title: Cortical-subcortical structural connections support transcranial magnetic stimulation engagement of the amygdala

abstract:
The amygdala processes valenced stimuli, influences affective states, and exhibits aberrant activity across anxiety disorders, depression, and PTSD. Interventions that modulate amygdala activity hold promise for treating transdiagnostic affective symptoms. We investigated (N=45) whether transcranial magnetic stimulation (TMS) elicits indirect changes in amygdala activity when applied to ventrolateral prefrontal cortex (vlPFC), a region important for affect regulation. Harnessing in-scanner interleaved TMS/functional MRI (fMRI), we reveal that vlPFC neurostimulation evoked acute, dose-dependent modulations of amygdala fMRI BOLD signal. Larger TMS-evoked changes in amygdala fMRI signal were associated with higher fiber density in a vlPFC-amygdala white matter pathway, suggesting this pathway facilitated stimulation-induced communication between cortex and subcortex. This work provides evidence of amygdala engagement by TMS, highlighting stimulation of vlPFC-amygdala circuits as a candidate treatment for affective psychopathology. More broadly, it indicates that targeting cortical-subcortical connections may enhance the impact of TMS on subcortical neural activity and, by extension, subcortex-subserved behaviors.

TeaserIndividualized, connectivity-guided transcranial magnetic stimulation modulates the amygdala, demonstrating therapeutic potential.


title: Hypnosis Measuring: The Pilot Study of Machine Learning Approach for Instrumental Control of the Trance Dynamics in Patients with Anxiety and Depressive Disorders

abstract: Having measurable physiological correlates, hypnosis should be measurable generally itself. The precise, continual, quantitative assessment (versus phenomenological one) of a current trance level (i.e., "depth") is possible only instrumentally. Weve shown that electrophysiological patterns of a trance are stable from session to session, but significantly vary among subjects. Hence, to measure the trance level individually we proposed the following Brain-Computer interface approach and tested it on the 27 video-EEG recordings of 8 outpatients with anxiety and depressive disorders: on the data of the first session using Common Spatial Pattern filtering and Linear Discriminant Analysis classification, we trained the predictive models to discriminate conditions of "a wakefulness" and "a deep trance" and applied them to the subsequent sessions to predict the deep trance probability (in fact, to measure the trance level). We obtained integrative individualized continuously changing parameter reflecting the hypnosis level graphically online, providing the trance dynamics control. The classification accuracy was high, especially while filtering the signal in 1.5-14 and 4-15 Hz. The applications and perspectives are being discussed.

category: neuroscience


title: Large-scale algorithmic search identifies stiff and sloppy dimensions in synaptic architectures consistent with murine neocortical wiring

abstract:
Complex systems can be defined by "sloppy" dimensions, meaning that their behavior is unmodified by large changes to specific parameter combinations, and "stiff" dimensions whose change results in considerable behavioral modification. In the neocortex, sloppiness in synaptic architectures would be crucial to allow for the maintenance of asynchronous irregular spiking dynamics with low firing rates despite a diversity of inputs, states, and both short- and long-term plasticity. Using simulations on neural networks with first-order spiking statistics matched to firing in murine visual cortex while varying connectivity parameters, we determined the stiff and sloppy parameters of synaptic architectures across three classes of input (brief, continuous, and cyclical). Algorithmically-generated connectivity parameter values drawn from a large portion of the parameter space reveal that specific combinations of excitatory and inhibitory connectivity are stiff and that all other architectural details are sloppy. Stiff dimensions are consistent across input classes with self-sustaining synaptic architectures following brief input occupying a smaller subspace as compared to the other input classes. Experimentally estimated connectivity probabilities from mouse visual cortex are consistent with the connectivity correlations found and fall in the same region of the parameter space as architectures identified algorithmically. This suggests that simple statistical descriptions of spiking dynamics are a sufficient and parsimonious description of neocortical activity when examining structure-function relationships at the mesoscopic scale. Additionally, coarse graining cell types does not prevent the generation of accurate, informative, and interpretable models underlying simple spiking activity. This unbiased investigation provides further evidence of the importance of the interrelationship of excitatory and inhibitory connectivity to establish and maintain stable spiking dynamical regimes in the neocortex.

Author SummaryConnections between neurons are continuously changing to allow learning and adaptation to new stimuli. However, the ability of neural networks to vary these connections while avoiding excessively high- or low-activity states is still not well understood. We tackled this question by studying how changes in the parameters of connectivity within and between different neuronal populations impacted network activity in computational models. We identified specific combinations of parameters, deemed "stiff", that must be maintained to observe activity consistent with recordings from murine visual cortex, while the rest of the parameters can be varied freely with minimal effects on activity. Our results agree with experimentally measured connectivity statistics demonstrating the importance of balancing opposing forces to maintain activity in a natural regime.


title: Patterns of regulatory divergence and gene expression in hybrids are associated with molecular evolution in species undergoing gene flow

abstract: The extent to which hybridization disrupts a genes pattern of expression likely governs its propensity for introgression, while its extent of molecular divergence can itself underlie such disruption. Together, these phenomena shape the landscape of sequence and transcriptional divergence across the genome as species diverge. To understand this process, we examine gene expression inheritance, regulatory and molecular divergences in the reproductive transcriptomes of species linked by gene flow. The fruit flies Anastrepha fraterculus and A. obliqua show evidence of gene flow despite clear evolutionary divergence and incomplete reproductive isolation. We find that their transcriptional patterns are a mosaic between those typically observed within and between allopatric species. Genes showing transgressive expression in hybrids or cis-regulatory divergence between species are associated with greater molecular divergence. This may reflect pleiotropic constraints that make them more resistant to gene flow or they may be more likely to experience divergent selection. However, while these highly divergent genes are likely to be important contributors to species differences, they are relatively rare. Instead, most differentially regulated genes, including those linked to reproduction, show high degrees of dominance in hybrids and trans-regulated divergence between species, suggesting widespread genetic compatibility that allowed for the identified introgression. These findings provide insights into how postzygotic isolating mechanisms might evolve in the presence of gene flow: regions showing cis-regulatory divergence or transgressive expression contribute to reproductive isolation, while regions with dominant expression and trans-regulatory divergence act as a buffer of hybrid breakdown, facilitating introgression, and leading to a genomic mosaic of expression and sequence divergence.

category: evolutionary biology


title: Deep Learning-based Automated Rare Sperm Identification from Testes Biopsies

abstract: Non-obstructive azoospermia (NOA), the most severe form of male infertility, is currently treated using microsurgical sperm extraction (microTESE) to retrieve sperm cells for in vitro fertilization via intracytoplasmic sperm injection (IVF-ICSI). The success rate of this procedure for NOA patients is currently limited by the ability of andrologists to identify a few rare sperm cells among millions of background testis cells. To improve this success rate, we developed a convolution neural network (CNN) to detect rare sperm from low-resolution microscopy images of microTESE samples. Our CNN uses the U-Net architecture to perform pixel-based classification on image patches from brightfield microscopy, which is followed by morphological analysis to detect individual sperm instances. This CNN is trained using microscopy images of fluorescently labeled sperm, which is fixed to eliminate their motility, and doped into testis biopsies obtained from NOA patients. We initially tested this algorithm using purified sperm samples at different imaging magnifications in order to determine the upper bounds of performance. We then tested this algorithm by doping rare sperm cells into testis biopsy samples from NOA patients and found a sperm detection F1 score of 85.2%. These results demonstrate the potential to use automated microscopy to dramatically increase the amount of testis biopsy tissue that could be comprehensively examined, which greatly increases the chance of finding rare viable sperm, and thereby increases the success rates of IVF-ICSI for couples with NOA.

category: bioengineering


title: Volumetric Tomographic 3D Bioprinting of Heterocellular Bone-like Tissues in Seconds

abstract: 3D bioprinting has emerged as a powerful tool for custom fabrication of biomimetic hydrogel constructs that support the differentiation of stem cells into functional bone tissues. Existing stem cell-derived in vitro bone models, however, often lack terminally differentiated bone cells named osteocytes which are crucial for bone homeostasis. Here, we report ultrafast volumetric tomographic photofabrication of centimeter-scale heterocellular bone models that enabled successful 3D osteocytic differentiation of human mesenchymal stem cells (hMSCs) within hydrogels after 42 days co-culture with human umbilical vein endothelial cell (HUVECs). It is hypothesized that after 3D bioprinting the paracrine signaling between hMSCs and HUVECs will promote their differentiation into osteocytes while recreating the complex heterocellular bone microenvironment. To this, we formulated a series of bioinks with varying concentrations of gelatin methacryloyl (GelMA) and lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP). A bioink comprising 5% GelMA and 0.05% LAP was identified as an optimal material with high cell viability (>90%) and excellent structural fidelity. Increasing LAP concentration led to much lower degree of cell spreading, presumably due to phototoxicity effects. Biochemical assays evidenced significantly increased expression of both osteoblastic markers (collagen-I, ALP, osteocalcin) and osteocytic markers (Podoplanin, PDPN; dentin matrix acidic phosphoprotein 1, Dmp1) after 3D co-cultures for 42 days. Additionally, we demonstrate volumetric 3D bioprinting of perfusable, pre-vascularized bone models where HUVECs self-organized into an endothelium-lined channel within 2 days. Altogether, this work leverages the benefits of volumetric tomographic bioprinting and 3D co-culture, offering a promising platform for scaled biofabrication of 3D bone-like tissues with unprecedented long-term functionality.


