title: The plant-specific SCL30a SR protein regulates ABA-dependent seed traits and salt stress tolerance during germination

abstract:
SR (serine/arginine-rich) proteins are conserved RNA-binding proteins best known as key regulators of splicing, which have also been implicated in other steps of gene expression. Despite mounting evidence for their role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain elusive. Here we show that the plant-specific SCL30a SR protein negatively regulates abscisic acid (ABA) signaling to control important seed traits and salt stress responses during germination in Arabidopsis. The SCL30a gene is upregulated during seed imbibition and germination, and its loss of function results in smaller seeds displaying enhanced dormancy and elevated expression of ABA-responsive genes as well as of genes repressed during the germination process. Moreover, the knockout mutant is hypersensitive to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA sensitivity and enhanced tolerance to salt stress during seed germination. An ABA biosynthesis inhibitor rescues the mutants enhanced sensitivity to stress, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the SR protein positively regulates stress tolerance during seed germination by reducing sensitivity to the phytohormone. Our results reveal a new key player in ABA-mediated control of early development and stress response, and underscore the role of plant SR proteins as important regulators of the ABA signaling pathway.

Author SummarySeed germination is a critical step in plant development determining the transition to aerial growth and exposure to a more challenging environment. As such, seeds have evolved mechanisms that prevent germination under adverse conditions, thereby increasing the chances of plant survival. As a general regulator of plant development and a key mediator of stress responses, the hormone abscisic acid (ABA) promotes a prolonged non-germinating state called dormancy, influences seed size and represses germination under environmental stress. Here, we show that an RNA-binding protein, SCL30a, controls seed size, dormancy, germination and tolerance to high salinity in the model plant Arabidopsis thaliana. Loss of SCL30a gene function results in smaller and more dormant seeds with reduced ability to germinate in a high-salt environment; by contrast, SCL30a overexpression produces larger seeds that germinate faster under salt stress. Using a large-scale gene expression analysis, we identify the ABA hormonal pathway as a putative target of SCL30a. We then use genetic and pharmacological tools to unequivocally demonstrate that the uncovered biological functions of SCL30a are achieved through modulation of the ABA pathway. Our study reveals a novel regulator of key seed traits and has biotechnological implications for crop improvement under adverse environments.


title: Haplotype mapping uncovers unexplored variation in wild and domesticated soybean at the major protein locus cqProt-003

abstract:
Here, we present association and linkage analysis of 985 wild, landrace and cultivar soybean accessions in a pan genomic dataset to characterize the major high-protein/low-oil associated locus cqProt-003 located on chromosome 20. A significant trait associated region within a 173 kb linkage block was identified and variants in the region were characterised, identifying 34 high confidence SNPs, 4 insertions, 1 deletion and a larger 304 bp structural variant in the high-protein haplotype. Trinucleotide tandem repeats of variable length present in the third exon of gene 20G085100 are strongly correlated with the high-protein phenotype and likely represent causal variation. Structural variation has previously been found in the same gene, for which we report the global distribution of the 304bp deletion and have identified additional nested variation present in high-protein individuals. Mapping variation at the cqProt-003 locus across demographic groups suggests that the high-protein haplotype is common in wild accessions (94.7%), rare in landraces (10.6%) and near absent in cultivated breeding pools (4.1%), suggesting its decrease in frequency primarily correlates with domestication and continued during subsequent improvement. However, the variation that has persisted in under-utilized wild and landrace populations holds high breeding potential for breeders willing to forego seed oil to maximise protein content. The results of this study include the identification of distinct haplotype structures within the high-protein population, and a broad characterization of the genomic context and linkage patterns of cqProt-003 across global populations, supporting future functional characterisation and modification.

Key messageThe major soy protein QTL, cqProt-003, was analysed for haplotype diversity and global distribution, results indicate 304bp deletion and variable tandem repeats in protein coding regions are likely causal candidates.

category: plant biology


title: Chemical inhibition of auxin inactivation pathway uncovers the metabolic turnover of auxin homeostasis

abstract: The phytohormone auxin, specifically indole-3-acetic acid (IAA) plays a prominent role in plant development. Cellular auxin concentration is coordinately regulated by auxin synthesis, transport, and inactivation to maintain auxin homeostasis; however, the physiological contribution of auxin inactivation to auxin homeostasis has remained elusive. The GH3 genes encode auxin amino acid conjugating enzymes that perform a central role in auxin inactivation. The chemical inhibition of GH3s in planta is challenging because the inhibition of GH3 enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we developed a potent GH3 inhibitor, designated as kakeimide (KKI), that selectively targets auxin-conjugating GH3s. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min), indicating auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.


