title: Purification and Characterization of Inorganic Pyrophosphatase for in vitro RNA Transcription

abstract:
Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and of high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size-exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering (SEC-MALS-DLS), analytical ultracentrifugation (AUC), and small-angle X-ray scattering (SAXS), we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for labs involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.

Statement of SignificanceWe show an easy two-step purification procedure to efficiently produce large quantities of iPPase, an expensive component of IVT reactions. Laboratory-produced iPPase can significantly reduce the financial strain on research labs that rely on large-scale RNA production for experiments. Furthermore, we show for the first time, using a combination of orthogonal biophysical techniques, that yeast iPPase assembles into higher-order oligomers similar to bacterial iPPase.


title: Model evolution in SARS-CoV-2 spike protein sequences using a generative neural network

abstract:
Modelling evolutionary elements inherent in protein sequences, emerging from one clade into another of the SARS-CoV-2 virus, would provide insights to augment our understanding of its impact on public health and may help in formulating better strategies to contain its spread. Deep learning methods have been used to model protein sequences for SARS-CoV-2 viruses. A few significant drawbacks in these studies include being deficient in modelling end-to-end protein sequences, modelling only those genomic positions that show high activity and upsampling the number of sequences at each genomic position for balancing the frequency of mutations. To mitigate such drawbacks, the current approach uses a generative model, an encoder-decoder neural network, to learn the natural progression of spike protein sequences through adjacent clades of the phylogenetic tree of Nextstrain clades. Encoder transforms a set of spike protein sequences from the source clade (20A) into its latent representation. Decoder uses the latent representation, along with Gaussian distributed noise, to generate a different set of protein sequences that are closer to the target clade (20B). The source and target clades are adjacent nodes in the phylogenetic tree of different evolving clades of the SARS-CoV-2 virus. Sequences of amino acids are generated, for the entire length, at each genomic position using the latent representation of the amino acid generated at a previous step. Using trained models, protein sequences from the source clade are used to generate sequences that form a collection of evolved sequences belonging to all children clades of the source clade. A comparison of this predicted evolution (between source and generated sequences) of proteins with the true evolution (between source and target sequences) shows a high pearson correlation (> 0.7). Moreover, the distribution of the frequencies of substitutions per genomic position, including high- and low-frequency positions, in source-target sequences and source-generated sequences exhibit a high resemblance (pearson correlation > 0.7). In addition, the model partially predicts a few substitutions at specific genomic positions for the sequences of unseen clades (20J (Gamma)) where they show little activity during training. These outcomes show the potential of this approach in learning the latent mechanism of evolution of SARS-CoV-2 viral sequences.

Codebasehttps://github.com/anuprulez/clade_prediction

category: bioinformatics


title: The C-type Lectin Receptor Dectin-2 is a receptor for  Aspergillus fumigatus  galactomannan

abstract:
Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly amongst immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient sera and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the innate immune system. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of purified Aspergillus galactomannan to identify a C-type lectin host receptor responsible for binding this fungal carbohydrate. We identified a novel interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2 dependent signaling including activation of spleen tyrosine kinase and potent TNFa production. Deficiency of Dectin-2 increased neutrophil recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for Aspergillus galactomannan which leads to a pro-inflammatory response.

SUMMARYThe Aspergillus carbohydrate galactomannan is the primary clinical biomarker for diagnosis of invasive aspergillosis. In this manuscript, we identify a novel interaction between galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan induces Dectin-2 dependent proinflammatory signaling in macrophages.

category: immunology


title: Heterotypic Influenza Infections Mitigate Susceptibility to Secondary Bacterial Infection

abstract: Influenza associated bacterial super-infections have devastating impacts on the lung and can result in increased risk of mortality. New strains of influenza circulate throughout the population yearly promoting the establishment of immune memory. Nearly all individuals have some degree of influenza memory prior to adulthood. Due to this we sought to understand the role of immune memory during bacterial super-infections. An influenza heterotypic immunity model was established using influenza A/PR/8/34 and A/X31. We report here that influenza experienced mice are more resistant to secondary bacterial infection with methicillin-resistant Staphylococcus aureus as determined by wasting, bacterial burden, pulmonary inflammation, and lung leak, despite significant ongoing lung remodeling. Multidimensional flow cytometry and lung transcriptomics revealed significant alterations in the lung environment in influenza-experienced mice compared with naive animals. These include changes in the lung monocyte and T cell compartments, characterized by increased expansion of influenza tetramer specific CD8+ T cells. The protection that was seen in memory experienced mouse model is associated with the reduction in inflammatory mechanisms making the lung less susceptible to damage and subsequent bacterial colonization. These findings provide insight into how influenza heterotypic immunity re-shapes the lung environment and the immune response to a re-challenge event, which is highly relevant to the context of human infection.


title: EstG is a novel esterase required for cell envelope integrity

abstract: Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the -proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell wall-targeting antibiotics, EstG does not demonstrate biochemical activity towards cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which is important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that, instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

category: microbiology


title: A recombinant murine rotavirus with Nano-Luciferase expression reveals tissue tropism, replication dynamics, and virus transmission

abstract: Rotaviruses (RVs) are one of the main causes of severe gastroenteritis, diarrhea, and death in children and young animals. Although suckling mice prove to be highly useful small animal models of RV infection and pathogenesis, direct visualization tools are lacking to track the temporal dynamics of RV replication and transmissibility in vivo. Here, we report the generation of the first recombinant murine RV that encodes a Nano-Luciferase reporter (NLuc) using a newly optimized RV reverse genetics system. The NLuc-expressing RV was replication-competent in cell culture and both infectious and virulent in neonatal mice in vivo. Strong luciferase signals were detected in the proximal and distal small intestines, colon, and mesenteric lymph nodes. We showed, via a noninvasive in vivo imaging system, that RV intestinal replication peaked at day 2 and day 5 post infection. Moreover, we successfully tracked RV transmission to uninoculated littermates as early as 3 days post infection, 1 day prior to clinically apparent diarrhea and 3 days prior to detectable fecal RV shedding in the uninoculated littermates. We also observed significantly increased viral replication in Stat1 knockout mice that lack the host interferon signaling. Our results suggest that the NLuc RV represents a non-lethal powerful tool for the studies of tissue tropism and host and viral factors that regulate RV replication and spread, as providing a new mechanism to facilitate the testing of prophylactic and therapeutic interventions in the future.


title: High levels of cyclic diguanylate interfere with beneficial bacterial colonization

abstract:
During colonization of the Hawaiian bobtail squid (Euprymna scolopes), Vibrio fischeri bacteria undergo a lifestyle transition from a planktonic motile state in the environment to a biofilm state in host mucus. Cyclic diguanylate (c-di-GMP) is a cytoplasmic signaling molecule that is important for regulating motility-biofilm transitions in many bacterial species. V. fischeri encodes 50 proteins predicted to synthesize and/or degrade c-di-GMP, but a role for c-di-GMP regulation during host colonization has not been investigated. We examined strains exhibiting either low or high levels of c-di-GMP during squid colonization and found that while a low c-di-GMP strain had no colonization defect, a high c-di-GMP strain was severely impaired. Expression of a heterologous c-di-GMP phosphodiesterase restored colonization, demonstrating that the effect is due to high c-di-GMP levels. In the constitutive high c-di-GMP state, colonizing V. fischeri exhibited reduced motility, altered biofilm aggregate morphology, and a transcriptional regulatory interaction between two key polysaccharides, cellulose and Syp. Our results highlight the importance of proper c-di-GMP regulation during beneficial animal colonization, illustrate multiple pathways regulated by c-di-GMP in the host, and uncover an interplay of multiple exopolysaccharide systems in host-associated aggregates.