title: Contrastive learning on protein embeddings enlightens midnight zone at lightning speed

abstract: Experimental structures are leveraged through multiple sequence alignments, or more generally through homology-based inference (HBI), facilitating the transfer of information from a protein with known annotation to a query without any annotation. A recent alternative expands the concept of HBI from sequence-distance lookup to embedding-based annotation transfer (EAT). These embeddings are derived from protein Language Models (pLMs). Here, we introduce using single protein representations from pLMs for contrastive learning. This learning procedure creates a new set of embeddings that optimizes constraints captured by hierarchical classifications of protein 3D structures defined by the CATH resource. The approach, dubbed ProtTucker, has an improved ability to recognize distant homologous relationships than more traditional techniques such as threading or fold recognition. Thus, these embeddings have allowed sequence comparison to step into the "midnight zone" of protein similarity, i.e., the region in which distantly related sequences have a seemingly random pairwise sequence similarity. The novelty of this work is in the particular combination of tools and sampling techniques that ascertained good performance comparable or better to existing state-of-the-art sequence comparison methods. Additionally, since this method does not need to generate alignments it is also orders of magnitudes faster. The code is available at https://github.com/Rostlab/EAT.

category: bioinformatics


title: Contrastive learning on protein embeddings enlightens midnight zone


title: Multi-Branch-CNN: classification of ion channel interacting peptides using parallel convolutional neural networks

abstract: Ligand peptides that have high affinity for ion channels are critical for regulating ion flux across the plasma membrane. These peptides are now being considered as potential drug candidates for many diseases, such as cardiovascular disease and cancers. There are several studies to identify ion channel interacting peptides computationally, but, to the best of our knowledge, none of them published available tools for prediction. To provide a solution, we present Multi-branch-CNN, a parallel convolutional neural networks (CNNs) method for identifying three types of ion channel peptide binders (sodium, potassium, and calcium). Our experiment shows that the Multi-Branch-CNN method performs comparably to thirteen traditional ML algorithms (TML13) on the test sets of three ion channels. To evaluate the predictive power of our method with respect to novel sequences, as is the case in real-world applications, we created an additional test set for each ion channel, called the novel-test set, which has little or no similarities to the sequences in either the sequences of the train set or the test set. In the novel-test experiment, Multi-Branch-CNN performs significantly better than TML13, showing an improvement in accuracy of 6%, 14%, and 15% for sodium, potassium, and calcium channels, respectively. We confirmed the effectiveness of Multi-Branch-CNN by comparing it to the standard CNN method with one input branch (Single-Branch-CNN) and an ensemble method (TML13-Stack). To facilitate applications, the data sets, script files to reproduce the experiments, and the final predictive models are freely available at https://github.com/jieluyan/Multi-Branch-CNN.


title: Reconstruction of sound driven, actively amplified and spontaneous motions within the tree cricket auditory organ

abstract: AO_SCPLOWBSTRACTC_SCPLOWHearing consists of a delicate chain of events. Sound is first captured by an eardrum or similar organ which is set into vibrations, these vibrations must then be transmitted to sensory cells in a manner that opens mechanosensory channels generating an electrical signal. Studying this process is challenging. Auditory vibrations are in the nano- to picometer-scale and occur at fast temporal scales of milli to microseconds. Finally, most of this process occurs within the body of the animal where it is inaccessible to conventional measurement techniques. For instance, even in crickets, a century-old auditory model system, it is unclear how sound evoked vibrations are transmitted to sensory neurons. Here, we use optical coherence tomography (OCT) to measure how vibrations travel within the auditory organ of the western tree cricket (Oecanthus californicus). We also measure the reversal of this process as mechanosensory cells generate spontaneous oscillations and amplify sound-evoked vibrations. Most importantly, we found that while the mechanosensory neurons were not attached to the peripheral sound collecting structures, they were mechanically well-coupled through acoustic trachea. Thus, the acoustic trachea are not merely conduits for sound but also perform a mechanical function. Our results generate several insights into the similarities between insect and vertebrate hearing, and into the evolutionary history of auditory amplification.

category: biophysics


title: Multiplexed Cell-Based Diagnostic Devices for Detection of Renal Biomarkers Using Genetic Circuits

abstract: The number of synthetic biology based solutions employed in the medical industry is growing every year. The whole cell biosensors being one of them, have been proven valuable tools for developing low-cost, portable, personalized medicine alternatives to conventional techniques. Based on this concept, we targeted one of the major health problems in the world, Chronic Kidney Disease (CKD). To do so, we developed two novel biosensors for the detection of two important renal biomarkers; urea and uric acid. Using advanced gene expression control strategies we improved the operational range and the response profiles of each biosensor to meet clinical specifications. We further engineered these systems to enable multiplexed detection as well as an AND-logic gate operating system. Finally, we tested the applicability of these systems and optimized their working dynamics inside complex medium human blood serum. This study could help the efforts to transition from labor-intensive and expensive laboratory techniques to widely available, portable, low cost diagnostic options.

category: synthetic biology


title: Mapping of individual sensory nerve axons from digits to spinal cord with the Transparent Embedding Solvent System

abstract: Understanding the connections and projections of neurons has been a fundamental issue for neuroscience. Although strategies have been developed to map the projection of individual axons within the mouse brain, high resolution mapping of individual peripheral nerve axons in peripheral organs or spinal cord has never been achieved. Here, we designed the Transparent Embedding Solvent System (TESOS) method and developed a technical pipeline for imaging, reconstructing and analyzing large samples containing various tissue types at sub-micron resolution. The mouse whole body was reconstructed at micron-scale resolution. We were able to image, reconstruct and analyze the complete axonal projection of individual sensory neurons within an intact mouse paw or spinal cord at sub-micron resolution. Furtherly, we imaged and reconstructed the entire projection course of individual sensory neurons from adult mouse digits to the spinal cord. The TESOS method provides an efficient tool for micron-scale connectome mapping of the peripheral nervous system.


title: Insightful inference compensates for distorted perception

abstract: Introspective agents can recognize the extent to which their internal perceptual experiences deviate from the actual states of the external world. This ability, also known as insight, is critically required for reality testing and is impaired in psychosis, yet very little is known about its cognitive underpinnings. We developed a Bayesian modeling framework and a novel psychophysics paradigm to quantitatively characterize this type of insight while participants experienced a motion after-effect illusion. Participants could compensate for the illusion when judging the actual direction of a motion stimulus. Furthermore, confidence, reaction-time, and pupil-dilation data all showed signatures consistent with inferential adjustments in the Bayesian insight model. Our results suggest that people can question the veracity of what they see and make insightful inferences that incorporate introspective knowledge about internal distortions.


title: Rapid reconstruction of neural circuits using tissue expansion and lattice light sheet microscopy

abstract: Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.


title: Repressing PTB is incapable to convert reactive astrocytes to dopaminergic neurons in a mouse model of Parkinson's disease

abstract:
Lineage reprograming of resident glia cells to induced dopaminergic neurons (iDAns) holds attractive prospect for cell-replacement therapy of Parkinsons disease (PD). Recently, whether repressing polypyrimidine tract binding protein 1 (PTBP1) could truly achieve efficient astrocyte-to-iDAn conversion in substantia nigra and striatum aroused widespread controversy. Although reporter positive iDAns were observed by two groups after delivering adeno-associated virus (AAV) expressing a reporter with shRNA or Crispr-CasRx to repress astroglial PTBP1, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage tracing method. By adopting stringent lineage tracing strategy, two other studies showed that neither knockdown nor genetic deletion of quiescent astroglial PTBP1 fails to obtain iDAns under physiological condition. However, the role of reactive astrocyte might be underestimated since upon brain injury, reactive astrocyte could acquire certain stem cell hallmarks which may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTBP1 repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2-mediated specific astrocyte-lineage tracing method to investigate whether reactive astrocytes could be converted to DAns in the 6-OHDA PD model. However, we found that no astrocyte-originated DAn was generated after effective knockdown of astroglial PTBP1 either in the substantia nigra or in the striatum, while AAV "leakage" to nearby neurons was observed. Our results further confirmed that repressing PTBP1 is unable to convert astrocytes to DAns no matter in physiological or PD-related pathological conditions.

Graphic abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/468309v2_ufig1.gif" ALT="Figure 1">
View larger version (17K):
org.highwire.dtl.DTLVardef@fa2fa5org.highwire.dtl.DTLVardef@14e328eorg.highwire.dtl.DTLVardef@d93faborg.highwire.dtl.DTLVardef@e32225_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIAAV-shPtbp1 rapidly and efficiently induces viral-reporter-labeled DAns in mouse brain under physiological condition
C_LIO_LIViral-reporter-positive DAns are not originated from PTBP1 repressed and lineage traced reactive astrocytes in a mouse PD model
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title: Repressing PTBP1 is incapable to convert reactive astrocytes to dopaminergic neurons in a mouse model of Parkinson's disease


title: In Silico Prediction and Comparison of Resistomes in Model Pseudomonas Strains by Resistance Gene Identifier (RGI)

abstract: 1.ABSTRACTSPseudomonas is a genus of bacteria including strains of human and plant pathogens, plant-growth promoting and biological control agents. While most Pseudomonas strains are known resistant to several antibiotics, their genetic elements conferring antimicrobial resistance (AMR) are largely unexplored systematically. The current study exploits a robust AMR gene predicting tool Resistance Gene Identifier of most recently updated version 5.2.0 based on newly curated database (the Comprehensive Antibiotic Research Database version 3.1.3) to detect AMR genes from thirteen genomes of Pseudomonas strains affiliated with seven species, including twelve pseudomonads as popularly studied model strains plus a well-known Pseudomonas protegens CHA0. A list of 281 AMR genes have been detected in five genomes of Pseudomonas aeruginosa, while 32 in the rest Pseudomonas spp. strains. Among the species, P. aeruginosa, P. fluorescens, P. protegens and P. stutzeri have the resistome of multi-drug resistance, while the rest is resistant to narrower spectrum of drugs. All Pseudomonas spp. investigated here have resistance genes to antibiotics classes of fluoroquinolone and tetracycline, which is consistent with an antibiotic resistance gene hit of adeF (ARO No. 3000777, resistant to fluoroquinolone, tetracycline) has found in high redundancy in almost all Pseudomonas species except P. aeruginosa and P. stutzeri, implying the limit of these classes of drugs for treating pseudomonads. While interspecies data were focused here, further analysis will be conducted to reveal the features of inter-strain level features of pseudomonads. The in silico analysis will complement wet-lab research for designing treating strategies of these bacteria.