title: Non-micronized and micronized curcumin do not prevent the behavioral and neurochemical effects induced by acute stress in zebrafish

abstract: Curcumin, a polyphenol extracted from the rhizome of Curcuma longa L. (Zingiberaceae), presents neuroprotective properties and can modulate neuronal pathways related to mental disorders. However, curcumin has low bioavailability, which can compromise its use. The micronization process can reduce the mean particle diameter and improve this compounds bioavailability and therapeutic potential. In this study, we compared the behavioral (in the open tank test, OTT) and neurochemical (thiobarbituric acid reactive substances (TBARS) and non-protein thiols (NPSH) levels) effects of non-micronized curcumin (CUR, 10 mg/kg, i.p.) and micronized curcumin (MC, 10 mg/kg, i.p.) in adult zebrafish subjected to 90-minute acute restraint stress (ARS). ARS increased the time spent in the central area and the number of crossings and decreased the immobility time of the animals. These results suggest an increase in locomotor activity and a decrease in thigmotaxis behavior in the OTT. Furthermore, ARS also induced oxidative damage by increasing TBARS and decreasing NPSH levels. ARS-induced behavioral and biochemical effects were not blocked by any curcumin preparation. Therefore, we suppose that curcumin does not have anti-stress effects on the ARS in zebrafish.

category: neuroscience


title: A Standardized Protocol for Quantifying the Behavioral Dynamics of Food-seeking in Mice

abstract: Food is generally hidden in a natural environment and require free-living animals to search for it. Although such food-seeking behaviors involve motivation and exploration, previous studies examined food-seeking simply by measuring the time spent in the food zone or the frequency of pursuing food-cued context. Moreover, after discovering food, animals need to taste and smell it in order to evaluate their nutritional value or possible toxicity. However, researchers could not easily distinguish food-seeking from food-evaluating behaviors because food was visible or accessible throughout each test. Herein, we describe a behavioral protocol that triggers animals to show the behavioral dynamics of food-seeking (e.g., navigation, nose-digging, and paw-digging) and that exclusively elicits food-seeking without provoking any other food-evaluating behaviors. First, we prepared an open-field box with the floor covered with bedding. After we hid foods under the bedding of each corner, the test mice were habituated in this arena for four days (pre-test phase). On the next day (test phase), they were placed under the same conditions, but the foods previously hidden were removed. This process enabled the mice to perceive their surroundings as a food-hidden environment, which induced the animal to exhibit sustained food-seeking. In conclusion, the protocol presented here is a powerful method for provoking multiple forms of food-seeking and quantifies food-seeking independently from other food-related behavioral stages.


title: Structural and functional asymmetry of the neonatal cerebral cortex

abstract: Features of brain asymmetry have been implicated in a broad range of cognitive processes; however, their origins are still poorly understood. Using a new left-right symmetric, spatiotemporal cortical surface atlas, we investigated cortical asymmetries in 442 healthy newborn infants soon after birth, using structural and functional magnetic resonance images from the Developing Human Connectome Project. We identified previously unrecognised structural and functional asymmetries in auditory, visual and sensorimotor cortices, which closely resemble known asymmetries in adults. These findings show that cortical asymmetries are largely determined prenatally and suggest that they may constrain the development of lateralised functions in later life. In adults, deviations in brain asymmetry have been implicated in a broad range of developmental and psychiatric disorders, some of which have been associated with abnormal perinatal neurodevelopment. To test the hypothesis that normal cortical asymmetry is disrupted in the perinatal period by severe environmental stress, we compared cortical asymmetries between the same group of term neonates and 103 preterm neonates imaged at term-equivalent age. No significant differences were seen between these two cohorts, showing that the development of cortical asymmetries proceeds largely unaffected by preterm birth.


