC-CMs in suspension showed a severe bleb-like morphology. However, BB-supplement led to significant improvements in morphology and INa recording, and we even obtained several CMs that showed spontaneous action potentials with typical morphology. Furthermore, APC faithfully recapitulated the single-cell electrophysiological phenotypes of iPSC-CMs derived from Brugada syndrome patients, as detected with MPC. Our study indicates that APC is capable of replacing MPC in the modeling of cardiac channelopathies using human iPSC-CMs by providing high-quality data with higher throughput. Contrasting resistance and resilience to light variation of the coupled oxic and anoxic componentsof an experimental microbial ecosystem Understanding how microbial communities of aquatic ecosystems respond to environmental change remains a critical challenge in microbial ecology. In this study, we used phototrophic oxic-anoxic micro-ecosystems to understand how the functioning and diversity of aerobic and anaerobic lake analog communities is affected by light deprivation. Continuous measurements were performed to describe oxygen dynamics (mean/min/max/amplitude) and time-series of full-length 16S rRNA sequencing were used to quantify changes in alpha- and beta-diversity. In the top oxic layer, oxygen concentration decreased significantly under light deprivation, but showed resilience in mean, minimum and maximum after light conditions were restored. Only the amplitude of diurnal fluctuations in oxygen concentrations did not recover fully, and instead tended to remain lower in treated ecosystems. Alpha-diversity of the top oxic layer communities showed a delayed increase after light conditions were restored, and was not resilient. In contrast, alpha-diversity of the anoxic bottom layer communities increased due to the stressor, but was resilient. Community composition changed significantly during light deprivation, showed resilience in the oxic layer and lack of resilience in the anoxic layer. Alpha-diversity and the amplitude of oxygen within and among treatments were strongly correlated, suggesting that higher diversity could lead to less variable oxygen concentrations, or vice versa. Our experiment showed that light deprivation induces multifaceted responses of community function and structure, hence focusing on a single stability component could potentially be misleading. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Diurnal activity budget and conservation implication of Hippopotamus amphibius at Bui National Park, Ghana Animals apportion time for their daily behavioral activities. We studied the activity budget of Hippopotamus amphibius at the Black Volta River in the Bui National Park, Ghana. We performed instantaneous scan sample using ground count survey between August 2020 to July 2021. We observed that H. amphibius spent most of their day time resting (54.75 %), followed by feeding (22.93 %), walking (19.2 %) and touching (3.12 %). We found significant difference in the time spent between the daily activities (H = 41.67; p < 0.0001). Except activities involving touching, we found no significant seasonal differences in feeding, resting and walking activities by H. amphibius. Understanding the behavioral activities of H. amphibius will enhance management and conservation of the animals. Combined docking and machine learning identifies key molecular determinants of ligand pharmacological activity on β2 adrenoceptor. G protein coupled receptors (GPCRs) are valuable therapeutic targets for many diseases. A central question of GPCR drug discovery is to understand what determines the agonism or antagonism of ligands which bind them. Ligands exert their action via the interactions in the ligand binding pocket. We hypothesised that there is a common set of receptor interactions made by ligands of diverse structures that mediate their action and that among a large dataset of different ligands, the functionally important interactions will be over-represented. We computationally docked ~2700 known {beta}2AR ligands to multiple {beta}2AR structures, generating ca 75,000 docking poses and predicted all atomic interactions between the receptor and the ligand. We used machine learning (ML) techniques to identify specific interactions that correlate with the agonist or antagonist activity of these ligands. The interpretation of ML analysis in human understandable form allowed us to construct an exquisitely detailed structure-activity relationship that identifies small changes to the ligands that invert their activity and thus helps to guide the drug discovery process. This approach can be readily applied to any drug target. The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the Functional Nanopore (FuN) screen now provides a quantitative and dynamic read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology. SPIRO - the automated Petri plate imaging platform designed by biologists, for biologists The imaging of plant seedlings, fungal mycelia and bacterial colonies grown on Petri plates is common in phenotyping assays, and is typically done manually despite the procedures being time-consuming and laborious. The main reason for this is the still limited availability of existing automated phenotyping tools and facilities. Additionally, constructing a custom-made automated solution is a daunting task for most research groups specializing in biology.

Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment. SPIRO was designed for biologists by biologists; thus, its assembly does not require experience in engineering or programming and its operation is sufficiently intuitive to be carried out without training. SPIRO has a small footprint optimal for fitting into standard incubators for plants and microbes, and is equipped with an LED light source for imaging in the dark, thus allowing acquisition of photos under optimal growth conditions. SPIROs web-based user interface allows setting up experiments and downloading data remotely, without interfering with samples growth. SPIROs excellent image quality is optimal for automated image processing, which we demonstrate with two semi-automated assays for analysis of commonly used phenotypic traits: seed germination and root growth.

Moreover, the robot can be easily customized for a specific use, as all information about SPIRO, including the models for 3D-printed structural components, control software, and scripts for image analysis, are released under permissive open-source licenses. Entorhinal cortex epigenome-wide association study highlights four novel loci showing differential methylation in Alzheimer's disease BackgroundStudies on DNA methylation (DNAm) in Alzheimers disease (AD) have recently highlighted several genomic loci showing association with disease onset and progression.

MethodsHere, we conducted an epigenome-wide association study (EWAS) using DNAm profiles in entorhinal cortex (EC) from 149 AD patients and control brains and combined these with two previously published EC datasets by meta-analysis (total n=337).