category: microbiology


title: Multi-marker metabarcoding resolves subtle variations in freshwater condition: Bioindicators, ecological traits, and trophic interactions

abstract: Freshwater systems are experiencing rapid biodiversity losses resulting from high rates of habitat degradation. Ecological condition is typically determined through identifying either macroinvertebrate or diatom bioindicator assemblages and comparing them to their known tolerance to stressors. These comparisons are typically conducted at family or genus levels depending on the availability of taxonomic keys and expertise for focal groups. The objective of this study was to test whether a more taxonomically comprehensive assessment of communities in benthic samples can provide a different perspective of ecological conditions. DNA metabarcoding was used to identify macroinvertebrates and diatoms from kick-net samples collected from sites with different habitat status. Sites with  good condition were associated with higher beta diversity as well as slightly higher directed connectance and modularity indicating higher resilience compared with  fair condition sites. Indicator value and correlation analyses used DNA metabarcoding data to detect 29 site condition indicator species consistent with known bioindicators and expected relative tolerances. DNA metabarcoding and trophic network analysis also recovered 11 keystone taxa. This study demonstrates the importance of taxonomic breadth across trophic levels for generating biotic data to study ecosystem status, with the potential to scale-up ecological assessments of freshwater condition, trophic stability, and resilience.

category: ecology


title: Movement ecology of vulnerable lowland tapirs across a gradient of human disturbance

abstract:
Animal movement is a key ecological process that is tightly coupled to local environmental conditions. While agriculture, urbanisation, and transportation infrastructure are critical to human socio-economic improvement, these have spurred substantial changes in animal movement across the globe with potential impacts on fitness and survival. Notably, however, human disturbance can have differential effects across species, and responses to human activities are thus largely taxa and context specific. As human disturbance is only expected to worsen over the next decade it is critical to better understand how species respond to human disturbance in order to develop effective, case-specific conservation strategies. Here, we use an extensive telemetry dataset collected over 22 years to fill a critical knowledge gap in the movement ecology of lowland tapirs (Tapirus terrestris) across a gradient of human disturbance within three biomes in southern Brazil: the Pantanal, Cerrado, and Atlantic Forest.

From these data we found that the mean home range size across all monitored tapirs was 8.31 km2 (95% CI: 6.53 - 10.42), with no evidence that home range sizes differed between sexes nor age groups. Interestingly, although the Atlantic Forest, Cerrado, and Pantanal vary substantially in habitat composition, levels of human disturbance, and tapir population densities, we found that lowland tapir movement behaviour and space use were consistent across all three biomes. Human disturbance also had no detectable effect on lowland tapir movement. Lowland tapirs living in the most altered habitats we monitored exhibited movement behaviour that was comparable to that of tapirs living in a near pristine environment.

Contrary to our expectations, we observed very little individual variability in lowland tapir space use and movement, and human impacts on the landscape also had no measurable effect on their movement. Lowland tapir movement behaviour thus appears to exhibit very little phenotypic plasticity. Crucially, the lack of any detectable response to anthropogenic disturbance suggests that human modified habitats risk being ecological traps for tapirs and this information should be factored into conservation actions and species management aimed towards protecting lowland tapir populations.


title: Correlational selection and genetic architecture promote the leaf economics spectrum in a perennial grass

abstract: The leaf economics spectrum (LES) is hypothesized to result from a trade-off between resource acquisition and conservation. Yet few studies have examined the evolutionary mechanisms behind the LES, perhaps because most species exhibit relatively specialized leaf economics strategies. In a genetic mapping population of the phenotypically diverse grass Panicum virgatum, we evaluate two interacting mechanisms that may drive LES evolution: 1) genetic architecture, where multiple traits are coded by the same gene (pleiotropy) or by genes in close physical proximity (linkage), and 2) correlational selection, where selection acts non-additively on combinations of multiple traits. We found evidence suggesting that shared genetic architecture (pleiotropy) controls covariation between two pairs of leaf economics traits. Additionally, at five common gardens spanning 17 degrees of latitude, correlational selection favored particular combinations of leaf economics traits. Together, these results demonstrate how the LES can evolve within species.


title: Numerous expansions in TRP ion channel diversity highlight widespread evolution of molecular sensors in animal diversification.

abstract: Transient Potential Receptor (TRP) ion channels are a diverse superfamily of multimodal molecular sensors that respond to a wide variety of stimuli, including mechanical, chemical, and thermal. TRP channels are present in most eukaryotes but best understood in mammalian, worm, and fly genetic models, where they are expressed in diverse cell-types and commonly associated with the nervous system. Here, we characterized TRP superfamily gene and genome evolution to better understand origins and evolution of molecular sensors, brains, and behavior in animals and help advance development of novel genetic technologies, like sonogenetics. We developed a flexible push-button bioinformatic and phylogenomic pipeline, GIGANTIC, that generated genome-based gene and species trees and enabled phylogenetic characterization of challenging remote homologs and distantly-related organisms deep in evolution. We identified complete sets of TRP superfamily ion channels, with over 3000 genes in 22 animal phyla and 70 species having publicly-available sequenced genomes, including 3 unicellular outgroups. We then identified clusters of TRP family members in genomes, evaluated gene models per cluster, and repaired split gene models. We also produced whole-organism PacBio transcriptomes for five species to independently validate our gene model assessment and model repairs. We find that many TRP families exhibited numerous and often extensive expansions in different phyla. Some expansions represent local clusters on respective genomes, a trend that is likely undercounted due to varied quality in genome assemblies and annotations of non-model organisms. Our work expands known TRP diversity across animals, including addition of previously uncharacterized phyla and identification of unrecognized homologs in previously characterized species.


title: Liver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts.

abstract: Three-dimensional scaffolds decellularized from native organs are a promising technique to establish engineered liver grafts and overcome the current shortage of donor organs. However, limited sources of bile duct cells and inappropriate cell distribution in bioengineered liver grafts have hindered their practical application. Organoid technology is anticipated to be an excellent tool for the advancement of regenerative medicine. In the present study, we reconstructed intrahepatic bile ducts in a rat decellularized liver graft by recellularization with liver ductal organoids. Using an ex vivo perfusion culture system, we demonstrated the biliary characteristics of repopulated mouse liver organoids, which maintained bile duct markers and reconstructed biliary tree-like networks with luminal structures. We also established a method for the co-recellularization with engineered bile ducts and primary hepatocytes, revealing the appropriate cell distribution to mimic the native liver. We then utilized this model in human organoids to demonstrate the reconstructed bile ducts. Our results show that liver ductal organoids are a potential cell source for bile ducts from bioengineered liver grafts using three-dimensional scaffolds.


title: Liver ductal organoids reconstruct intrahepatic biliary trees in decellularized liver grafts


title: CLMB: deep contrastive learning for robust metagenomic binning

abstract: The reconstruction of microbial genomes from large metagenomic datasets is a critical procedure for finding uncultivated microbial populations and defining their microbial functional roles. To achieve that, we need to perform metagenomic binning, clustering the assembled contigs into draft genomes. Despite the existing computational tools, most of them neglect one important property of the metagenomic data, that is, the noise. To further improve the metagenomic binning step and reconstruct better metagenomes, we propose a deep Contrastive Learning framework for Metagenome Binning (CLMB), which can efficiently eliminate the disturbance of noise and produce more stable and robust results. Essentially, instead of denoising the data explicitly, we add simulated noise to the training data and force the deep learning model to produce similar and stable representations for both the noise-free data and the distorted data. Consequently, the trained model will be robust to noise and handle it implicitly during usage. CLMB outperforms the previous state-of-the-art binning methods significantly, recovering the most near-complete genomes on almost all the benchmarking datasets (up to 17% more reconstructed genomes compared to the second-best method). It also improves the performance of bin refinement, reconstructing 8-22 more high-quality genomes and 15-32 more middle-quality genomes than the second-best result. Impressively, in addition to being compatible with the binning refiner, single CLMB even recovers on average 15 more HQ genomes than the refiner of VAMB and Maxbin on the benchmarking datasets. On a real mother-infant microbiome dataset with 110 samples, CLMB is scalable and practical to recover 365 high-quality and middle-quality genomes (including 21 new ones), providing insights into the microbiome transmission. CLMB is open-source and available at https://github.com/zpf0117b/CLMB/.


title: Determination of nuclear position by the arrangement of actin filaments using deep generative networks

abstract: The cell nucleus is a dynamic structure that changes locales during cellular processes such as proliferation, differentiation, or migration1, and its mispositioning is a hallmark of several disorders2,3. As with most mechanobiological activities of adherent cells, the repositioning and anchoring of the nucleus are presumed to be associated with the organization of the cytoskeleton, the network of protein filaments providing structural integrity to the cells4. However, this correlation between cytoskeleton organization and nuclear position has not, to date, been demonstrated, as it would require the parameterization of the extraordinarily intricate cytoskeletal fiber arrangements. Here, we show that this parameterization and demonstration can be achieved outside the limits of human conceptualization, using generative network and raw microscope images, relying on machine-driven interpretation and selection of parameterizable features. The developed transformer-based architecture was able to generate high-quality, completed images of more than 8,000 cells, using only information on actin filaments, predicting the presence of a nucleus and its exact localization in more than 70 per cent of instances. Our results demonstrate one of the most basic principles of mechanobiology with a remarkable level of significance. They also highlight the role of deep learning as a powerful tool in biology beyond data augmentation and analysis, capable of interpreting--unconstrained by the principles of human reasoning--complex biological systems from qualitative data.