IMPORTANCEThere is substantial interest in studying cyclic diguanylate (c-di-GMP) in pathogenic and environmental bacteria, which has led to an accepted paradigm in which high c-di-GMP levels promote biofilm formation and reduce motility. However, considerably less focus has been placed on understanding how this compound contributes to beneficial colonization. Using the Vibrio fischeri-Hawaiian bobtail squid study system, we took advantage of recent genetic advances in the bacterium to modulate c-di-GMP levels and measure colonization and track c-di-GMP phenotypes in a symbiotic interaction. Studies in the animal host revealed a c-di-GMP-dependent interaction between two distinct biofilm polysaccharides, Syp and cellulose, that was not evident in culture-based studies: elevated c-di-GMP altered the composition and abundance of the in vivo biofilm by decreasing Syp transcription due to increased cellulose synthesis. This study reveals important parallels between pathogenic and beneficial colonization and additionally identifies c-di-GMP-dependent regulation that occurs specifically in the squid host.


title: Structural basis of CHMP2A-CHMP3 ESCRT-III polymer assembly and membrane cleavage

abstract: The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo electron microscopy structures of membrane-coated CHMP2A-CHMP3 filaments of two different diameters at 3.3 and 3.6 [A] resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence towards the tube interior. Inter-filament interactions are electrostatic, which facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high speed atomic force microscopy imaging corroborate that CHMP2A-CHMP3 polymers and VPS4 can constrict and cleave narrow membrane tubes, thus acting as a minimal membrane fission machinery.

category: cell biology


title: Piezo mechanosensory channels regulate centrosome integrity

abstract: Piezo1 and 2 are evolutionarily conserved mechanosensory cation channels known to function on the cell surface by responding to external pressure and transducing a mechanically activated Ca2+ current. Here we show that both Piezo1 and 2 also exhibit concentrated intracellular localization at centrosomes. Both Piezo1 and 2 loss-of-function and Piezo1 activation by the small molecule Yoda1 produced supernumerary centrosomes due to inappropriate centriole disengagement. Using a centrosome-localized GCaMP Ca2+-sensitive reporter, we show that perturbations in Piezo modulate Ca2+ local concentration at centrosomes. We designed a photoactivable Yoda1 analog (caged-Yoda1) and revealed that its photoactivation specifically at centrosomes leads to rapid premature centriole disengagement within minutes. We identified the sorting nexin Snx5, which is involved in endocytic uptake and trafficking, as a protein-interactor with the conserved Piezo C-terminal domain, and Snx5 also co-localizes with Piezo1 and 2 at centrosomes. Moreover, inhibition of Polo-like-kinase 1 (PLK1) abolishes Yoda1-induced centriole disengagement. Collectively, these data suggest that Piezo1 and 2 in pericentrosomal endosomes control centrosome integrity, likely by maintaining local Ca2+ within a defined range through mechanotransduction of cell intrinsic forces from microtubules.


title: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites.

abstract: The precise control of phagosome maturation is critical for innate and adaptive immunity, determining whether phagocytosed material is destroyed or used to present antigens. We observed previously that non-fusogenic contacts between the endoplasmic reticulum (ER) and phagosomes, called membrane contact sites (MCS), are tethered by the calcium regulator STIM1 and fine-tune phagosomal maturation. The secretory pathway SNARE protein Sec22b has been implicated in controlling phagocytosis, phagosome maturation and antigen presentation, though its effects are controversial, and its mechanism of action poorly understood. Recently, Sec22b was shown to tether MCS at the plasma membrane without mediating membrane fusion. Here, we show that Sec22b localizes to and regulates the frequency of ER-phagosome contacts independently of STIM proteins. Sec22b knockdown and overexpression of a an MCS-disrupting mutant Sec22b-P33 induced only mild or no effect on global and local calcium signalling. However, Sec22b knockdown altered phagosomal phospholipids including PI(3)P, PI(4)P and PS, but not PI(4,5)P2. Increased PI(4)P in shSec22b cells was rescued by re-expression of Sec22b or the artificial MCS tether MAPPER but not the P33 mutant. Moreover, Sec22b co-precipitated and was co-recruited to phagosomes with the PS/PI(4)P lipid exchange protein ORP8. Expression of wild-type, but not mutant ORP8, also rescued phagosomal PI(4)P. Concordantly, Sec22b, MAPPER and ORP8 but not P33 or the ORP8 mutant decreased phagolysosome fusion in shSec22b cells. These results clarify a novel mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.


title: Phylogenomics and body shape morphometrics reveal recent origin and diversification in the goatfishes (Syngnatharia: Mullidae)

abstract: Clades of marine fishes exhibit many patterns of diversification, ranging from relatively constant throughout time to rapid changes in the rates of speciation and extinction. The goatfishes (Syngnatharia: Mullidae) are a family of marine, reef associated fishes with a relatively recent origin, distributed globally in tropical and temperate waters. Despite their abundance and economic importance, the goatfishes remain one of the few coral reef families for which the species level relationships have not been examined using genomic techniques. Here we use phylogenomic analysis of ultra-conserved elements (UCE) and exon data to resolve a well-supported, time-calibrated phylogeny for 72 species of goatfishes, supporting a recent origin of the goatfishes at 21.9 million years ago. We used this framework to test hypotheses about the associations among body shape morphometrics, taxonomy, and phylogeny, as well as to explore relative diversification rates across the phylogeny. Body shape was strongly associated with generic-level taxonomy of goatfishes, with morphometric analyses showing evidence for high phylogenetic signal across all morphotypes. Rates of diversification in this clade reveal a recent sharp increase in lineage accumulation, with 92% of the goatfish species sampled across all clades and major body plans having originated in just the past 5 million years. We suggest that habitat diversity in the early Pliocene oceans and the generalist ecology of goatfishes are key factors in the unusual evolutionary tempo of the family Mullidae.

category: evolutionary biology


title: Karyorelict ciliates use an ambiguous genetic code with context-dependent stop/sense codons

abstract: In ambiguous stop/sense genetic codes, the stop codon(s) not only terminate translation but can also encode amino acids. Such codes have evolved at least four times in eukaryotes, twice among ciliates (Condylostoma magnum and Parduczia sp.). These have appeared to be isolated cases whose next closest relatives use conventional stop codons. However, little genomic data have been published for the Karyorelictea, the ciliate class that contains Parduczia sp., and previous studies may have overlooked ambiguous codes because of their apparent rarity. We therefore analyzed single-cell transcriptomes from four of the six karyorelict families to determine their genetic codes. Reassignment of canonical stops to sense codons was inferred from codon frequencies in conserved protein domains, while the actual stop codon was predicted from full-length transcripts with intact 3-UTRs. We found that all available karyorelicts use the Parduczia code, where canonical stops UAA and UAG are reassigned to glutamine, and UGA encodes either tryptophan or stop. Furthermore, a small minority of transcripts may use an ambiguous stop-UAA instead of stop-UGA. Given the ubiquity of karyorelicts in marine coastal sediments, ambiguous genetic codes are not mere marginal curiosities but a defining feature of a globally distributed and abundant group of eukaryotes.

category: genomics


title: Tuning aromatic contributions by site-specific encoding of fluorinated phenylalanine residues in bacterial and mammalian cells

abstract:
The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a new group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools will enable deconstruction of biological mechanisms which utilize aromatic-pi interactions in structural and cellular contexts.

Statement of SignificanceThe aromatic side-chains of phenylalanine, tyrosine, and tryptophan are crucial for protein function and pharmacology due to their hydrophobic and electrostatic contributions to catalytic centers and ligand-binding pockets. However, few experimental approaches can chemically assess the functional roles of aromatics in cellular environments. The accepted computational method for aromatic interrogation is via serial fluorination, which lacks an experimental correlate in bacterial or mammalian cell systems. We have identified a family of synthetases to encode multiple different types of fluorinated phenylalanine residues in E. coli and HEK cells via nonsense suppression. The efficiency of these synthetases is sufficient to support biochemical characterization and structural determination of proteins with site-specific incorporation of unnatural phenylalanine analogs.

category: biochemistry


title: Goldilocks calcium and the mitochondrial respiratory chain: too much, too little, just right

abstract: Calcium (Ca2+) is a key regulator in diverse intracellular signaling pathways, and has long been implicated in metabolic control and mitochondrial function. Mitochondria can actively take up large amounts of Ca2+, thereby acting as important intracellular Ca2+ buffers and affecting cytosolic Ca2+ transients. Excessive mitochondrial matrix Ca2+ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture and cell death. But moderate Ca2+ within the organelle can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. However, in vitro studies involving the regulation of mitochondrial enzymes by Ca2+ may not uncover its full effects on oxidative phosphorylation. Here, we aimed to determine if extra or intramitochondrial Ca2+ modulate oxidative phosphorylation in mouse liver mitochondria and intact hepatocytes. We found that isolated mitochondria present increased respiratory control ratios (a measure of oxidative phosphorylation efficiency) when incubated with low and medium Ca2+ concentrations in the presence of complex I-linked substrates pyruvate plus malate and -ketoglutarate, respectively, but not complex II-linked succinate. In intact hepatocytes, both low and high cytosolic Ca2+ led to decreased respiratory rates, while ideal rates were present under physiological conditions. High Ca2+ decreased mitochondrial respiration in cells in a substrate-dependent manner, mediated by mPTP. Overall, our results uncover a Goldilocks effect of Ca2+ on liver mitochondria, with specific "just right" concentrations that activate oxidative phosphorylation.


title: Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

abstract:
The tumor microenvironment (TME) consists of different cell types that secrete proteins and also control the extracellular concentration of ions and metabolites. Changes in these intra-tumoral analytes and conditions, including K+, glucose, and pH, have been described to alter the metabolic activity of cancer cells, promote tumor cell growth, and impair anti-tumor immunity. However, the mechanisms regulating ion and metabolite levels and their effects on certain characteristics of the TME are still poorly understood. Therefore, accurate determination and visualization of analyte or state changes in real time within the TME is desired.