ResultsWe identified 12 cytosine-phosphate-guanine (CpG) sites showing epigenome-wide significant association with either case-control status or Braaks tau-staging. Four of these CpGs, located in proximity to CNFN/LIPE, TENT5A, PALD1/PRF1, and DIRAS1, represent novel findings. Integrating DNAm levels with RNA sequencing-based mRNA expression data generated in the same individuals showed significant DNAm-mRNA correlations for 6 of the 12 significant CpGs. Lastly, by calculating rates of epigenetic age acceleration using two recently proposed "epigenetic clock" estimators we found a significant association with accelerated epigenetic aging in AD patients vs. controls.

ConclusionIn summary, our study represents the hitherto most comprehensive EWAS in AD using EC and highlights several novel differentially methylated loci with potential effects on gene expression. Temporal and Spatial Dynamics of Hepatic Stellate Cell Activation in Intrahepatic Cholangiocarcinoma Intrahepatic cholangiocarcinoma (iCCA) is an aggressive type of primary liver cancer characterized by its highly desmoplastic stroma. Compared to the ample knowledge on cancer-associated fibroblast residing within iCCA tumor mass, little is known about the fibrotic response from the tumor-surrounding liver and its role in iCCA development. In this study, we find a significant accumulation of peritumoral myofibroblasts (pMFs) in both patient iCCA and tumors from an iCCA orthotopic allograft mouse model. Using tumor and liver spheroid coculture we show that iCCA-liver interaction induces rapid pMF accumulation at the interface. We find pMFs placed around iCCA spheroids exert a strong suppressive effect on tumor cells growth, in contrast to the pro-proliferative effect of MFs mixed within tumor spheroids. However, prolonged iCCA-pMF interaction elicits tumor cell dissemination in vitro. We find an upregulation of Vcam1 in tumor cells in the early phase of iCCA-pMF interaction both in vitro and in vivo which is downregulated when tumor cells disseminate. Blocking Vcam1 activity in iCCA allograft mouse models slows primary tumor growth but lead to increased tumor metastasis. Our data suggest that pMFs are beyond simple pro- or anti-tumorigenic in iCCA, with the ability to suppress tumor growth but elicit tumor cell dissemination. Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies Dementia with Lewy bodies is pathologically defined by the cytoplasmic accumulation of alpha-synuclein within neuronal cells in the brain. Alpha-synuclein is predominately pre-synaptic, but has been reported present in various subcellular compartments in cell and animal models. In particular, nuclear alpha-synuclein is evident in-vitro and in disease models and has been associated with altered DNA integrity, gene transcription, nuclear homeostasis. However, owing to various factors, the presence of alpha-synuclein in the nuclei of human brain cells remains controversial, as does its role in synucleinopathies.

Here, we close this gap and provide a unique demonstration confirming the presence of nuclear alpha-synuclein in post-mortem brain tissue obtained from cases of dementia with Lewy bodies as well as from controls via immunohistochemistry, immunoblot, and label-free mass-spectrometry.

Discrete intra-nuclear alpha-synuclein puncta reactive against phosphorylated serine 129-alpha-synuclein and pan-alpha-synuclein antibodies were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations from Dementia with Lewy bodies cases and matched controls. Subsequent biochemical analysis of subcellular fractionated tissue confirmed alpha-synuclein as present in a nuclear fraction at levels ~ 10-fold lower than in the cytoplasm. Critically, however, an increase in monomeric nuclear alpha-synuclein phosphorylated as serine 129 was observed in cases of dementia with Lewy bodies alongside higher molecular weight pan- and phosphorylation reactive alpha-synuclein species, consistent with the formation of intranuclear phosphorylated alpha-synuclein oligomers. Furthermore, the presence of nuclear alpha-synuclein was confirmed via label free mass spectrometry, as 6 unique alpha-synuclein derived peptide sequences were identified in nuclear fractions (71.4% sequence coverage).

Collectively, our data confirm the presence of nuclear alpha-synuclein in human brain tissue and describe nuclear pathology associated with dementia with Lewy bodies. These findings address a major controversy in the synucleinopathy field by confirming the presence of nuclear alpha-synuclein in autoptic human brain tissue and, for the first time, identify that alpha-synuclein is aggregated into novel and potentially pathological assemblies in the nucleus as part of the disease process associated with dementia with Lewy bodies and thus may contribute to the disease phenotype. The 55K SNP-Based Exploration of QTL for Spikelet Number Per Spike in a Tetraploid Wheat (Triticum turgidum L.) Population: Chinese Landrace Ailanmai X Wild Emmer The spikelet number per spike (SNS) is a primary factor determining wheat yield. Common wheat breeding reduces the genetic diversity among elite germplasm resources, leading to a detrimental effect on future wheat production. It is, therefore, necessary to explore new genetic resources for SNS to increase wheat yield. A tetraploid landrace  Ailanmai x wild emmer wheat recombinant inbred line (RIL) population was used to construct a genetic map using the wheat 55K single nucleotide polymorphism (SNP) array. The linkage map containing 1150 bin markers with a total genetic distance of 2411.8 cM was obtained. Based on phenotypic data from eight environments and best linear unbiased prediction (BLUP) values, five quantitative trait loci (QTL) for SNS were identified, explaining 6.71-29.40% of the phenotypic variation. Two of them, QSns.sau-AM-2B.3 and QSns.sau-AM-3B.2, were detected as the major and novel QTL. Their effects were further validated in two additional F2 populations using tightly linked Kompetitive Allele-Specific PCR (KASP) markers. Potential candidate genes within the physical intervals of corresponding QTL were predicted to participate in inflorescence development and spikelet formation. Genetic associations between SNS and other agronomic traits were also detected and analyzed. This study demonstrates the feasibility of wheat 55K SNP array developed for common wheat in genetic mapping of tetraploid population and shows the potential application of wheat related species in wheat improvement programs. Induced antigen-binding polyreactivity in human serum IgA Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.