title: Ca2+-inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM

abstract: Activation of the intracellular Ca2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca2+ surge, while elevated cytosolic Ca2+ inhibits the channel in a negative feedback mechanism. Cryo-EM carried out under partially inactivating Ca2+ conditions revealed two conformations of RyR1, an open state and an inactivated state, resolved at 4.0 and 3.3 [A] resolution, respectively. RyR1s were embedded in nanodiscs with two lipids resolved at each inter-subunit crevice. Ca2+ binding to the high affinity site engages the central (CD) and C-terminal domains (CTD) into a quasi-rigid unit, which separates the S6 four-helix bundle and opens the channel. Further out-of-plane rotation of the quasi-rigid unit pushes S6 towards the central axis, closing (inactivating) the channel. The inactivated conformation is characterized by a downward conformation of the cytoplasmic assembly, a tightly-knit subunit interface contributed by a fully occupied and partially remodeled Ca2+ activation site, and two salt bridges between the EF hand domain and the S2-S3 loop of the neighboring subunit validated by naturally-occurring diseasecausing mutations. Ca2+ also bound to ATP, mediating a tighter interaction between S6 and CTD. Our study suggests that the closed-inactivated is a distinctive state of the RyR1 and its transition to the closed-activable state is not a simple reverse of the Ca2+ mediated activation pathway.

category: physiology


title: Shade suppresses wound-induced leaf repositioning through a mechanism involving PHYTOCHROME KINASE SUBSTRATE (PKS) genes

abstract:
Shaded plants challenged with herbivores or pathogens prioritize growth over defense. However, most experiments have focused on the effect of shading light cues on defense responses. To investigate the potential interaction between shade-avoidance and wounding-induced Jasmonate (JA)-mediated signaling on leaf growth and movement, we used repetitive mechanical wounding of leaf blades to mimic herbivore attacks. Phenotyping experiments with combined treatments on Arabidopsis thaliana rosettes revealed that shade strongly inhibits the wound effect on leaf elevation. By contrast, petiole length is reduced by wounding both in the sun and in the shade. Thus, the relationship between the shade and wounding/JA pathways varies depending on the physiological response, implying that leaf growth and movement can be uncoupled. Using RNA-sequencing, we identified genes with expression patterns matching the hyponastic response (opposite regulation by both stimuli, interaction between treatments with shade dominating the wound signal). Among them were genes from the PKS (Phytochrome Kinase Substrate) family, which was previously studied for its role in phototropism and leaf positioning. Interestingly, we observed reduced shade suppression of the wounding effect in pks2pks4 double mutants while a PKS4 overexpressing line showed constitutively elevated leaves and was less sensitive to wounding. Our results indicate a trait-specific interrelationship between shade and wounding cues on Arabidopsis leaf growth and positioning. Moreover, we identify PKS genes as integrators of external cues in the control of leaf hyponasty further emphasizing the role of these genes in aerial organ positioning.

Author summaryPlants face different types of stressful situations without the ability to relocate to favorable environments. For example, increasing plant density reduces access to sunlight as plants start to shade each other. Foliar shading represents a stress that many plants cope with by changing their morphology. This includes elongation of stem-like structures and repositioning of leaves to favor access to unfiltered sunlight. Plants also defend themselves against various pathogens including herbivores. Defense mechanisms include the production of deterrent chemical and morphological adaptations such as stunted growth and downwards leaf repositioning. Here we studied the morphological response of plants when simultaneously facing shade and herbivore stress. When facing both stresses petiole growth was intermediate between the shade-enhanced and wound-repressed response. In contrast, the shade cue overrides the wounding cue leading to a similar upwards leaf repositioning in the combined treatments or in the response to shade alone. Using gene expression analyses and genetics we identified two members of the Phytochrome Kinase Substrate family as playing a signal integration role when plants simultaneously faced both stresses. This contributes to our understanding of the mechanisms underlying plant morphological adaptations when facing multiple stresses.

category: plant biology


title: Word structure tunes electrophysiological and hemodynamic responses in the frontal cortex

abstract: To date, it is still unclear how word structure might impact lexical processing in the brain for morphological impoverished language like Chinese. In this study, concurrent EEG and fNIRS recordings were performed to inspect the temporal and spatial brain activity that are related to the morphological priming effect (compound/derivation constitute priming vs. non-morphological priming) and word structure (compound vs. derivation) modulation. Interestingly, it was discovered that the morphological priming effect was mainly detected by the behavioral performance and spatial brain activation in the left prefrontal cortex, while word structure effect was revealed by the behavioral data as well as the temporal and spatial brain activation patterns. In addition, Chinese derivations exhibited significantly enhanced brain activation in the frontal cortex and involved more brain networks as compared to lexicalized compounds. The results were interpreted by the differing connection patterns between constitute morphemes within a given word structure from spreading activation perspective. More importantly, we demonstrated that Chinese word structure effect showed a distinct brain activation pattern as compared to that from the dual-route mechanism in alphabetic languages. Therefore, this pilot work paves a new avenue for comprehensively understanding the underlying cognitive neural mechanism associated with Chinese derivations and coordinate compounds.


title: Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

abstract:
The Streptococcus pyogenes cell envelope protease (SpyCEP) is a vital virulence factor in streptococcal pathogenesis. Despite its key role in disease progression and strong association with invasive disease, little is known about the enzymatic function beyond the ELR+ CXC chemokine substrate range. We utilised multiple SpyCEP constructs to interrogate the protein domains and catalytic residues necessary for enzyme function. We leveraged high-throughput mass spectrometry to describe the Michaelis-Menton parameters of active SpyCEP, revealing a Michaelis-Menton constant (KM) of 53.49 nM and a turnover of 1.34 molecules per second, for the natural chemokine substrate CXCL8.

Unexpectedly, we found that an N-terminally-truncated SpyCEP C-terminal construct consisting of only the H279 and S617 catalytic dyad had specific CXCL8 cleaving activity, albeit with a reduced substrate turnover of 2.45 molecules per hour, representing a ~2000-fold reduction in activity. In contrast, the KM of the C-terminal SpyCEP construct and full-length enzyme did not differ. We conclude that the SpyCEP C-terminus plays a key role in substrate binding and recognition with key implications for both current and future streptococcal vaccine designs.


title: Reassembling a cannon in the DNA defense arsenal: genetics of StySA, a BREX phage exclusion system in Salmonella lab strains

abstract: Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX systems, RM-like activities that employ host protection by DNA modification but replication arrest without evident nuclease action on unmodified phage DNA. We show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a BREX homolog. The stySA29 allele of the hybrid strain LB5000 carries a mutated version of the ancestral LT2 BREX system. Surprisingly, both a restriction and a methylation defect are observed for this lineage despite lack of mutations in brxX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations () in C-terminal domains. To avoid plasmid artifacts and potential stoichiometric interference, we chose to investigate this system in situ, replacing the mutated pglZ and brxC genes with wild type (WT). PglZ-WT supports methylation in the presence of either BrxC or BrxC-WT but not in the presence of a deletion/insertion allele, {Delta}brxC::cat. Restriction of phage L requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. Disruption of four other CDS with cat cassettes still permitted modification, suggesting that BrxC, PglZ and BrxX are principal components of the modification activity. BrxL is required for restriction only. A partial disruption of brxL disrupts transcription globally.

category: genetics


title: Single-sample proteome enrichment enables missing protein recovery and phenotype association

abstract: Proteomic studies characterize the protein composition of complex biological samples. Despite recent developments in mass spectrometry instrumentation and computational tools, low proteome coverage remains a challenge. To address this, we present Proteome Support Vector Enrichment (PROSE), a fast, scalable, and effective pipeline for scoring protein identifications based on gene co-expression matrices. Using a simple set of observed proteins as input, PROSE gauges the relative importance of proteins in the phenotype. The resultant enrichment scores are interpretable and stable, corresponding well to the source phenotype, thus enabling reproducible recovery of missing proteins. We further demonstrate its utility via reanalysis of the Cancer Cell Line Encyclopedia (CCLE) proteomic data, with prediction of oncogenic dependencies and identification of well-defined regulatory modules. PROSE is available as a user-friendly Python module from https://github.com/bwbio/PROSE.


title: Genome-wide H3K9 Acetylation Level Increases with Age-Dependent Senescence of Flag Leaf in Rice (Oryza sativa)

abstract:
Flag leaf senescence is an important biological process that drives the remobilization of nutrients to the growing organs of rice. Leaf senescence is controlled by genetic information via gene expression and epigenetic modification, but the precise mechanism is as of yet unclear. Here, we analyzed genome-wide acetylated lysine residue 9 of histone H3 (H3K9ac) enrichment by chromatin immunoprecipitation-sequencing (ChIP-seq) and examined its association with transcriptomes by RNA-seq during flag leaf aging in rice (Oryza sativa). We found that genome-wide H3K9 acetylation levels increased with age-dependent senescence in rice flag leaf, and there was a positive correlation between the density and breadth of H3K9ac and gene expression and transcript elongation. A set of 1,249 up-regulated, differentially expressed genes (DEGs) and 996 down-regulated DEGs showing a strong relationship between temporal changes in gene expression and gain/loss of H3K9ac was observed during rice flag leaf aging. We produced a landscape of H3K9 acetylation-modified gene expression targets that includes known senescence-associated genes, metabolism-related genes, as well as miRNA biosynthesis-related genes. Our findings reveal a complex regulatory network of metabolism- and senescence-related pathways mediated by H3K9ac and also elucidate patterns of H3K9ac-mediated regulation of gene expression during flag leaf aging in rice.