title: Mast cell regranulation involves a metabolic switch promoted by the interaction between mTORC1 and a glucose-6-phosphate transporter

abstract: Mast cells (MCs) are highly granulated tissue resident hematopoietic cells and because of their capacity to degranulate and release many proinflammatory mediators, they are major effectors of chronic inflammatory disorders including asthma and urticaria. As MCs have the unique capacity to reform their granules following degranulation in vitro, their potential to undergo multiple cycles of degranulation and regranulation in vivo has been linked to their pathogenesis. However, it is not known what factors regulate MC regranulation let alone if MC regranulation occurs in vivo. Here, we report that IgE-sensitized mice can undergo multiple bouts of regranulation, following repeated anaphylactic reactions. mTORC1, a critical nutrient sensor that activates protein and lipid synthesis, was found necessary for MC regranulation. mTORC1 activity in MCs was regulated by a glucose-6-phosphate transporter, Slc37a2, which was found to be necessary for increased glucose-6-phosphate and ATP levels during regranulation, two upstream signals of mTOR. Slc37a2 is highly expressed at the cell periphery early during regranulation where it appears to colocalize with mTORC1. Additionally, this transporter was found to concentrate extracellular metabolites within endosomes which are trafficked directly into nascent granules. Thus, the metabolic switch associated with MC regranulation is mediated by the interactions of a cellular metabolic sensor and a transporter of extracellular metabolites into MC granules.

category: cell biology


title: Environmentally dependent and independent control of cell shape determination by Rho GTPase regulators in melanoma

abstract: In order to invade 3D tissues, cancer cells dynamically change cell morphology in response to geometric and mechanical cues in the environment. But how cells determine their shape in 3D versus 2D environments is poorly understood. Studying 2D versus 3D single cell shape determination has historically been technically difficult due to the lack of methodologies to directly compare the two environments. We developed an approach to study cell shape in 2D versus 3D by measuring cell shape at different depths in collagen using stage-scanning oblique plane microscopy (ssOPM). We find characteristic shape changes occur in melanoma cells depending on whether a cell is attached to a 2D surface or 3D environment, and that these changes can be modulated by Rho GTPase regulatory proteins. Our data suggest that regulation of cell protrusivity undergoes a  switch of control between different Rho GTPase regulators depending on the physical microenvironment.


title: On the Use of Z-Scores for Fine-Mapping with Related Individuals

abstract: Using summary statistics from genome-wide association studies (GWAS) has been widely used for fine-mapping complex traits in humans. The statistical framework was largely developed for unrelated samples. Though it is possible to apply the framework to fine-mapping with related individuals, extensive modifications are needed. Unfortunately, this has often been ignored in summary-statistics-based fine-mapping with related individuals. In this paper, we show in theory and simulation what modifications are necessary to extend the use of summary statistics to related individuals. The analysis also demonstrates that though existing summary-statistics-based fine-mapping methods can be adapted for related individuals, they appear to have no computational advantage over individual-data-based methods.

category: genetics


title: New markers for qPCR detection of the dinoflagellate Alexandrium catenella in Chile

abstract: Alexandrium catenella (Whedon & Kofoid) is a dinoflagellate known as a primary source of paralytic shellfish poisoning in Chile. The distribution range of harmful algal blooms generated by this species has extended during the last decades, and the frequency of these events has increased. In this work, we developed TaqMan markers from Chilean strains that can be used to identify and quantify through qPCR, which can be implemented in monitoring programs for the early detection of this species.


title: The fungicide Tebuconazole induces electromechanical cardiotoxicity in murine hearts

abstract: Tebuconazole (TEB) is an important fungicide that belongs to the triazole family. It is largely applied in agriculture and its use has increased in the last decade. Since TEB is stable in water and soil, long-term exposure of humans to this pesticide is a real threat. Acute toxicological studies to uncover the toxicity of TEB are limited, and there is evidence of an association between long-term exposure to TEB and damage of several biological systems, including hepatotoxicity and cardiotoxicity. In this paper, the effects of acute exposure of cardiomyocytes and murine hearts to TEB were addressed to elucidate its impact on electromechanical properties of the cardiac tissue. In whole-cell patch-clamp records, TEB inhibited both the total outward potassium current (IC50=5.7{+/-}1.5 mol.l-1) and the L-type calcium current (IC50=33.2{+/-}7.4 mol.l-1). Acute exposure to TEB at 30 mol.l-1 prolonged the action potential duration as well as an induced out-of-pace action potential, and increased the sodium/calcium exchanger current in its forward and reverse modes. Moreover, sarcomere shortening and calcium transient in isolated cardiomyocytes was enhanced when cells were exposed to TEB at 30 mol.l-1. In ex vivo experiments, TEB 30 mol.l-1 caused significant electrocardiogram remodeling with prolonged PR, QRS, and QT interval duration. Accordingly, TEB exposure was prone to the appearance of arrhythmias. Combined, our results demonstrate that acute TEB exposure affects the cardiomyocytes electro-contractile properties and triggers the appearance of ECG abnormalities, including conduction defects and arrhythmias.