In this study, we genetically combined FRET-based fluorescent biosensors with nanobodies (Nbs) and used them for targeted visualization and monitoring of extracellular changes in K+, pH, and glucose on cell surfaces. We demonstrated that these recombinant biosensors quantitatively visualize extracellular K+ alterations on multiple cancer and non-cancer cell lines and primary neurons. By implementing a HER2 specific Nb, we generated K+ and pH sensors, which retain their functionality and specifically stained HER2 positive breast cancer cells. Based on the successful technical development of several Nb-biosensor combinations, we anticipate that this approach can be easily extended to design other targeted biosensors. Such versatile probes will open new possibilities for the reliable study of extracellular analytes in advanced 3D cell models or even in vivo systems.


title: Base editor scanning charts the DNMT3A activity landscape

abstract: DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulation of this process is known to cause aberrant development and oncogenesis, yet how DNMT3A is regulated holistically by its three domains remains challenging to study. Here we integrate base editing with a DNA methylation reporter to perform in situ mutational scanning of DNMT3A in cells. We identify mutations throughout the protein that perturb function, including ones at an interdomain interface that block allosteric activation. Unexpectedly, we also find mutations in the PWWP domain, a histone reader, that modulate enzyme activity despite preserving histone recognition and protein stability. These effects arise from altered PWWP domain DNA affinity, which we show is a noncanonical function required for full activity in cells. Our findings highlight mechanisms of interdomain crosstalk and demonstrate a generalizable strategy to probe sequence-activity relationships of nonessential chromatin regulators.


title: Expanding adult tubular microvessels on stiff substrates with endothelial cells and pericytes from the same tissue

abstract: Microvessels are essential for tissue engineering and regeneration. In current methods, endothelial cells are usually cultured in commercially available media and form a monolayer of cell sheets on stiff substrates and a tubular structure when cultured with soft hydrogels. To mimic the microvessels in vivo, researchers usually coculture the endothelial cells and pericytes from different adult tissues or derived from pluripotent stem cells in a three-dimensional hydrogel. However, there is a challenge for these models to reflect tissue-specific characteristics due to the vascular heterogeneity throughout the body. Here, we established a culture model for expanding adult tubular microvessels on stiff substrates with endothelial cells and pericytes derived from the same tissue. We isolated microvessels from adult rat subcutaneous soft connective tissue and cultured them on regular plastic dishes. We performed a series of screenings and formulated a custom-made medium (Medium-X), containing mainly antioxidants and three small molecules, Chir99021, A83-01, and Y27632. Medium-X significantly promoted adult microvessel growth while maintaining their characteristic tubular morphology up to 8 weeks in vitro, contrary to the monolayer of endothelial cell sheets in the commercially available medium EGM2MV. Transcriptomic analysis showed that Medium-X maintained the tubular morphology of microvessels by promoting angiogenesis and vascular remodeling while suppressing oxidation and lipid metabolic pathways. The model presented in this study can be applied to other organs for expanding organ-specific microvessels for tissue engineering and vascular regeneration.

category: bioengineering


title: Individual activator and repressor transcription factors induce global changes in chromatin mobility

abstract: The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. Yet how nucleus-wide chromatin structures respond dynamically to the activity of specific TFs remains unclear. Here we asked how TFs with opposite effects on modulating local chromatin accessibility impact nucleus-wide chromatin dynamics in living cells. We combine High-resolution Diffusion mapping (Hi-D) and Dense Flow reConstruction and Correlation (DFCC) to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs in live cells. We show that expression of individual activator and repressor TFs increases nucleus-wide chromatin mobility and induces opposite coherent chromatin motions at the micron scale. Our quantitative analysis thus reveals the broad and differential impact of TFs opening and closing chromatin in nucleus-wide chromatin dynamics.

category: biophysics


title: Exploring generality of experimental conformational changes with AlphaFold predictions

abstract: Structural predictions have matched the accuracy of experimental structures in the case of close homologues, outperformed docking methods for multimeric complexes and helped sampling the conformational landscape of transporters and receptors. Such successes prompt the question whether predictions can be used to relate experimental structures in the context of available knowledge. LysR-type transcriptional regulators (LTTR) constitute the most common family of bacterial regulators. Intriguingly, their experimental structures are remarkably diverse. The active species, composed of flexible monomers dimerizing through their N- and C-terminal domains in a circular arrangement, differ across LTTR, due to intrinsic sequence differences or because crystals stabilize diverse snapshots of a common dynamic mechanism. We have used AlphaFold2 (AF) to interrogate the experimental AtzR structure in the context of predictions guided towards the different hetero-multimeric conformations known for other LTTR. Our approach drives AF prediction with the structure-based selection of the information input through sequence alignment and template conformation, linked to examination of the energy with PISA and interactions with ALEPH.


title: Hit-and-run silencing of endogenous DUX4 by targeting DNA hypomethylation on D4Z4 repeats in facioscapulohumeral muscular dystrophy

abstract:
Facioscapulohumeral muscular dystrophy (FSHD), a progressive skeletal muscle disorder, is epigenetically characterized by DNA hypomethylation of the D4Z4 repeats in the 4q35 region, which enables aberrant DUX4 expression. Sustainable DUX4 suppression is thus a promising therapeutic strategy by which to prevent disease progression, but most of the supposed methods to achieve this depend on the expression of a mediator biochemical entity that would potentially narrow the quality of life of individuals with FSHD in the clinical context. In this study, we report that by applying hit-and-run silencing with dCas9-mediated epigenetic editing targeting DNA hypomethylation on D4Z4 repeats, we could achieve the suppression of endogenous DUX4 in our FSHD patient-derived iPSC model. Notably, DNA methylation was significantly upregulated in FSHD cells and suppression effects were observed for at least two weeks after intervention, which was not the case with transient treatments of typical dCas9-KRAB alone. Off-target analysis showed that despite the potential genome-wide risk for DNA methylation, the impact on the transcriptome was limited. We propose that hit-and-run silencing could be a promising option to prevent disease progression with minimum intervention for individuals with FSHD, motivating further study for clinical development.

Graphical abstract

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category: pathology


title: Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis

abstract: Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1-4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12-80%) and specificities (14-100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.


title: Proteolytic Analysis of Epsilon 34 Phage Tailspike protein indicates Partial Sensitivity to Proteinase K

abstract: Purified bacteriophage {varepsilon}34 tailspike protein ({varepsilon}34 TSP) can bind to Salmonella newington (S. newington) via the binding site of the protein, which is the O antigens of the LPS of the bacterium. We demonstrated else-where that purified {varepsilon}34 TSP possessed bacteria lytic property on S. newington. The {varepsilon}34 TSP has been shown via computational prediction to consist of parallel {beta}-helices like that of P22 TSP. These protein moieties are among the simplest repetitive structural elements in proteins. There exist extensive research on the folding behavior of {beta}-helix proteins, which also provides insight on how amyloid fibrils are generated since these proteins consist of similar parallel {beta}-helix motifs. One of the most significantly studied system for investigating protein folding is the from the Salmonella bacteriophage P22. The major component of this protein is a right-handed parallel {beta}-helix with 13 rungs. Initial in silico analysis of the {varepsilon}34 phage TSP indicates similar structural similarity to the P22 TSP. Our previous studies indicated that despite the similarities of the two proteins, P22 TSP shows higher resistance to proteases (e.g. trypsin) and heat compared to {varepsilon}34 TSP. In this study we further proof that {varepsilon}34 TSP is partially sensitive to proteinase K, whereas P22 TSP is completely resistant to this protein. Detailed analysis indicates that specific structural motifs of {varepsilon}34 TSP is insensitive to the protease, whereas other regions of the protein showed susceptibility to it.


title: Silent recognition of flagellins from human gut commensal bacteria by Toll-like receptor 5

abstract:
Flagellin, the protein unit of the bacterial flagellum, stimulates the innate immune receptor Toll-like receptor (TLR)5 following pattern recognition, or evades TLR5 through lack of recognition. This binary response fails to explain the weak agonism of flagellins from commensal bacteria, raising the question of how TLR5 response is tuned. Here, we describe a novel class of flagellin-TLR5 interaction, termed silent recognition. Silent flagellins are weak agonists despite high affinity binding to TLR5. This dynamic response is tuned by TLR5-flagellin interaction distal to the site of pattern recognition. Silent flagellins are produced primarily by the abundant gut bacteria Lachnospiraceae and are enriched in non-Western populations. These findings provide a mechanism for the innate immune system to tolerate commensal-derived flagellins.