Significance statementGenome-wide H3K9 acetylation levels increased with age-dependent senescence in rice flag leaf, and positively correlation the density and breadth of H3K9ac with transcript elongation and expression. Identified numerous H3K9 acetylation-modified gene expression targets reveal a complex regulatory network and metabolism-mediated senescence network that are associated with H3K9ac during leaf aging in rice.


title: pGlycoQuant with a deep residual network for precise and minuscule-missing-value quantitative glycoproteomics enabling the functional exploration of site-specific glycosylation

abstract: Interpreting large-scale glycoproteomic data for intact glycopeptide identification has been tremendously advanced by software tools. However, software tools for quantitative analysis of intact glycopeptides remain lagging behind, which greatly hinders exploring the differential expression and functions of site-specific glycosylation in organisms. Here, we report pGlycoQuant, a generic software tool for accurate and convenient quantitative intact glycopeptide analysis, supporting both primary and tandem mass spectrometry quantitation for multiple quantitative strategies. pGlycoQuant enables intact glycopeptide quantitation with very low missing values via a deep residual network, thus greatly expanding the quantitative function of several powerful search engines, currently including pGlyco 2.0, pGlyco3, Byonic and MSFragger-Glyco. The pGlycoQuant-based site-specific N-glycoproteomic study conducted here quantifies 6435 intact N-glycopeptides in three hepatocellular carcinoma cell lines with different metastatic potentials and, together with in vitro molecular biology experiments, illustrates core fucosylation at site 979 of the L1 cell adhesion molecule (L1CAM) as a potential regulator of HCC metastasis. pGlycoQuant is freely available at https://github.com/expellir-arma/pGlycoQuant/releases/. We have demonstrated pGlycoQuant to be a powerful tool for the quantitative analysis of site-specific glycosylation and the exploration of potential glycosylation-related biomarker candidates, and we expect further applications in glycoproteomic studies.

category: systems biology


title: Hypoxia classifier for transcriptome datasets

abstract:
Molecular gene signatures are useful tools to characterize the physiological state of cell populations, but most have developed under a narrow range of conditions and cell types and are often restricted to a set of gene identities.

Focusing on the transcriptional response to hypoxia, we aimed to generate widely applicable classifiers sourced from the results of a meta-analysis of 69 differential expression datasets which included 425 individual RNA-seq experiments from 33 different human cell types exposed to different degrees of hypoxia (0.1-5%O2) for 2-48h.

The resulting decision trees include both gene identities and quantitative boundaries, allowing for easy classification of individual samples without control or normoxic reference. Each tree is composed by 3-5 genes mostly drawn from a small set of just 8 genes (EGLN1, MIR210HG, NDRG1, ANKRD37, TCAF2, PFKFB3, BHLHE40, and MAFF). In spite of their simplicity, these classifiers achieve over 95% accuracy in cross validation and over 80% accuracy when applied to additional challenging datasets. Our results indicate that the classifiers are able to identify hypoxic tumor samples from bulk RNAseq and hypoxic regions within tumor from spatially resolved transcriptomics datasets. Moreover, application of the classifiers to histological sections from normal tissues suggest the presence of a hypoxic gene expression pattern in the kidney cortex not observed in other normoxic organs. Finally, tree classifiers described herein outperform traditional hypoxic gene signatures when compared against a wide range of datasets. This work describes a set of hypoxic gene signatures, structured as simple decision tress, that identify hypoxic samples and regions with high accuracy and can be applied to a broad variety of gene expression datasets and formats.


title: #COVIDisAirborne: AI-Enabled Multiscale Computational Microscopy of Delta SARS-CoV-2 in a Respiratory Aerosol

abstract:
We seek to completely revise current models of airborne transmission of respiratory viruses by providing never-before-seen atomic-level views of the SARS-CoV-2 virus within a respiratory aerosol. Our work dramatically extends the capabilities of multiscale computational microscopy to address the significant gaps that exist in current experimental methods, which are limited in their ability to interrogate aerosols at the atomic/molecular level and thus ob-scure our understanding of airborne transmission. We demonstrate how our integrated data-driven platform provides a new way of exploring the composition, structure, and dynamics of aerosols and aerosolized viruses, while driving simulation method development along several important axes. We present a series of initial scientific discoveries for the SARS-CoV-2 Delta variant, noting that the full scientific impact of this work has yet to be realized.

ACM Reference FormatAbigail Dommer1{dagger}, Lorenzo Casalino1{dagger}, Fiona Kearns1{dagger}, Mia Rosenfeld1, Nicholas Wauer1, Surl-Hee Ahn1, John Russo,2 Sofia Oliveira3, Clare Morris1, AnthonyBogetti4, AndaTrifan5,6, Alexander Brace5,7, TerraSztain1,8, Austin Clyde5,7, Heng Ma5, Chakra Chennubhotla4, Hyungro Lee9, Matteo Turilli9, Syma Khalid10, Teresa Tamayo-Mendoza11, Matthew Welborn11, Anders Christensen11, Daniel G. A. Smith11, Zhuoran Qiao12, Sai Krishna Sirumalla11, Michael OConnor11, Frederick Manby11, Anima Anandkumar12,13, David Hardy6, James Phillips6, Abraham Stern13, Josh Romero13, David Clark13, Mitchell Dorrell14, Tom Maiden14, Lei Huang15, John McCalpin15, Christo- pherWoods3, Alan Gray13, MattWilliams3, Bryan Barker16, HarindaRajapaksha16, Richard Pitts16, Tom Gibbs13, John Stone6, Daniel Zuckerman2*, Adrian Mulholland3*, Thomas MillerIII11,12*, ShantenuJha9*, Arvind Ramanathan5*, Lillian Chong4*, Rommie Amaro1*. 2021. #COVIDisAirborne: AI-Enabled Multiscale Computational Microscopy ofDeltaSARS-CoV-2 in a Respiratory Aerosol. In Supercomputing  21: International Conference for High Perfor-mance Computing, Networking, Storage, and Analysis. ACM, New York, NY, USA, 14 pages. https://doi.org/finalDOI


title: Mutagenic distinction between the receptor-binding and fusion subunits of the SARS-CoV-2 spike glycoprotein

abstract: We observe that a residue R of the spike glycoprotein of SARS-CoV-2 which has mutated in one or more of the current Variants of Concern or Interest or under Monitoring rarely participates in a backbone hydrogen bond if R lies in the S1 subunit and usually participates in one if R lies in the S2 subunit. A partial explanation for this based upon free energy is explored as a potentially general principle in the mutagenesis of viral glycoproteins. This observation could help target future vaccine cargos for the evolving coronavirus as well as more generally. A study of the Delta and Omicron variants suggests that Delta was an energetically necessary intermediary in the evolution from Wuhan-Hu-1 to Omicron.

category: immunology


title: Mutagenic distinction between the receptor-binding and fusion subunits of the SARS-CoV-2 spike glycoprotein and its upshot


title: Full protection against all four SARS-CoV-2 variants of concern (VOC) in hamsters requires revision of spike antigen used for vaccination.

abstract:
Current first-generation COVID-19 vaccines are based on prototypic spike sequences from ancestral 2019 SARS-CoV-2 strains. However, the ongoing pandemic is fueled by variants of concern (VOC) that threaten to escape vaccine-mediated protection. Here we show in a stringent hamster model that immunization using prototypic spike expressed from a potent YF17D viral vector (1) provides vigorous protection against infection with ancestral virus (B lineage) and VOC Alpha (B.1.1.7), however, is insufficient to provide maximum protection against the Beta (B.1.351) variant. To improve vaccine efficacy, we created a revised vaccine candidate that carries an evolved spike antigen. Vaccination of hamsters with this updated vaccine candidate provides full protection against intranasal challenge with all four VOCs Alpha, Beta, Gamma (P.1) and Delta (B.1.617.2) resulting in complete elimination of infectious virus from the lungs and a marked improvement in lung pathology. Vaccinated hamsters did also no longer transmit the Delta variant to non-vaccinated sentinels. Hamsters immunized with our modified vaccine candidate also mounted marked neutralizing antibody responses against the recently emerged Omicron (B.1.1.529) variant, whereas the old vaccine employing prototypic spike failed to induce immunity to this antigenically distant virus. Overall, our data indicate that current first-generation COVID-19 vaccines need to be urgently updated to cover newly emerging VOCs to maintain vaccine efficacy and to impede virus spread at the community level.