category: pharmacology and toxicology


title: Topological Constraints on Noise Propagation in Gene Regulatory Networks

abstract: Gene expression, the production of protein from DNA and mRNA in the biological cell, is inherently stochastic. Cells with identical DNA exhibit fluctuations or  noise in gene expression. This noise propagates over gene regulatory networks (GRNs), which encode gene-gene interactions. The propagated  extrinsic noise interacts and combines with  intrinsic noise to affect biological decisions. Consequently, it is essential to understand how GRN topology affects total noise. Recently, uncertainty principles were established for noise propagation over GRN. In particular, in ring GRNs, exactly one node can have noise reduction below the intrinsic limit. We establish necessary and sufficient conditions for noise reduction in ring GRN. Specifically, for two- and three-node rings, an odd number of negative regulations is necessary for noise reduction. Further, sufficiency is ensured if sensitivities to input for feedforward and feedback regulations are bounded from below and above, respectively. These constraints are valid even if the ring GRN are regulated by an upstream gene. Finally, we use graph theory to decompose noise propagation in a general directed network over its strongly connected components. The combination of graph theory and stochastic processes may be a general framework for studying noise propagation.

category: systems biology


title: Fetal cerebellar development: 3D morphometric analysis of fetal postmortem MRI among Chinese fetuses.

abstract:
The development of the cerebellum starts from early gestational period and extends postnatal. Because of its protracted development, the cerebellum is susceptible to developmental anomalies such as Dandy-Walker malformations, Blakes pouch cysts and vermin hypoplasia. Measurements of fetal cerebellar parameters of a normal growing fetus in each week of gestation is important for setting up morphometric standards and hence used as clinical reference data. Any deviation from the normal cerebellar parameters alerts the clinicians for the possibility of presence of cerebellar malformations.

Study objectiveThe objective of this study was to assess the fetal cerebellar growth by quantifying the following parameters; fetal cerebellar volume, anterior-posterior diameter and superior-inferior diameter.

MethodsWe used 3T and 7T MRI to scan the postmortem fetal brains at different stages of development and subsequently analyze the images using ITK-SNAP software.

ResultsThe mean superior-inferior cerebellar diameter was found to be 19.12{+/-}2. 70mm.The linear(y=bo+b1t) model was the best fit (r2=0.996, F=32022.961) to describe the relationship between the gestational age and the superior-inferior diameter(y=5.89+0.49t). There was significant correlation between the superior-inferior cerebellar diameter and the gestation age, Pearson correlation coefficient of 0.999, r=0.001. The median cerebellar volume was 8607.7mm, the mean rank high among males(78.12) as compared to female(68.25).There was no statistically significant difference of the cerebellar volume between males and females (u=2193.5,p=0.16).The quadratic(y=bo+b1t+b2t2) model was the best fit regression equation (r2=0.994,F=10791.157) describing the relationship between the cerebellar volume and the gestational age. The median anterior- posterior diameter was 12.45 mm. There was significant correlation between anterior-posterior diameter and the gestational age with Spearmans rho of (0.997, p=0.01). The linear model was the best fit the best fit model (y=bo+b1t) describing the relationship between anterior-posterior diameter and the gestational age(y=3.31+0.5t) r2=0.998, F=70646.838

ConclusionSignificant correlation between the superior-inferior cerebellar diameters, the anterior-posterior cerebellar diameter and the gestational age was found. These two linear parameters follow the first-degree polynomial in relation to the gestational age. The cerebellar volume follows the second-degree polynomial as it increases with the gestational age and correlate significantly with the gestational age. This study has provided new insight to the development of the cerebellum, and setup a benchmark data of which the deviation from it will alert the clinicians for the possibility of presence of cerebellar malformations.


title: Long-range migration of centrioles to the apical surface of the olfactory epithelium

abstract: Olfactory sensory neurons (OSNs) in vertebrates detect odorants using multiple cilia, which protrude from the end of the dendrite and require centrioles for their formation. In mouse olfactory epithelium, the centrioles originate in progenitor cells near the basal lamina, often 50 to 100 m from the apical surface. It is unknown how centrioles traverse this distance or mature to form cilia. Using high-resolution expansion microscopy, we found that centrioles migrate together, with multiple centrioles per group and multiple groups per OSN, during dendrite outgrowth. Centrioles were found by live imaging to migrate slowly, with a maximum rate of 0.18 m/min. Centrioles in migrating groups were associated with microtubule nucleation factors, but acquired rootletin and appendages only in mature OSNs. The parental centriole had preexisting appendages, formed a single cilium prior to other centrioles, and retained its unique appendage configuration in the mature OSN. We developed an air-liquid interface explant culture system for OSNs and used it to show that centriole migration can be perturbed ex vivo by stabilizing microtubules. We consider these results in the context of a comprehensive model for centriole formation, migration, and maturation in this important sensory cell type.