One-Sentence SummaryTLR5 sensitively recognizes, but responds weakly to, flagellins from gut commensal bacteria.


title: The cellular poly(rC)-binding protein 2 prevents the early steps of hepatitis C virus virion assembly

abstract:
Poly(rC)-binding protein 2 (PCBP2) was previously shown to bind to the hepatitis C virus (HCV) genome; however, its precise role in the viral life cycle remained unclear. Herein, we found that PCBP2 does not directly affect viral entry, translation, genome stability, replication, or virion egress. Rather, our data suggests that endogenous PCBP2 normally limits virion assembly, thereby indirectly promoting translation and replication by increasing the translating/replicating pool of viral RNAs. Additionally, we found that an alternative RNA conformation (SLIIalt) was important for efficient virion assembly, but functions in a PCBP2-independent manner. The latter may explain why the Japanese fulminant hepatitis 1 isolate is able to produce infectious particles in cell culture, while other HCV isolates are lost in translation. Taken together, our results suggest that PCBP2 and SLIIalt independently modulate HCV genome packaging and alter the balance of viral RNAs in the translating/replicating pool and those engaged in virion assembly.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

category: molecular biology


title: Supramolecular architecture of the ER-mitochondria encounter structure in its native environment

abstract: The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis which rely on lipid exchange via membrane contact sites, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the ER-mitochondria encounter structure ERMES, a four-subunit complex of unclear stoichiometry and architecture. We determined the molecular organization of ERMES within cells using integrative structural biology, combining quantitative live-imaging, cryo-correlative microscopy, subtomogram averaging and molecular modeling. ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three lipid-binding SMP domains arranged in zig-zag fashion. Our molecular model of ERMES reveals an unconventional restrained pathway for lipids. These findings resolve a supramolecular architecture controlling interorganelle lipid fluxes.


title: An Inhibitory Function of TRPA1 Channels in TGF-β1-driven Fibroblast to Myofibroblast Differentiation

abstract: Transient receptor potential ankyrin 1 (TRPA1) is a non-selective Ca2+ permeable cation channel, which was originally cloned from human lung fibroblasts (HLFs). TRPA1-mediated Ca2+ entry is evoked by exposure to several chemicals, including allyl isothiocyanate (AITC), and a protective effect of TRPA1 activation in the development of cardiac fibrosis has been proposed. Yet, the function of TRPA1 in transforming growth factor {beta}1 (TGF-{beta}1)-driven fibroblast to myofibroblast differentiation and the development of pulmonary fibrosis remains elusive. TRPA1 expression and function was analyzed in cultured primary HLFs, and mRNA levels were significantly reduced after adding TGF-{beta}1. Expression of genes encoding fibrosis markers, e.g. alpha smooth muscle actin (ACTA2), plasminogen activator inhibitor 1 (SERPINE1), fibronectin (FN1) and type I collagen (COL1A1) was increased after siRNA-mediated down-regulation of TRPA1-mRNA in HLFs. Moreover, AITC-induced Ca2+ entry in HLFs was decreased after TGF-{beta}1 treatment and by application of TRPA1 siRNAs, while AITC treatment alone did not reduce cell viability or enhanced apoptosis. Cell barrier function increased after addition of TRPA1 siRNAs or TGF-{beta}1 treatment. Most interestingly, AITC-induced TRPA1 activation augmented ERK1/2 phosphorylation, which might inhibit TGF-{beta}-receptor signaling. Our results suggest an inhibitory function of TRPA1 channels in TGF-{beta}1-driven fibroblast to myofibroblast differentiation. Therefore, activation of TRPA1 channels might be protective during the development of pulmonary fibrosis in patients.


title: Viral origin of eukaryotic type IIA DNA topoisomerases

abstract: Type II DNA topoisomerases of the family A (Topo IIA) are present in all bacteria (DNA gyrase) and eukaryotes. In eukaryotes, they play a major role in transcription, DNA replication, chromosome segregation and modulation of chromosome architecture. The origin of eukaryotic Topo IIA remains mysterious since they are very divergent from their bacterial homologues and have no orthologues in Archaea. Interestingly, eukaryotic Topo IIA have close homologues in viruses of the phylum Nucleocytoviricota, an expansive assemblage of large and giant viruses formerly known as the nucleocytoplasmic large DNA viruses (NCLDV). Topo IIA are also encoded by some bacterioviruses of the class Caudoviricetes (tailed bacteriophages). To elucidate the origin of the eukaryotic Topo IIA, we performed in-depth phylogenetic analyses combining viral and cellular Topo IIA homologs. Topo IIA encoded by bacteria and eukaryotes form two monophyletic groups nested within Topo IIA encoded by Caudoviricetes and Nucleocytoviricota, respectively. Importantly, Nucleocytoviricota remained well separated from eukaryotes after removing both bacteria and Caudoviricetes from the dataset, indicating that the separation of Nucleocytoviricota and eukaryotes is probably not due to long branch attraction artefact. The topology of our tree suggests that the eukaryotic Topo IIA was probably acquired from an ancestral member of the Nucleocytoviricota of the class Megaviricetes, before the emergence of the last eukaryotic common ancestor (LECA). This result further highlights a key role of these viruses in eukaryogenesis and suggests that early proto-eukaryotes used a Topo IIB instead of a Topo IIA for solving their DNA topological problems.


title: Long-term balancing selection and the genetic load linked to the self-incompatibility locus in Arabidopsis halleri and A. lyrata.

abstract: Balancing selection is a form of natural selection maintaining diversity at the sites it targets and at linked nucleotide sites. It is generally expected to facilitate the accumulation of a "sheltered" genetic load of linked deleterious mutations, but the overall extent of its genomic impact remains poorly documented. Taking advantage of plant self-incompatibility as one of the best-understood and most intense examples of long-term balancing selection, we provide the most highly resolved picture of the effect of balancing selection on the sheltered genetic load in any plant genome. We used targeted genome resequencing to evaluate the intensity of indirect selection on the genomic region flanking the self-incompatibility locus in three sample sets in each of the two closely related plant species Arabidopsis halleri and A. lyrata, and used 100 control regions from throughout the genome to factor out differences in demographic histories and/or sample structure. Nucleotide polymorphism increased strongly around the S-locus in all sample sets, but only over a limited genomic extent, as it became indistinguishable from the genomic background beyond the first 25kb. Genes around the S-locus carried more mutations than those in the control regions, but in contrast to the classical model for the accumulation of the sheltered load we observed no relaxation of the efficacy of purifying selection. Overall, our results challenge our understanding of how natural selection in one genomic region affects the evolution of the adjacent genomic regions, and have consequences for how systems of mating that enforce outcrossing in populations are maintained.


title: Human and murine Cryptococcus neoformans infection selects for common genomic changes in an environmental isolate.

abstract: A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from the cerebral spinal fluid (CSF) of an immunocompromised patient with cryptococcosis based on the molecular analyses available in 2000. Here we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MAT strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including 8 nonsynonymous changes involving 7 genes. To ascertain whether changes in these genes are selected during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frame-shift mutation in one of the seven genes altered in the human sample, a gene predicted to encode a SWI-SNF chromatin-remodeling complex protein. Both cockatoo and patient strains as well as mouse passaged isolates obtained from brain tissue had a premature stop codon in a homolog of ZFC3, a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and appears to have reverted to a full-length protein in the mouse passaged isolates obtained from lung tissue. The patient strain and mouse passaged isolates show variability in the expression of virulence factors, with differences in capsule size, melanization, and rates on non-lytic expulsion from macrophages observed. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.


title: A chromosome-scale genome assembly of a Bacillus thuringiensis Cry1Ac insecticidal protein resistant strain of Helicoverpa zea

abstract:
Helicoverpa zea (Lepidoptera: Noctuidae) is an insect pest of major cultivated crops in North and South America. The species has adapted to different host plants and developed resistance to several insecticidal agents, including Bacillus thuringiensis (Bt) insecticidal proteins in transgenic cotton and maize. H. zea populations persist year-round in tropical and subtropical regions, but seasonal migrations into temperate zones increase the geographic range of associated crop damage. To better understand the genetic basis of these physiological and ecological characteristics, we generated a high-quality chromosome-level assembly for a single H. zea male from Bt resistant strain, HzStark_Cry1AcR. Hi-C data were used to scaffold an initial 375.2 Mb contig assembly into 30 autosomes and the Z sex chromosome (scaffold N50 = 12.8 Mb and L50 = 14). The scaffolded assembly was error-corrected with a novel pipeline, polishCLR. The mitochondrial genome was assembled through an improved pipeline and annotated. Assessment of this genome assembly indicated 98.8% of the Lepidopteran Benchmark Universal Single-Copy Ortholog set were complete (98.5% as complete single-copy). Repetitive elements comprised approximately 29.5% of the assembly with the plurality (11.2%) classified as retroelements. This chromosome-scale reference assembly for H. zea, ilHelZeax1.1, will facilitate future research to evaluate and enhance sustainable crop production practices.