Significance StatementSARS-CoV-2 keeps mutating rapidly, and the ongoing COVID-19 pandemic is fueled by new variants escaping immunity induced by current first-generation vaccines. There is hence an urgent need for universal vaccines that cover variants of concern (VOC). In this paper we show that an adapted version of our vaccine candidate YF-S0* provides full protection from infection, virus transmission and disease by VOCs Alpha, Beta, Gamma and Delta, and also results in markedly increased levels of neutralizing antibodies against recently emerged Omicron VOC in a stringent hamster model. Our findings underline the necessity to update COVID-19 vaccines to curb the pandemic, providing experimental proof on how to maintain vaccine efficacy in view of an evolving SARS-CoV-2 diversity.


title: Universal COVID-19 vaccine with updated spike antigen confers full protection against all SARS-CoV-2 variants of concern.


title: Updated vaccine protects from infection with SARS-CoV-2 variants, prevents transmission and is immunogenic against Omicron in hamsters


title: Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2

abstract: Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

category: molecular biology


title: Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro

abstract: In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like (3CLpro) protease can cleave human immune signaling proteins, like NF-{kappa}B Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.14 [A] resolution crystal structure of 3CLpro C145S bound to NEMO226-235 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro- NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for in the pathology of COVID-19.


title: Deep embedding and alignment of protein sequences

abstract: Protein sequence alignment is a key component of most bioinformatics pipelines to study the structures and functions of proteins. Aligning highly divergent sequences remains, however, a difficult task that current algorithms often fail to perform accurately, leaving many proteins or open reading frames poorly annotated. Here, we leverage recent advances in deep learning for language modelling and differentiable programming to propose DEDAL, a flexible model to align protein sequences and detect homologs. DEDAL is a machine learning-based model that learns to align sequences by observing large datasets of raw protein sequences and of correct alignments. Once trained, we show that DEDAL improves by up to two- or three-fold the alignment correctness over existing methods on remote homologs, and better discriminates remote homologs from evolutionarily unrelated sequences, paving the way to improvements on many downstream tasks relying on sequence alignment in structural and functional genomics.


title: Built on sand: the shaky foundations of simulating single-cell RNA sequencing data

abstract:
With the emergence of hundreds of single-cell RNA-sequencing (scRNA-seq) datasets, the number of computational tools to analyse aspects of the generated data has grown rapidly. As a result, there is a recurring need to demonstrate whether newly devel-oped methods are truly performant - on their own as well as in comparison to existing tools. Benchmark studies aim to consolidate the space of available methods for a given task, and often use simulated data that provide a ground truth for evaluations. Thus, demanding a high quality standard for synthetically generated data is critical to make simulation study results credible and transferable to real data.

Here, we evaluated methods for synthetic scRNA-seq data generation in their ability to mimic experimental data. Besides comparing gene- and cell-level quality control summaries in both one- and two-dimensional settings, we further quantified these at the batch- and cluster-level. Secondly, we investigate the effect of simulators on clustering and batch correction method comparisons, and, thirdly, which and to what extent quality control summaries can capture reference-simulation similarity.

Our results suggest that most simulators are unable to accommodate complex designs without introducing artificial effects; they yield over-optimistic performance of integration, and potentially unreliable ranking of clustering methods; and, it is generally unknown which summaries are important to ensure effective simulation-based method comparisons.


title: A live attenuated influenza virus-vectored intranasal COVID-19 vaccine provides rapid, prolonged, and broad protection against SARS-CoV-2 infection

abstract: Remarkable progress has been made in developing intramuscular vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, they are limited with respect to eliciting local immunity in the respiratory tract, which is the primary infection site for SARS-CoV-2. To overcome the limitations of intramuscular vaccines, we constructed a nasal vaccine candidate based on an influenza vector by inserting a gene encoding the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, named CA4-dNS1-nCoV-RBD (dNS1-RBD). A preclinical study showed that in hamsters challenged 1 day and 7 days after single-dose vaccination or 6 months after booster vaccination, dNS1-RBD largely mitigated lung pathology, with no loss of body weight, caused by either the prototype-like strain or beta variant of SARS-CoV-2. Lasted data showed that the animals could be well protected against beta variant challenge 9 months after vaccination. Notably, the weight loss and lung pathological changes of hamsters could still be significantly reduced when the hamster was vaccinated 24 h after challenge. Moreover, such cellular immunity is relatively unimpaired for the most concerning SARS-CoV-2 variants. The protective immune mechanism of dNS1-RBD could be attributed to the innate immune response in the nasal epithelium, local RBD-specific T cell response in the lung, and RBD-specific IgA and IgG response. Thus, this study demonstrates that the intranasally delivered dNS1-RBD vaccine candidate may offer an important addition to fight against the ongoing COVID-19 pandemic, compensating limitations of current intramuscular vaccines, particularly at the start of an outbreak.

category: pathology


title: Learning a new class of multisensory associations: High-density electrophysiological mapping of the temporal course of audio-visual object processing.

abstract: Multisensory objects that are frequently encountered in the natural environment lead to strong associations across a distributed sensory cortical network, with the end result experience of a unitary percept. Remarkably little is known, however, about the cortical processes sub-serving multisensory object formation and recognition. To advance our understanding in this important domain, the present study investigated the brain processes involved in learning and identification of novel visual-auditory objects. Specifically, we introduce and test a rudimentary three-stage model of multisensory object-formation and processing. Thirty adults were remotely trained for a week to recognize a novel class of multisensory objects (3D shapes paired to complex sounds), and high-density event related potentials (ERPs) were recorded to the corresponding unisensory (shapes or sounds only) and multisensory (shapes and sounds) stimuli, before and after intensive training. We identified three major stages of multisensory processing: 1) an early, multisensory, automatic effect (<100 ms) in occipital areas, related to the detection of simultaneous audiovisual signals and not related to multisensory learning 2) an intermediate object-processing stage (100-200 ms) in occipital and parietal areas, sensitive to the learned multisensory associations and 3) a late multisensory processing stage (>250 ms) that appears to be involved in both object recognition and possibly memory consolidation. Results from this study provide support for multiple stages of multisensory object learning and recognition that are subserved by an extended network of cortical areas.


title: Spatially regulated protease activity in lymph nodes renders B cell follicles a sanctuary for retention of intact antigens

abstract: The structural integrity of vaccine antigens is critical, as antigen degradation in vivo could eliminate neutralizing epitopes and create competing B cell responses against irrelevant breakdown products. Using FRET imaging and imaging zymography, we found that protease activity and antigen breakdown are spatially heterogeneous in lymph nodes. Following protein immunization, antigens are rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions of the tissue. By contrast, the follicles and follicular dendritic cell (FDC) networks exhibit low protease activity and antigen degradation rates. Immunization regimens targeting antigen rapidly to FDCs led to germinal centers (GCs) where responses to intact antigen were highly dominant, while traditional bolus immunizations led to weaker GC responses where more GC B cells bound to breakdown products than intact antigen. Thus, spatially-compartmentalized antigen proteolysis impacts humoral immunity and can be exploited to enhance vaccine-induced production of antibody responses against key pathogen structural epitopes.


title: Ly6Chi monocytes are metabolically reprogrammed in the blood during inflammatory stimulation allowing for macrophage lineage commitment

abstract:
Acute inflammation is a rapid and dynamic process involving the recruitment and activation of multiple cell types in a co-ordinated and precise manner. Using cell tracking, linage tracing and single cell transcriptomics we investigated the origin and transcriptional reprogramming of monocytes and macrophages in acute inflammation.

Monocyte trafficking and adoptive transfer experiments revealed that monocytes undergo rapid phenotypic change as they exit the blood and give rise to monocyte-derived macrophages that persist during the resolution of inflammation. Single cell transcriptomics revealed significant heterogeneity within the surface marker defined CD11b+Ly6G-Ly6Chi monocyte population within the blood and at the site of inflammation. Lineage trajectory analysis revealed that Ly6Chi monocytes in the blood are re-programmed into a defined differentiation pathway following inflammatory stimulus. We show that two major transcriptional reprogramming events occur during the initial 6 h of Ly6Chi monocyte mobilisation, one in the blood priming monocytes for migration and a second at the site of inflammation. Pathway analysis revealed an important role for oxidative phosphorylation (OxPhos) during both these reprogramming events in a subset of M2-like cells. Experimentally we also demonstrate that OxPhos is essential for murine and human monocyte chemotaxis. These new findings opening up the possibility that altering monocyte metabolic capacity towards OxPhos could facilitate enhanced macrophage M2-like polarisation to aid inflammation resolution and tissue repair.


title: Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture

abstract:
The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded characterization. Polysaccharides remain attached to the cell as capsular polysaccharide (CPS) or are shed into the surroundings in the form of exopolysaccharide (EPS). While a great deal of study has been done examining the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and removed capsular material. Sonication and glucanex digestion yield soluble CPS preparations, while French Press and modified glucanex digestion plus vortexing remove the capsule and cell wall producing polysaccharide aggregates that we call  capsule ghosts. The existence of capsule ghosts implies an inherent organization that allows it to exist independent of the cell wall surface. As sonication and glucanex digestion were noncytotoxic, it was possible to observe the cryptococcal cells rebuilding their capsule, revealing new insights into capsule architecture and synthesis consistent with a model in which the capsule is assembled from smaller polymers, which are then assemble into larger ones.