title: The mechanochemical origins of the microtubule sliding motility within the kinesin-5 domain organization

abstract: The conserved kinesin-5 bipolar tetrameric motors slide apart microtubules during mitotic spindle assembly and elongation. Kinesin-5 bipolar organization originates from its conserved tetrameric helical minifilament, which position the C-terminal tail domains of two subunits near the N-terminal motor domains of two anti-parallel subunits (Scholey et al, 2014). This unique tetrameric structure enables kinesin-5 to simultaneously engage two microtubules and transmit forces between them, and for multiple kinesin-5 motors to organize via tail to motor interactions during microtubule sliding (Bodrug et al, 2020). Here, we show how these two structural adaptations, the kinesin-5 tail-motor domain interactions and the length of the tetrameric minifilament, determine critical aspects of kinesin-5 motility and sliding mechanisms. An x-ray structure of the 34-nm kinesin-5 minifilament reveals how the dual dimeric N-terminal coiled-coils emerge from the tetrameric central bundle. Using this structure, we generated active bipolar mini-tetrameric motors from Drosophila and human orthologs, which are half the length of native kinesin-5. Using single-molecule motility assays, we show that kinesin-5 tail domains promote mini-tetramers static pauses that punctuate processive motility. During such pauses, kinesin-5 mini-tetramers form multi-motor clusters mediated via tail to motor domain cross-interactions. These clusters undergo slow and highly processive motility and accumulate at microtubule plus-ends. In contrast to native kinesin-5, mini-tetramers require tail domains to initiate microtubule crosslinking. Although mini-tetramers are highly strained in initially aligning microtubules, they slide microtubules more efficiently than native kinesin-5, due to their decreased minifilament flexibility. Our studies reveal that the conserved kinesin-5 motor-tail mediated clustering and the length of the tetrameric minifilament are key features for sliding motility and are critical in organizing microtubules during mitotic spindle assembly and elongation.


title: A stress-reduced passaging technique improves the viability of human pluripotent cells

abstract:
Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, yet some problems, such as the reproducibility among the experiments, remain. Here we describe an improved method for the subculture of human PSCs. The revised method significantly enhanced the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing, gene delivery, and directed differentiation. Furthermore, the method did not alter PSC characteristics after long-term culture and attenuated the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and reduces the rate of PSCs failing quality control of human PSC research and application.

HighlightsO_LIThe revised passaging method significantly increases the viability of human PSCs.
C_LIO_LIThe method triggers less DNA damage and apoptosis signals compared to the conventional method.
C_LIO_LIThe stress-reduced method improves the results of downstream applications.
C_LIO_LIThe method does not alter PSC characters and attenuates the overgrowth of abnormal subpopulations.
C_LI


title: Superresolution microscopy reveals partial preassembly and subsequent bending of the clathrin coat during endocytosis

abstract:
Eukaryotic cells use clathrin-mediated endocytosis to take up a large range of extracellular cargos. During endocytosis, a clathrin coat forms on the plasma membrane, but it remains controversial when and how it is remodeled into a spherical vesicle.

Here, we use 3D superresolution microscopy to determine the precise geometry of the clathrin coat at large numbers of endocytic sites. Through pseudo-temporal sorting, we determine the average trajectory of clathrin remodeling during endocytosis. We find that clathrin coats assemble first on flat membranes to 50% of the coat area, before they become rapidly and continuously bent, and confirm this mechanism in three cell lines. We introduce the cooperative curvature model, which is based on positive feedback for curvature generation. It accurately describes the measured shapes and dynamics of the clathrin coat and could represent a general mechanism for clathrin coat remodeling on the plasma membrane.