SignificanceWe established a chromosome-level reference assembly for Helicoverpa zea, an insect pest of multiple cultivated crops in the Americas. This assembly of a Bacillus thuringiensis insecticidal protein resistant strain, HzStark_Cry1AcR, will facilitate future research in areas such as population genomics and adaptations to agricultural control practices.


title: A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

abstract: Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We developed a novel tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3,000 (>9,500) phosphopeptides from 1 (10) {micro}g of cell lysate using the label-free method without a spectral library. It also enabled precise quantification of [~]600 phosphopeptides from 100 cells sorted by FACS (single-cell level input for the enriched phosphopeptides) and [~]700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 {micro}m (equivalent to [~]100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.


title: Cryo-EM structure of an atypical proton-coupled peptide transporter: Di- and tripeptide permease C

abstract: Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The E. coli genome codes for four different POTs, known as Di- and Tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 [A]. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.


title: Proteomic study of differentially expressed proteins in seeds between parents and offspring of castor bean (Ricinus communis L.)

abstract: Castor bean (Ricinus communis L.), one of the top 10 oilseed crops in the world, has high economic value. Hybridization is the most direct and effective method to breed new varieties with high yield, high oil content, and strong stress resistance. Therefore, prediction of desired traits in castor hybrid offspring is particularly important. In this study, proteomic analysis was performed to identify differentially expressed proteins (DEPs) in seeds between castor hybrid offspring and their female (Lm female line aLmAB2) and male parents (CSR*181). Among the DEPs upregulated in the seeds of hybrid offspring, the majority were related to seed yield and stress resistance, while some were related to oil synthesis and fatty acid synthesis and metabolism in seeds. In other words, the hybrid offspring showed heterosis for seed yield, stress resistance, oil synthesis, and fatty acid synthesis and metabolism when compared with their parents. Further, real-time quantitative polymerase chain reaction assays were performed on 12 genes encoding DEPs involved in oil synthesis, pollen abortion, yield, and stress resistance of seeds. The results showed that the expression levels of the 12 genes were consistent with those of the DEPs.


title: Deciphering the mRNA-lncRNA-miRNA interaction landscape in human pluripotency

abstract:
Human embryonic stem cells offer a huge potential for the study of early human development and for application in biomedical sciences. Growing evidence has shown that besides the protein coding genes, the non-coding elements of the human genome play a crucial role in maintaining its property of self renewal and in cell fate determination. However, a clear understanding of this regulatory mechanism and the landscape of interactions between the coding and non-coding elements was still lacking. To fill in this void, we use transcriptomic data from RNA-seq and small RNA-seq experiments to reconstruct the core pluripotency circuitry involving mRNAs, lncRNAs and miRNAs. The overall interaction landscape revealed an alternate circuit for the maintenance of pluripotency devoid of the classic pluripotency transcription factors NANOG, SOX2 and POU5F1. We also identified networks specific to the naive and primed states of human pluripotency revealing a new set of transcriptomic markers that could not only be used to differentiate pluripotent state from non-pluripotent state but also to identify the intra-pluripotency state. The lncRNA DANT1 was found to be crucial in determination to the two pluripotency states as it formed a bridge between the naive and primed state specific pluripotency networks. Further, we also identified and computationally validated putative ceRNA mechanism involving DANT1, the miRNAs hsa-miR-30c-2-3p, hsa-miR-210-3p and hsa-let-7b-5p, and several key pluripotency related genes including PTPRZ1, SALL2, TOX3, ZNF695, and ZYG11A which warrants further experimental validation.

HIGHLIGHTSO_LIReconstructed the key MLMi circuitry underlying human pluripotency by combining RNA-Seq data and known interaction information.
C_LIO_LIIdentified an alternate pluripotency circuit devoid of classic pluripotency transcription factors NANOG, SOX2 and POU5F1.
C_LIO_LIPredicted a new set of markers that can not only distinguish between pluripotent and non-pluripotent states but also identify the intra-pluripotency state.
C_LIO_LIIdentified novel putative function of lncRNA DANT1 as ceRNA in formation of naive and primed state pluripotency.
C_LIO_LIDANT1 harbours binding sites for miRNAs hsa-miR-30c-2-3p, hsa-miR-210-3p and hsa-let-7b-5p.
C_LI


title: Machine learning-assisted fluoroscopy of bladder function in awake mice

abstract: Understanding the lower urinary tract (LUT) and development of highly needed novel therapies to treat LUT disorders depends on accurate techniques to monitor LUT (dys)function in preclinical models. We recently developed videocystometry in rodents, which combines intravesical pressure measurements with X-ray-based fluoroscopy of the LUT, allowing the in vivo analysis of the process of urine storage and voiding with unprecedented detail. Videocystometry relies on the precise contrast-based determination of the bladder volume at high temporal resolution, which can readily be achieved in anesthetized or otherwise motion-restricted mice but not in awake and freely moving animals. To overcome this limitation, we developed a machine-learning method, in which we trained a neural network to automatically detect the bladder in fluoroscopic images, allowing the automatic analysis of bladder filling and voiding cycles based on large sets of time-lapse fluoroscopic images (>3 hours at 30 images/second) from behaving mice and in a non-invasive manner. With this approach, we found that urethane, an injectable anesthetic that is commonly used in preclinical urological research, has a profound, dose-dependent effect on urethral relaxation and voiding duration and that implantation of a suprapubic catheter, as is standardly performed for cystometric analyses, leads to a [~]4-fold reduction in bladder capacity. Our findings provide a paradigm for the non-invasive, in vivo monitoring of a hollow organ in behaving animals and pinpoint important limitations of the current gold standard techniques to study the LUT in mice.

category: physiology


title: Genotype-dependent responses to long-term water stress in Chenopodium quinoa Willd

abstract:
Within the current climate context, freshwater resources have become scarce. Agriculture, especially in rain-fed conditions, should deal with the need for increasing yields to contribute to food security under limiting water availability. Exploring underutilized crops such as Chenopodium quinoa (quinoa) has become a unique opportunity as some of these crops possess the ability to tolerate several abiotic stresses, including drought. In line with this, this work aimed at evaluating the genotype-dependent response to drought by comparing the performance of different European-adapted cultivars (F14, F15, F16, and Titicaca). The results show that the cultivars here evaluated presented different mechanisms to cope with long-term water stress, including changes in phenology, morphology, or physiology. Among them, the cultivar F16 might be the most promising genotype to grow under water-limiting conditions as it was able to increase Water Use Efficiency (WUE), reducing the stomatal conductance and keeping CO2 assimilation rates similar to well-watered conditions, maintaining seed yield and increasing harvest index (HI) under water deficit conditions. Furthermore, based on these results, we propose a model in which differences between a tolerant and a sensitive genotype are presented. Altogether, we believe that this work will significantly contribute to broadening our understanding regarding how quinoa responds to long-term water stress highlighting genotype-related differences that will allow the selection of the best adapted genotypes for water-limiting environments.

HighlightQuinoa physiological, phenological, and morphological distinct responses to long-term water stress depending on the genotype.

category: plant biology


title: Control and coding of pupil size by hypothalamic orexin neurons

abstract: Brain orexin (hypocretin) neurons are implicated in sleep-wake switching and reward-seeking, but their roles in rapid arousal dynamics and reward perception remain unclear. Here, cellspecific stimulation, deletion, and in vivo recordings revealed strong correlative and causal links between pupil dilation - a quantitative arousal marker - and orexin cell activity. Coding of arousal and reward was distributed across orexin cells, indicating that they specialize in rapid, multiplexed communication of momentary arousal and reward states.

category: neuroscience


title: Calibration allows accurate estimation of the axonal volume fraction with diffusion MRI

abstract: Biophysical models enable the non-invasive estimation of microstructural tissue features of the central nervous system such as the axonal volume fraction using diffusion weighted imaging (DWI)-data. However, these models trade accuracy with complexity and demands for time-efficient data acquisition. In this study, we hypothesise that their accuracy can be improved substantially through biophysically motivated, linear calibration. We test this hypothesis in the context of axonal volume fraction estimation in four different DWI-models of different complexity using multi-modal data including ex-vivo diffusion MRI- and electron microscopy (EM)-data in mice with broad dynamic range, whereby the latter served as gold standard. We found that two calibration parameters, an offset accounting for the fraction of unmyelinated axons in severely hypomyelinated mice and a scaling accounting for the compartment-specific relaxation, substantially improved the accuracy of axonal volume fraction. Furthermore, we theoretically predict the scaling parameter, and demonstrate that similar accuracy improvement can be achieved for a subset of DWI-models, if the scaling parameter is fixed to the predicted value instead of estimating it on basis of data. This is of practical relevance because it allows to estimate the remaining offset calibration parameter from a limited amount of multi-modal data and thus makes the proposed method usable in human brain data.


title: Binding of different substrate molecules at the docking site and the active site of γ-secretase can trigger toxic events in sporadic and familial Alzheimer's disease

abstract:
Significancepathogenic changes in {gamma}-secretase activity, and its response to different drugs, can be greatly affected by changes in saturation of {gamma}-secretase with its substrate. The molecular mechanism is unclear.