ImportanceCharacterization of the cryptococcal polysaccharide capsule relies on methods of isolation for its in vitro study. This study demonstrates that the capsule is susceptible to physical and enzymatic removal. The application of new methods yields insights into the anatomy, modular nature, and architecture of the capsule with both soluble CPS preparations and  capsule ghosts. Together these insights inform on a long-standing debate modeling capsular assembly wherein our data shows that the capsule is assembled by smaller polymers added distally rather than by proximal addition or by polymers spanning the entire capsule radius.


title: Bacteriophage cocktails displayed protective effects against soft rot-causing Pectobacterium sp.

abstract: Soft rot caused by Pectobacterium sp. is responsible for significant losses in vegetable production worldwide. Methods for the effective control of soft rot are limited and are primarily based on good agricultural practices. The use of bacteriophages as biocontrol agents appears to be a promising alternative to combat phytopathogens. In this study, we investigated the efficacy of lytic phages recovered from symptomatic tissues and environmental samples against soft rot caused by Pectobacterium carotovorum subsp. carotovorum. Three bacteriophage isolates, designated as vB_PcaP-A3, vB_PcaM-D1, and vB_PcaM-J3, were observed to effectively lyse P. carotovorum subsp. carotovorum. Phage vB_PcaP-A3 exhibited virion morphology similar to the members of the podovirus group, while phages vB_PcaM-D1 and vB_PcaM-J3 showed myovirus morphology based on transmission electron microscopy. The optimal multiplicity of infection (MOI) differed greatly among the three phages. All three phages survived incubations at 30{degrees}C, 40{degrees}C and 50{degrees}C and pH conditions ranging from 3.0 to 9.0, but were all inactivated at 60{degrees}C and at pH 12. Both monophage and cocktail preparations were effective in inhibiting the growth of P. carotovorum subsp. carotovorum in the in vitro challenge tests. In the semi-in planta assays, while treatment with cocktail preparations completely inhibited the development of soft rot in tissue slices, monophage treatments not only resulted in significant reduction of tissue maceration in slices, but also showed protective effect against soft rot in tubers. Overall, these results demonstrate the efficacy of phages vB_PcaP-A3, vB_PcaM-D1, and vB_PcaM-J3 for the biocontrol of soft rot caused by P. carotovorum subsp. carotovorum.


title: Lytic phages displayed protective effects against soft rot-causing Pectobacterium sp.


title: Nieman-Pick Type C2 proteins in Aedes aegypti: Their Structure-Function relationships and Expression in Uninfected versus Virus-infected Mosquitos

abstract: Aedes aegypti is a major vector that transmits arboviruses through the saliva injected into the host. Salivary proteins help in uninterrupted blood intake and enhance the transmission of pathogens. We studied Nieman-Pick Type C2 (NPC2) proteins, a superfamily of saliva proteins that play important role in arbovirus infections. In vertebrates, a single conserved gene encodes for the NPC2 protein that functions in cholesterol trafficking. Arthropods, in contrast, have several genes that encode for divergent NPC2 proteins. We compared the sequences of 20 A. aegypti NPC2 proteins to the cholesterol-binding residues of human and bovine, and fatty acid-binding residues of ant NPC2 proteins. We identified four and one mosquito NPC2 proteins as potential sterol- and fatty acid-binding proteins, respectively. From the published data, we analysed the expression of NPC2 genes in various tissues and their differential expression in midgut and salivary gland post-arbovirus infections. NPC2 genes are downregulated rather than upregulated in virus-infected tissues. Interestingly, AAEL012064 is the only gene that is downregulated in both the midgut and salivary gland in all virus infections. In addition, AAEL001650 is downregulated in the salivary gland infected with CHIKV, DENV2 or ZIKV. This gene in the midgut is downregulated infected with DENV1 but upregulated with DENV2. We studied the variation in cis elements in the promoter regions of two groups of closely related NPC2 genes and the expression of relevant transcription factors (TFs). In the midgut infected with DENV1 or DENV2, six TFs (CRE-BP1, AP1, c-Jun, c-Fos, Odd, and NF-kB) appear to play an opposing role in the expression of AAEL006854. Two TFs (RXR-beta/alpha and USF) have a potential role in the downregulation of AAEL009556 in CHIKV-infected midgut and salivary gland. Similarly, two TFs (COUP and Ftz) may have a key role in the downregulation of AAEL009555 and AAEL009556 in DENV2-infected salivary gland.


title: Genome editing of Capsaspora owczarzaki suggests an ancestral function of the Hippo signaling effector YAP/TAZ/Yorkie in cytoskeletal dynamics but not proliferation

abstract: Animal development is mediated by a surprisingly small set of canonical signaling pathways such as Wnt, Hedgehog, TGF-beta, Notch and Hippo pathways. Although once thought to be present only in animals, recent genome sequencing has revealed components of these pathways in the closest unicellular relatives of animals. These findings raise questions about the ancestral functions of these developmental pathways and their potential role in the emergence of animal multicellularity. Here, we provide the first functional characterization of any of these developmental pathways in unicellular organisms by developing techniques for genetic manipulation in Capsaspora owczarzaki, a close unicellular relative of animals that displays aggregative multicellularity. We then use these tools to characterize the Capsaspora ortholog of the Hippo signaling nuclear effector YAP/TAZ/Yorkie (coYki), a key regulator of tissue size in animals. In contrast to what might be expected based on studies in animals, we show that coYki is dispensable for cell proliferation but regulates cytoskeletal dynamics and the three-dimensional (3D) shape of multicellular structures. We further demonstrate that the cytoskeletal abnormalities of individual coYki mutant cells underlie the abnormal 3D shape of coYki mutant aggregates. Taken together, these findings implicate an ancestral role for the Hippo pathway in cytoskeletal dynamics and multicellular morphogenesis predating the origin of animal multicellularity, which was co-opted during evolution to regulate cell proliferation.

category: cell biology


title: Genome editing in the unicellular holozoan Capsaspora owczarzaki suggests a premetazoan role for the Hippo pathway in multicellular morphogenesis


title: Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1a.

abstract:
Endoplasmic reticulum (ER) to nucleus homeostatic signalling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1. While IRE1 can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1 complexes as well as the targeting mechanism that recruits XBP1 to IRE1 oligomers, remain unknown.

Here, we used a single molecule imaging approach to directly monitor the recruitment of individual XBP1 transcripts to the ER surface. We confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2-dependent targeting and relies on active translation. In addition, we show that IRE1-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1 clusters, which only assemble upon overexpression of fluorescently-tagged IRE1 during ER stress. Our findings support a model where ribosome-engaged, ER-poised XBP1 mRNA is processed by functional IRE1 assemblies that are homogenously distributed throughout the ER membrane.


title: Structures of the Cyanobacterial Phycobilisome

abstract: The phycobilisome is an elaborate antenna that is responsible for light-harvesting in cyanobacteria and red-algae. This large macromolecular complex captures incident sunlight and transfers the energy via a network of pigment molecules called bilins to the photosynthetic reaction centers. The phycobilisome of the model organism Synechocystis PCC 6803 consists of a core to which six rods are attached but its detailed molecular architecture and regulation in response to environmental conditions is not well understood. Here we present cryo-electron microscopy structures of the 6.2 MDa phycobilisome from Synechocystis PCC 6803 resolved at 2.1 [A] (rods) to 2.7 [A] (core), revealing three distinct conformations, two previously unknown. We found that two of the rods are mobile and can switch conformation within the complex, revealing a layer of regulation not described previously. In addition, we found a novel linker protein in the structure, that may represent a long-sought subunit that tethers the phycobilisome to the thylakoid membrane. Finally, we show how excitation energy is transferred within the phycobilisome and correlate our structures with known spectroscopic properties. Together, our results provide detailed insights into the biophysical underpinnings of cyanobacterial light harvesting and lay the foundation for bioengineering of future phycobilisome variants and artificial light harvesting systems.

category: biochemistry


title: Structure and functionality of a multimeric human COQ7:COQ9 complex

abstract: Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined  complex Q metabolon. Here we present a structure and functional analyses of a substrate- and NADH-bound oligomeric complex comprised of two complex Q subunits: the hydroxylase COQ7, which performs the penultimate step in CoQ biosynthesis, and the prenyl lipid-binding protein COQ9. We reveal that COQ7 adopts a modified ferritin-like fold with an extended hydrophobic access channel whose substrate binding capacity is enhanced by COQ9. Using molecular dynamics simulations, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for CoQ intermediates to translocate from within the bilayer to the proteins lipid-binding sites. Two such tetramers assemble into a soluble octamer, closed like a capsid, with lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors and coordinate subsequent synthesis steps toward producing mature CoQ.


title: A Cryptic K48 Ubiquitin Chain Binding Site on UCH37 is Required for its Role in Proteasomal Degradation

abstract: Degradation by the 26S proteasome is an intricately regulated process fine-tuned by the precise nature of ubiquitin modifications attached to a protein substrate. By debranching ubiquitin chains composed of K48 linkages, the proteasome-associated ubiquitin C-terminal hydrolase UCHL5/UCH37 serves as a positive regulator of protein degradation. How UCH37 achieves specificity for K48 chains is unclear. Here, we use a combination of hydrogen-deuterium mass spectrometry, chemical crosslinking, small-angle X-ray scattering, NMR, molecular docking, and targeted mutagenesis to uncover a cryptic K48 ubiquitin chain specific binding site on the opposite face of UCH37 relative to the canonical S1 ubiquitin-binding site. Biochemical assays demonstrate the K48 chain-specific binding site is required for chain debranching and proteasome-mediated degradation of proteins modified with branched chains. Using quantitative proteomics, translation shutoff experiments, and linkage-specific affinity tools, we then identify specific proteins whose degradation depends on the debranching activity of UCH37. Our findings suggest that UCH37 and potentially other DUBs could use more than one S1 site to perform different biochemical functions.


title: Structure of the Quenched Cyanobacterial OCP-Phycobilisome Complex

abstract: Photoprotection is an essential mechanism in photosynthetic organisms to balance the harvesting of light energy against the risks of photodamage. In cyanobacteria, photoprotective non-photochemical quenching relies on the interaction between a photoreceptor, the Orange Carotenoid Protein (OCP), and the antenna, the phycobilisome (PBS). Here we report the first structure of the OCP-PBS complex at 2.7 [A] overall resolution obtained by cryo-electron microscopy. The structure shows that the 6.2 MDa PBS is quenched by four 34 kDa OCP organized as two dimers. The complex also reveals that the structure of the active form of the OCP is drastically different than its resting, non-quenching form, with an [~]60 [A] displacement of its regulatory domain. These results provide a high-resolution blueprint of the structural basis of the protective quenching of excess excitation energy that enables cyanobacteria to harvest light energy and fix CO2 across environmentally diverse and dynamic surface of our planet.