Resultsmultiscale molecular dynamics studies show that saturation of {gamma}-secretase with its substrate can result in parallel binding of different substrate molecules at the docking site and the active site. C-terminal domain of the second substrate can bind at cytosolic end of presenilin subunit while {gamma}-secretase is still processing its first substrate. Such interactions can disrupt dynamic presenilin structures that regulate proteolytic steps. Similar disruptions in dynamic presenilin structures can be produced by different drugs and by different disease-causing mutations. Thus, the presented two-substrate mechanism, can explain toxic inhibition of {gamma}-secretase activity and toxic increase in production of the longer, more hydrophobic, A{beta}-proteins. Toxic aggregation between N-terminal domains of the two substrates is controlled by nicastrin ectodomain. Such aggregation is more likely to happen with C99-{beta}CTF-APP than with C83-CTF-APP substrate, which can explain, why {beta}-secretase path is more pathogenic than -secretase path. The binding of C99-{beta}CTF-APP substrate to {gamma}-secretase can be controlled by substrate-channeling between nicastrin ectodomain and {beta}-secretase.

ConclusionsThe presented two-substrate mechanism can explain why different studies consistently show that increase in saturation of {gamma}-secretase with its substrate can support pathogenic changes in different sporadic and familiar cases of the disease. Future drug-development strategies can target different physiological mechanisms that control the balance between cellular levels of {gamma}-secretase activity and the total amyloid metabolism.


title: Representation of illusory shapes within the topographic areas of the posterior parietal cortex

abstract:
The human visual system consists of multiple topographic maps that extend from the early visual cortex along the dorsal and ventral processing streams. Responses to illusory shapes within these maps have been demonstrated in the ventral stream areas, in particular the lateral occipital complex. Recently, the intraparietal sulcus of the dorsal stream has been linked to the processing of illusory shapes defined by motion. It therefore remains unclear whether the topographically organized parietal areas also respond to static illusory shapes, which would suggest their generic role in representing illusory content. Here we measured brain responses using fMRI while human participants observed flickering inducers around the fixation task. The inducers either formed an illusory diamond in the center, a triangle in the left or in the right hemifield, or were inverted such that no illusory figure was formed. We compared responses of parietal regions IPS0-IPS5 and SPL1 to each illusory figure with the non-illusory condition. To determine the role of attention in illusory shape responses we manipulated the difficulty of the fixation task. Our results show that all IPS areas responded to illusory shapes. The more posterior areas IPS0-IPS3 additionally displayed a preference towards the contralateral shapes, while the more anterior areas IPS4 and IPS5 showed response attenuation with increased task difficulty. We suggest that the IPS can represent illusory content irrespective of the perceptual mechanism that generated it. These responses may serve as a potential feedback signal that drives illusory shape responses in early and ventral visual areas.

Significance statementThe traditional view of the ventral visual pathway being solely responsible for representation of objects has recently been challenged by demonstrating illusory shape representation within the dorsal visual pathway with moving bistable stimuli. Our results provide evidence for the dorsal stream contribution to representing not only moving, but also static illusory shapes. Our results also show a functional subdivision along the topographic maps, with spatially specific shape responses in the more posterior, and attention-dependent responses in the more anterior areas. IPS areas of the dorsal stream should thus be considered in the theoretical accounts and neural models of how subjective content is generated in the brain.


title: Stably bound adaptor proteins modulate directionality of RNP transport

abstract: Kinesin-1 and cytoplasmic dynein are molecular motors that mediate long range transport of cargoes along the microtubule cytoskeleton. oskar RNA has been documented to switch between the motors during its localization in the Drosophila germline syncytium. oskar RNA undergoes dynein-mediated transport from the transcriptionally active nurse cells into the oocyte, following which the RNA translocates via kinesin to the posterior pole. Adaptor proteins link the RNA to its motors: the Egalitarian-Bicaudal-D complex links dynein to oskar RNA for the initial phase of transport, whereas atypical Tropomyosin 1 (aTm1) links kinesin-1 to oskar RNA for the latter phase. Components of the Exon Junction Complex (EJC) as well as the SOLE, a stem loop formed upon splicing of oskar RNA, have also been found to be necessary for kinesin-mediated transport of oskar RNA. In this study, to dissect the minimal elements required for kinesin-based transport, we tethered aTm1 or kinesin-1 to oskar RNA constructs lacking the SOLE. Our results suggest that stably bound aTm1 can indeed bypass the SOLE and EJC to mediate kinesin-1 activity, but the effects of tethered aTm1 are less potent than that of tethered kinesin-1. We also tethered Bicaudal-D to oskar RNA, to test whether this would affect kinesin-directed transport of oskar RNA, and found that tethered Bicaudal-D directs dynein mediated localization. Our results show that activated Bicaudal-D, along with the recruited dynein, is sufficient for dynein activity. We also show that stable binding of kinesin-1 to the RNA cargo is sufficient for strong kinesin-1 activity. Stably bound aTm1, meanwhile, can only mediate mild kinesin activity, suggesting that other factors may be required to stabilize the binding of kinesin-1 to the RNA cargo.


title: An updated C. elegans body muscle transcriptome for studies in muscle formation and function

abstract:
The body muscle is an important tissue used in organisms for proper viability and locomotion. The contractile unit of the muscle is the sarcomere, which is ultimately responsible for the contraction reaction leading to movement. Although this tissue is generally well studied and characterized, and many pathways have been elucidated throughout the years, we still lack a comprehensive understanding of its transcriptome, and how it controls muscle development and function. Here, we have updated a nuclear FACS sorting-based approach to isolate and sequence a high-quality muscle transcriptome from C. elegans mixed stage animals. We have identified 2,848 muscle-specific protein-coding genes, including 78 transcription factors and 206 protein-coding genes containing an RNA binding domain. We studied their interaction network, performed a detailed promoter analysis, and identified novel muscle-specific cis-acting elements. We have also identified 16 high-quality muscle-specific miRNAs, studied their function in vivo using fluorochrome-based analyses, and developed the first high-quality miRNA Interactome in a living organism, incorporating other muscle-specific datasets produced by our lab and others.

Our study expands our understanding of how muscle tissue functions in C. elegans and in turn, provide results that can in the future be applied to humans to study muscular-related diseases.

category: genetics


title: Differential gene expression underlying the biosynthesis of Dufour's gland signals in Bombus impatiens

abstract: Pheromones regulating social behavior are one of the most explored phenomena in social insects. However, compound identity, biosynthesis and their genetic regulation are known in only a handful of species. Here we conducted chemical and RNA-seq analyses of the Dufours gland in the bumble bee Bombus impatiens and examined the signals and the pathways regulating signal production in queens and workers. Across Hymenopterans, the Dufours gland contains mostly long-chained hydrocarbons and esters that signal reproductive and social status in several bee species. In bumble bees, the Dufours gland contains queen- and worker-specific esters, in addition to terpenes and terpene-esters only found in gynes and queens. These compounds are assumed to be synthesized de novo in the gland, however, their genetic regulation is unknown. A whole transcriptome gene expression analysis of the gland in queens, gynes, queenless and queenright workers showed distinct transcriptomic profiles, with thousands of differentially expressed genes between the groups. Workers and queens express genes associated with key enzymes in the biosynthesis of wax esters, while queens and gynes preferentially express key genes in terpene biosynthesis. In contrast, no genes were differentially expressed in queenless and queenright workers, despite differences in their Dufours gland chemistry and reproductive state, suggesting the quantitative differences in worker secretion are not regulated at the level of production. Overall, our data demonstrate gland-specific regulation of chemical signals associated with social behavior and identifies genes and pathways regulating caste-specific chemical signals in social insects.


title: A systematic examination of learning in the invasive ant Linepithema humile reveals very rapid development of short and long-term memories

abstract: The Argentine ant (Linepithema humile) is one of the most damaging and widespread invasive ant species worldwide. However, control attempts often fail due to insufficient bait uptake, or bait abandonment. Increasing preference for, and consumption of, is thus an important requirement for successful control. Associative learning and within-nest information transfer might be a potential tool for achieving this goal. Here, we conducted a detailed and systematic investigation of olfactory learning and side learning in Argentine ants. The ants showed very strong and rapid side learning, choosing the correct arm in a Y-maze 65% of time after just one visit, and 84% correct after two. Odour learning was even more rapid, with just one visit to a flavoured food source, reached by a scented runway, leading to 85% choices for the corresponding scent on a Y-maze. Further experiments demonstrated that having two cues (runway odour and food flavour) does not improve learning significantly over just one cue. This rapid learning is long-lasting, with one exposure to a runway odour associated with a reward resulting in a strong preference (73%) for this odour even after 48 hours. Food flavour information is transferred efficiently between nestmates in the nest, driving preference: naive ants housed with ants fed on flavoured food show a strong preference (77%) for that odour after 24 hours. Our results demonstrate the impressive learning abilities of Linepithema humile, which coupled with efficient intranidal information transfer and strong use of pheromonal recruitment may help explain their ability to discover and then dominate resources. However, these strengths could potentially be used against them, by exploiting learning and information transfer to increase toxic bait uptake during control efforts. Steering ant preference by leveraging learning might be an underappreciated tool in invasive alien species control.

category: animal behavior and cognition


title: Inverse Material Parameter Estimation of Patient Specific Finite Element Models at the Carotid Bifurcation: The Impact of Excluding the Zero Pressure Configuration and Residual Stress

abstract:
Abstract SummaryThe carotid bifurcation experiences a complex loading environment due to its anatomical structure. Previous in-vivo material parameter estimation methods often use simplified model geometries, isotropic hyperelastic constitutive equations or neglect key aspects of the vessel, such as the zero-pressure configuration or residual stress. These factors have independently been shown to alter the stress environment of the vessel wall. Characterising the location of high stress in the vessel wall has often been proposed as a potential indicator of structural weakness. However, excluding the afore-mentioned zero-pressure configuration, residual stress and patient specific material parameters can lead to an incorrect estimation of the true stress values observed, meaning stress alone as a risk indicator of rupture is insufficient. In this study, we investigate how the estimated material parameters and overall stress distributions in geometries of carotid bifurcations, extracted from in-vivo MR images, alter with the inclusion of the zero-pressure configuration and residual stress.

This approach consists of the following steps: (1) geometry segmentation and hexahedral meshing from in-vivo MRI images at two known phases; (2) computation of the zero-pressure configuration and the associated residual stresses; (3) minimisation of an objective function built on the difference between the stress states of an " almost true" stress field at two known phases and a "deformed" stress field by altering the input material parameters to determine patient specific material properties; and (4) comparison of the stress distributions throughout these carotid bifurcations for all cases with estimated material parameters. This numerical approach provides insights into the need for estimation of both the zero-pressure configuration and residual stress for accurate material property estimation and stress analysis for the carotid bifurcation, establishing the reliability of stress as a rupture risk metric.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/487823v1_ufig1.gif" ALT="Figure 1">
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title: Proximal Exploration for Model-guided Protein Sequence Design

abstract: Designing protein sequences with a particular biological function is a long-lasting challenge for protein engineering. Recent advances in machine-learning-guided approaches focus on building a surrogate sequence-function model to reduce the demand for expensive in-lab experiments. In this paper, we study the exploration mechanism of model-guided sequence design. We leverage a natural property of protein fitness landscape that a concise set of mutations upon the wild-type sequence are usually sufficient to enhance the desired function. By utilizing this property, we propose Proximal Exploration (PEX) algorithm that conducts a proximal optimization framework to prioritize the search for advantageous mutants with low mutation counts. In addition, we construct a specialized model architecture to predict low-order mutational effects, which further improves the sample efficiency of model-guided evolutionary search. We extensively evaluate our method on a suite of in-silico protein sequence design tasks and demonstrate substantial improvement over baseline algorithms.


title: Cell-free, methylated DNA in blood samples reveals tissue-specific, cellular damage from radiation treatment

abstract:
Radiation therapy is an effective cancer treatment although damage to healthy tissues is common. Here we establish sequencing-based, cell-type specific DNA methylation reference maps of human and mouse tissues to infer the origins of cell-free DNA fragments released from dying cells into the circulation. We find cell-type specific DNA blocks mostly hypomethylated and located within genes intrinsic to cellular identity. In a mouse model, thoracic radiation-induced tissue damages were reflected by dose-dependent increases in lung endothelial, cardiomyocyte and hepatocyte methylated DNA in serum. The analysis of serum samples from breast cancer patients undergoing radiation treatment revealed distinct tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation indicating the impact on liver tissues. Thus, cell-free methylated DNA in serum can uncover cell-type specific effects of radiation on healthy tissues and inform treatment.

Graphical Abstract

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category: cancer biology


title: Physiological insight into the conserved properties of Caenorhabditis elegans acid-sensing DEG/ENaCs

abstract: Acid sensing ion channels (ASICs) are members of the diverse family of degenerin/epithelial sodium channels (DEG/ENaCs). They perform a wide range of physiological roles in healthy organisms, including in gut function and synaptic transmission, but also play important roles in disease, as acidosis is a hallmark of painful inflammatory and ischaemic conditions. We performed a screen for acid-sensitivity on all 30 subunits of the C. elegans DEG/ENaC family using Two-Electrode Voltage Clamp (TEVC) in Xenopus oocytes. We found two groups of acid-sensing DEG/ENaCs characterised by being inhibited or activated by increasing proton concentrations. Three of these acid-sensitive C. elegans DEG/ENaCs were activated by acidic pH, making them functionally similar to the vertebrate ASICs. We also identified four new members of the acid-inhibited DEG/ENaC group, giving a total of seven additional acid-sensitive channels. We observed sensitivity to the anti-hypertensive drug amiloride as well as modulation by the trace element zinc. Acid-sensitive DEG/ENaCs were found to be expressed in both neurons and non-neuronal tissue, highlighting the likely functional diversity of these channels. Our findings provide a framework to exploit the C. elegans channels as models to study the function of these acid-sensing channels in vivo, as well as to study them as potential targets for anti-helminthic drugs.


title: Modulation of Arabidopsis growth by volatile organic compounds from a root-derived bacterial community

abstract: Plant roots are surrounded by fluctuating biotic and abiotic factors. The living component - the microbiota - is actively shaped by the plant and plays an important role in overall plant health. While it has been shown that specialized metabolites exuded from the plant are involved in shaping host interactions with the microbiota, it is unclear how underground volatile organic compounds (VOCs) influence this communication. This is especially true for root-associated bacteria which are known to release VOCs that can influence plant growth. Using a simplified synthetic bacterial community (SynCom) representing the phylogenetic diversity of bacteria in the root microbiome, we set out to characterize plant growth and defense metabolites when subjected to bacterial VOCs (bVOCs). Moreover, by profiling the SynCom community composition after co-cultivation with the plant, we explored how members of the community influenced each other in our growth setup. Our findings reveal that plant growth promotion can occur via VOCs from a bacterial SynCom, but that the plant response differs for individual community members. In addition, we find that bVOCs are able to repress chemical defense responses in the plant, possibly to facilitate colonization. By removing key species from the SynCom, we find that complex bacteria-bacteria interactions are likely to underlie this phenomenon, and that bVOC-induced modulation of plant responses in the rhizosphere may be an emergent property of bacterial communities rather than depending on individual species.


title: Two stages of bandwidth scaling drives efficient neural coding of natural sounds

abstract: Theories of efficient coding propose that the auditory system is optimized for the statistical structure of natural sounds, yet the transformations underlying optimal acoustic representations are not well understood. Using a database of natural sounds including human speech and a physiologically-inspired auditory model, we explore the consequences of peripheral (cochlear) and mid-level (auditory midbrain) filter tuning transformations on the representation of natural sound spectra and modulation statistics. Whereas Fourier-based spectrographic decompositions with equal resolution filters can preserve spectral details, cochlear filters with bandwidth scaling, sacrifice spectral information while producing a more robust temporal representation. Cochlear bandwidth scaling produces a frequency-dependent gain that counteracts the tendency of natural sound power to decrease with frequency, resulting in a whitened output representation. Mid-level auditory filter scaling further enhance the representation of natural sounds by producing a whitened modulation power spectrum (MPS) with higher modulation entropy than both the cochlear outputs and the conventional Fourier MPS. These findings suggest that the tuning characteristics of the peripheral and mid-level auditory system together produce a whitened output representation in three dimensions (frequency, temporal and spectral modulation) that reduces redundancies and allows for a more efficient use of neural resources. This hierarchical multi-stage tuning strategy is thus likely optimized to extract available information and may underlies perceptual sensitivity to natural sounds.


title: Gestational Ethanol Exposure Induces Sex-specific Striatal Acetylcholine and Dopamine Deficits

abstract: Fetal alcohol exposure has deleterious consequences on the cognitive abilities and motor skills of patients affected by Fetal Alcohol Spectrum Disorder (FASD) and in pre-clinical models of gestational ethanol exposure (GEE). Deficits in striatal cholinergic and dopamine function impair action learning and execution, yet the effects of GEE on striatal acetylcholine and dopamine release remain unexplored. Here, we report that ethanol (EtOH) exposure during the first ten postnatal days (GEEP0-P10), which mimics EtOH consumption during the last gestational trimester in humans, induces enduring sex-specific anatomical and motor learning deficits in female mice. These behavioral impairments were accompanied by increased evoked-dopamine levels in the dorsolateral striatum (DLS) of GEEP0-P10 female, but not male, mice. Further experiments revealed impaired striatal {beta}2-containing nicotinic acetylcholine receptor (nAChRs)-modulation of electrically evoked dopamine release. Using a genetically encoded acetylcholine sensor (GACh3.0), we found a reduced decay of acetylcholine transients in DLS of GEEP0-P10 females in the presence of an acetylcholinesterase inhibitor galantamine. Finally, we showed that this effect is associated with decreased excitability of striatal cholinergic interneurons (CINs), pointing to activity-dependent defects in acetylcholine release. Altogether, these data shed a new light on striatal deficits that might underlie cognitive and motor learning symptoms of patients affected by FASD.