title: Structure and mechanism of oxalate transporter OxlT in an oxalate-degrading bacterium in the gut microbiota

abstract: Oxalobacter formigenes is an oxalate-degrading bacterium in the gut microbiota that absorbs food-derived oxalate to use this as a carbon and energy source and thereby helps reduce the risk of kidney stone formation of the host animals 1-4. The bacterial oxalate transporter OxlT uptakes oxalate from the gut to bacterial cells and excrete formate as a degradation product, with a strict discrimination from other carboxylates that serve as nutrients 5-7. Nevertheless, the underlying mechanism remains unclear. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two different conformations, occluded and outward-facing states. The oxalate binding site contains two basic residues that form salt bridges with a dicarboxylate substrate while preventing the conformational switch to the occluded state without an acidic substrate, a  disallowed state for an antiporter 8, 9. The occluded ligand-binding pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the binding pocket are completely blocked by extensive interdomain hydrophobic and ionic interactions. Nevertheless, a molecular dynamics simulation showed that a flip of a single side chain neighbouring the substrate is sufficient to trigger the gate opening. The OxlT structure indicates the underlying metabolic interactions enabling favourable symbiosis at a molecular level.


title: Cryo-EM of the yeast VO complex reveals distinct binding sites for macrolide V-ATPase inhibitors

abstract: Vacuolar-type adenosine triphosphatases (V-ATPases) are proton pumps found in almost all eukaryotic cells. These enzymes consist of a soluble catalytic V1 region that hydrolyzes ATP and a membrane-embedded VO region responsible for proton translocation. V-ATPase activity leads to acidification of endosomes, phagosomes, lysosomes, secretory vesicles, and the trans-Golgi network, with extracellular acidification occurring in some specialized cells. Small molecule inhibitors of V-ATPase have played a crucial role in elucidating numerous aspects of cell biology by blocking acidification of intracellular compartments, while therapeutic use of V-ATPase inhibitors has been proposed for treatment of cancer, osteoporosis, and some infections. Here, we determine structures of the isolated VO complex from Saccharomyces cerevisiae bound to two well-known macrolide inhibitors: bafilomycin A1 and archazolid A. The structures reveal different binding sites for the inhibitors on the surface of the proton-carrying c ring, with only a small amount of overlap between the two sites. Binding of both inhibitors is mediated primarily through van der Waals interactions in shallow pockets and suggests that the inhibitors block rotation of the ring. Together, these structures indicate the existence of a large chemical space available for V-ATPase inhibitors that block acidification by binding the c ring.


title: Bacteria but not fungi communities differ among perennial grassland ecotypic rhizosphere under abiotic environmental stress

abstract: Environmental change, especially frequent droughts, is predicted to detrimentally impact the North American perennial grasslands. Consistent dry spells will affect plant communities as well as their associated rhizobiomes, possibly altering the plant host performance under environmental stress. Therefore, there is a need to understand the impact of drought on the rhizobiome, and how the rhizobiome may modulate host performance and ameliorate its response to drought stress. In this study, we analyzed bacterial and fungal communities in the rhizospheres of three ecotypes (dry, mesic, and wet) of a dominant prairie grass, Andropogon gerardii. The ecotypes were established in 2010 in a common garden design and grown for a decade under persistent dry conditions at the arid margin of the species range in Colby Kansas. The experiment aimed to answer whether and to what extent do the different ecotypes maintain or recruit distinct rhizobiomes after ten years in an arid climate. In order to answer this question, we screened the bacterial and fungal rhizobiome profiles of the ecotypes under the arid conditions of western KS as a surrogate for future climate environmental stress using 16S rRNA and ITS2 metabarcoding sequencing. Under these conditions, bacterial communities differed compositionally among the A. gerardii ecotypes, whereas the fungal communities did not. The ecotypes were instrumental in driving the differences among bacterial rhizobiomes, as the ecotypes maintained distinct bacterial rhizobiomes even after ten years at the edge of the host species range. This study will aid us to optimize plant productivity through the use of different ecotypes under future abiotic environmental stress, especially drought.


title: Bacterial but not fungal rhizosphere communities differ among perennial grass ecotypes under abiotic environmental stress


title: The effect of sequencing and assembly on the inference of horizontal gene transfer on chromosomal and plasmid phylogenies

abstract: The spread of antibiotic resistance genes on plasmids is a threat to human and animal health. Phylogenies of bacteria and their plasmids contain clues regarding the frequency of plasmid transfer events, as well as the co-evolution of plasmids and their hosts. However, whole genome sequencing data from diverse ecological or clinical bacterial samples is rarely used to study plasmid phylogenies and resistance gene transfer. This is partially due to the difficulty to extract plasmids from short-read sequencing data. Here, we use both short- and long-read sequencing data of 24 clinical extended-spectrum {beta}-lactamase producing Escherichia coli to estimate chromosomal and plasmid phylogenies. We compare the impact of different sequencing and assembly methodologies on these phylogenies and on the inference of horizontal gene transfer. We find chromosomal phylogenies can be estimated robustly with all methods, whereas plasmid phylogenies have more variable topology and branch lengths across the methods used. Specifically, hybrid methods that use long reads to resolve short-read assemblies (HybridSPAdes and Unicycler) perform better than those that started from long-reads during assembly graph generation (Canu). In contrast, the inference of plasmid and antibiotic resistance gene transfer using a parsimony-based criterion is mostly robust to the choice of sequencing and assembly method.


title: MICU1 occludes MCU in the mitochondrial calcium uniporter complex

abstract: The mitochondrial calcium uniporter imports cytoplasmic Ca2+ into the mitochondrial matrix to regulate cell bioenergetics, Ca2+ signaling, and apoptosis. The uniporter contains the pore-forming MCU subunit, an EMRE protein that binds to MCU, and the regulatory MICU1/MICU2 subunits. Structural and biochemical studies have suggested that MICU1 gates MCU by blocking and unblocking the Ca2+ pore. However, mitoplast patch-clamp experiments argue that MICU1 does not block Ca2+ transport but instead potentiates MCU. To address this direct clash of proposed MICU1 function, we applied purified MICU1 to Ca2+-conducting MCU-EMRE subcomplexes in outside-out patches excised from Xenopus oocytes. MICU1 strongly inhibits Ca2+ currents, and the inhibition is abolished by mutating an MCU-interacting K126 residue in MICU1. Further experiments show that MICU1 block was not observed in mitoplasts because MICU1 dissociates from the uniporter complex. These results firmly establish that MICU1 shuts the uniporter in resting cellular conditions.


title: Engraftment of allotransplantated tumour cells in adult rag2 mutant Xenopus tropicalis

abstract: Modelling human genetic diseases and cancer in lab animals has been greatly aided by the emergence of genetic engineering tools such as TALENs and CRISPR/Cas9. We have previously demonstrated the ease with which genetically engineered Xenopus models (GEXM) can be generated. This included the induction of autochthonous tumour formation by injection of early embryos with Cas9 recombinant protein loaded with sgRNAs targeting multiple tumour suppressor genes. What has been lacking so far is the possibility to propagate the induced cancers via transplantation. In this paper we describe the generation of a rag2-/- knock-out line in Xenopus tropicalis that is deficient in functional T- and B-cells. This line was validated by means of an allografting experiment with a primary tp53-/- donor tumour. In addition, we optimized available protocols for sub-lethal gamma irradiation of X. tropicalis froglets. Irradiated animals also allowed stable, albeit transient, engraftment of transplanted tp53-/- tumour cells. The novel X. tropicalis rag2-/- line and the irradiated wild type froglets will further expand the experimental toolbox in this diploid amphibian, and help to establish it as a versatile and relevant model for exploring human cancer.

category: cancer biology


title: Loss of soluble guanylyl cyclase in platelets contributes to atherosclerotic plaque formation and vascular inflammation

abstract:
AimThe role of platelets in atherosclerosis remains incompletely understood. Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here we sought to investigate the contribution of platelet sGC to atherosclerosis and the therapeutic potential of targeting sGC in atherosclerosis.

Methods and ResultsWe genetically deleted sGC in platelets of atherosclerosis-prone Ldlr-/- mice. By intravital fluorescence microscopy such Pf4-Cre+Gucy1b1flox/floxLdlr-/- mice displayed enhanced leukocyte adhesion to atherosclerotic plaques in comparison with their litter mates. Moreover, histological and flow cytometry analyses revealed more numerous inflammatory leukocytes and larger plaque sizes in aortic tissue of Ldlr-/- mice lacking sGC in platelets. In vitro, supernatant from activated platelets lacking sGC promoted leukocyte adhesion to endothelial cells (EC) via enhanced EC activation. Using cytokine profiling, we identified reduced angiopoietin-1 release by Pf4-Cre+Gucy1b1flox/flox and human GUCY1A1 risk allele carrier platelets to be responsible for enhanced activation of EC and subsequent leukocyte adhesion. Pharmacological sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced recruitment of adoptively transferred leukocytes in Ldlr-/- mice fed a Western diet. Pharmacological sGC stimulation further reduced atherosclerotic plaque formation and vascular inflammation.

ConclusionLoss of sGC in platelets contributes to atherosclerotic plaque formation via reduced release of the soluble factor angiopoietin-1 and, subsequently, enhanced leukocyte recruitment. Pharmacological sGC stimulation might represent a novel therapeutic strategy to prevent and treat CAD.

Translational perspectiveReduced platelet soluble guanylyl cyclase activity contributes to atherosclerotic plaque formation and vascular inflammation. Stimulators of the soluble guanylyl cyclase, an emerging class of drugs already used in pulmonary hypertension and heart failure, are able to reduce atherosclerosis and inflammation in this preclinical model. Together with evidence from human genetics, our findings suggest a promising role of soluble guanylyl cyclase stimulation to prevent coronary artery disease.