title: Astrocyte-mediated transduction of muscle fiber contractions synchronizes hippocampal neuronal network development

abstract: Exercise supports brain health in part through enhancing hippocampal function. The leading hypothesis is that muscles release factors when they contract (e.g., lactate, myokines, growth factors) that enter circulation and reach the brain where they enhance plasticity (e.g., increase neurogenesis and synaptogenesis). However, it remains unknown how the muscle signals are transduced by the hippocampal cells to modulate network activity and synaptic development. Thus, we established an in vitro model in which the media from contracting primary muscle cells (CM) is applied to developing primary hippocampal cell cultures on a microelectrode array. We found that the hippocampal neuronal network matures more rapidly (as indicated by synapse development and synchronous neuronal activity) when exposed to CM than regular media (RM). This was accompanied by a fourfold increase in the proliferation of astrocytes. Further, experiments established that the astrocytes release factors that inhibit neuronal excitability and facilitate network development. Results provide new insight into how exercise may support hippocampal function through regulating astrocyte proliferation and subsequent taming of neuronal activity into an integrated network.


title: Speech prosody supports speaker selection and auditory stream segregation in a multi-talker situation

abstract: To process speech in a multi-talker environment, listeners need to segregate the mixture of incoming speech streams and focus their attention on one of them. Potentially, speech prosody could aid the segregation of different speakers, the selection of the desired speech stream, and detecting targets within the attended stream. For testing these issues, we recorded behavioral responses and extracted event-related potentials and functional brain networks from electroencephalographic signals recorded while participants listened to two concurrent speech streams, performing a lexical detection and a recognition memory task in parallel. Prosody manipulation was applied to the attended speech stream in one group of participants and to the ignored speech stream in another group. Naturally recorded speech stimuli were either intact, synthetically F0-flattened, or suppressed by the speaker. Results show that prosody - especially the parsing cues mediated by speech rate - facilitates stream selection, while playing a smaller role in auditory stream segmentation and target detection.


title: Deep Brain Magnetothermal Silencing of Dopaminergic Neurons via Endogenous TREK1 Channels Abolishes Place Preference in Mice

abstract:
Reversible silencing of neuronal activity in freely behaving animals provides a powerful tool to determine the roles of specific neuronal populations in circuit dynamics and behavior. Here, we show how magnetothermal silencing using endogenous TREK-1 channels can suppress the dopaminergic reward in the ventral tegmental area and the place preference in mice.

Magnetothermal silencing uses alternating magnetic fields penetrating tissue undiminished without harmful effects to heat superparamagnetic nanoparticles at the target cells. The induced sudden, small increase in temperature inhibits neuronal firing rapidly and reversibly, and without the need of genetic modification of the neurons. We demonstrate that two-pore potassium channel TREK-1 channels are responsible for this thermal neuronal silencing. Using fiber-based optogenetics we measure the heating and neuronal silencing in the ventral tegmental area (VTA) brain of the animals. We show that in a place preference assay magnetothermal neuronal silencing of the dopaminergic neurons in the VTA is sufficient to abolished the place preference. TREK1 knock-out mice are immune to the magnetothermal silencing and behave as if the magnetic field were not applied.

HighlightsO_LITREK1 is a highly efficient, thermally activated neuronal silencer
C_LIO_LIFirst magnetothermal neuronal silencing in behaving mice
C_LIO_LIFiber photometry determination of local heating and silencing of target neurons in the ventral tegmental area
C_LIO_LIMagnetothermal suppression of dopaminergic reward response in the ventral tegmental area is sufficient to abolish place preference
C_LI


title: Age-related changes in the neural mechanics of dynamic auditory attention in time

abstract:
Listening in everyday life requires attention to be deployed dynamically - when listening is expected to be difficult and when relevant information is expected to occur - to conserve mental resources. Conserving mental resources may be particularly important for older adults who often experience difficulties understanding speech. We use electro- and magnetoencephalography to investigate the neural and behavioral mechanics of dynamic attention regulation during listening and the effects that aging may have on these. We show that neural alpha oscillatory activity indicates when in time attention is deployed (Experiment 1) and that deployment depends on listening difficulty (Experiment 2). Older adults also show successful attention regulation, although younger adults appear to utilize timing information a bit differently compared to older adults. We further show that the recruited brain regions differ between age groups. Superior parietal cortex is involved in attention regulation in younger adults, whereas posterior temporal cortex is more involved in older adults (Experiment 3). This difference in the sources of alpha activity across age groups was only observed when a task was performed, and not for alpha activity during resting-state recordings (Experiment S1). In sum, our study suggests that older adults employ different neural control strategies compared to younger adults to regulate attention in time under listening challenges.

Significance statementListening requires attention to be deployed dynamically in time to conserve mental resources. Conserving mental resources may be especially important for older adults who often experience listening difficulties. We investigate the neural and behavioral mechanics of dynamic attention during listening in younger and older adults. We show that neural alpha oscillatory activity indicates when in time attention is deployed and that deployment depends on listening difficulty. Older adults appear to employ more reactive than proactive listening behavior and recruit different brain regions than younger adults. Superior parietal cortex is involved in younger adults, whereas posterior temporal cortex in older adults. In sum, older adults may use different strategies compared to younger adults to regulate attention in time during challenging listening.


title: Light targeting the melanopic system suppresses melatonin, but does not alter sleepiness, vigilance, sensory processing, or sleep

abstract:
Study objectivesPre-sleep exposure to short-wavelength light acutely suppresses melatonin, increases vigilance, and decrease sleepiness with the activating effects extending to sleep. The effects have mainly been attributed to melanopic effects of light, but mechanistic insights are missing. Thus, we investigated whether two metameric light conditions designed to only differ in their melanopic effects (123 vs. 59 lux melanopic EDI) also differentially affect sleep besides melatonin effects. Beyond this, we also studied whether the light conditions modulate sensory processing during wakefulness and sleep.

MethodsIn a preregistered study, twenty-nine healthy participants aged 18-30 y (15 women) were exposed to two metameric light conditions (high-vs. low-melanopic, differing by a factor of 2x, 7-day washout period) for 1 hour prior to their habitual bed time. This was followed by an 8-h sleep opportunity with polysomnography. Objective sleep measurements were complemented by self-reported sleep evaluation after wake-up. Salivary melatonin levels, subjective sleepiness, and behavioural vigilance were sampled in regular intervals. Sensory processing was evaluated using an oddball paradigm participants completed during the light exposure in the evening and the following sleep episode. Specifically, we were interested in event-related potentials (ERPs) and time-frequency responses related to violations of expectations, that is, the mismatch response.

ResultsDespite {approx}14% melatonin suppression in the high-compared to the low-melanopic condition, light conditions did not differentially affect objectively assessed sleep, self-perceived sleep quality, sleepiness, or vigilance. A neural mismatch response was evident during all sleep stages but not differentially modulated by pre-sleep light.

ConclusionsSuppression of melatonin by light targeting the melanopic system does not necessarily translate to acutely altered levels of vigilance or sleepiness and neither to changes in sleep, sleep quality, or basic sensory processing. This may suggest that an interaction between melanopsin and cone-rod signals could be at work for such effects to occur.


title: Carbohydrates complement high protein diets to maximize the growth of an actively hunting predator

abstract:
In nature, food is often variable in composition and availability. As a consequence, predators may need to seek non-prey food sources. Some predators are known to feed on nectar when food is limited. Nectar and other carbohydrate resources could also be beneficial when prey are more abundant if it helps predators balance protein-biased diets. We tested if an actively hunting predator, the jumping spider, Phidippus audax, benefited from liquid carbohydrates when prey were not limited. We also tested if the benefit of carbohydrates varied with the nutrient content of prey (i.e., from protein to lipid-biased). Spiders were reared on one of six live prey, Drosophila melanogaster, treatments that ranged from high protein to high lipid. Half of the spiders were given access to a 20% sucrose solution. After two months, we measured spider mass, cephalothorax width, instar duration, percent body fat, survival, and estimated number of prey eaten. Spiders reared on high protein diets with carbohydrates were larger and heavier than spiders on other treatments. Access to carbohydrates also increased percent body fat and survival across prey treatments. Our results suggest that carbohydrates may be a valuable component of spider diets, especially when prey have high protein and low lipid content as is commonly observed in prey in the field. Our results highlight the importance of diet balancing for predators, and that liquid carbohydrates can be an important nutrient to supplement a diet of prey rather than just being an energy supplement during periods of starvation.

Significance statementProtein and lipid are thought to be the primary nutrients used by predators, including spiders. Yet, some spiders have been observed feeding on carbohydrate-rich nectar from flowers. We tested if the addition of carbohydrates to a high protein or high lipid diet affected the growth of the North American jumping spider, Phidippus audax. Spiders grew largest on high protein diets with carbohydrates, demonstrating that plant-based foods rich in carbohydrates can be important for some predators.

category: ecology