title: De novo Prediction of Cell-Drug Sensitivities Using Deep Learning-based Graph Regularized Matrix Factorization

abstract: Application of artificial intelligence (AI) in precision oncology typically involves predicting whether the cancer cells of a patient (previously unseen by AI models) will respond to any of a set of existing anticancer drugs, based on responses of previous training cell samples to those drugs. To expand the repertoire of anticancer drugs, AI has also been used to repurpose drugs that have not been tested in an anticancer setting, i.e., predicting the anticancer effects of a new drug on previously unseen cancer cells de novo. Here, we report a computational model that addresses both of the above tasks in a unified AI framework. Our model, referred to as deep learning-based graph regularized matrix factorization (DeepGRMF), integrates neural networks, graph models, and matrix-factorization techniques to utilize diverse information from drug chemical structures, their impact on cellular signaling systems, and cancer cell cellular states to predict cell response to drugs. DeepGRMF learns embeddings of drugs so that drugs sharing similar structures and mechanisms of action (MOAs) are closely related in the embedding space. Similarly, DeepGRMF also learns representation embeddings of cells such that cells sharing similar cellular states and drug responses are closely related. Evaluation of DeepGRMF and competing models on Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE) datasets show its superiority in prediction performance. Finally, we show that the model is capable of predicting effectiveness of a chemotherapy regimen on patient outcomes for the lung cancer patients in The Cancer Genome Atlas (TCGA) dataset.*

category: bioinformatics


title: Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization

abstract: The microtubule (MT) cytoskeleton is central to cellular processes including axonal growth, intracellular transport, and cell division, all of which rely on precise spatiotemporal control of MT organization. Kinesin-8s play a key role in regulating MT length by combining highly processive directional motility with MT-end disassembly. However, how kinesin-8 switches between these two apparently opposing activities remains unclear. Here, we define the structural features underlying this molecular switch through cryo-EM analysis of the yeast kinesin-8, Kip3 bound to MTs, and molecular dynamics simulations to approximate the complex of Kip3 with the curved tubulin state found at the MT plus-end. By integrating biochemical and single-molecule biophysical assays, we identified specific intra- and intermolecular interactions that modulate processive motility and MT disassembly. Our findings suggest that Kip3 undergoes conformational changes in response to tubulin curvature that underlie its unique ability to interact differently with the MT lattice than with the MT-end.

category: biophysics


title: Recording of cellular physiological histories along optically readable self-assembling protein chains

abstract: Observing cellular physiological histories is key to understanding normal and disease-related processes, but longitudinal imaging is laborious and equipment-intensive. A tantalizing possibility is that cells could record such histories in the form of digital biological information within themselves, for later high-throughput readout. Here we show that this concept can be realized through information storage in the form of growing protein chains made out of multiple self-assembling subunits bearing different labels, each corresponding to a different cellular state or function, so that the physiological history of the cell can be visually read out along the chain of proteins. Conveniently, such protein chains are fully genetically encoded, and easily readable with simple, conventional optical microscopy techniques, compatible with visualization of cellular shape and molecular content. We use such expression recording islands (XRIs) to record gene expression timecourse downstream of pharmacological and physiological stimuli, in cultured neurons and in living mouse brain.

category: synthetic biology


title: Tissue-wide coordination of calcium signaling regulates the epithelial stem cell pool during homeostasis

abstract: Skin epidermal homeostasis is maintained via constant regeneration by stem cells, which must communicate to balance their self-renewal and differentiation. A key molecular pathway, Ca2+ signaling has been implicated as a signal integrator in developing and wounded epithelial tissues[1, 2, 3, 4]. Yet how stem cells carry out this signaling across a regenerative tissue remains unknown due to significant challenges in studying signaling dynamics in live mice, limiting our understanding of the mechanisms of stem cell communication during homeostasis. To interpret high dimensional signals that have complex spatial and temporal patterns, we combined optimized imaging of Ca2+ signaling in thousands of epidermal stem cells in living mice with a new machine learning tool, Geometric Scattering Trajectory Homology (GSTH). Using a combination of signal processing, data geometry, and topology, GSTH captures patterns of signaling at multiple scales, either between direct or distant stem cell neighbors. Here we show that epidermal stem cells display dynamic intercellular Ca2+ signaling among neighborhoods of up to 10 cells that is surprisingly coordinated and directed through time across a pool of thousands of stem cells. We find that this collective coordination is an emergent property of the stem cell compartment, distinct from excitatory quiescent neuronal tissues. We demonstrate that cycling stem cells, specifically G2 cells, govern homeostatic patterns of Ca2+ signaling. Stem cells in different cell cycle stages dynamically regulate localization of the gap junction component Connexin43 (Cx43). Lastly, we uncouple global from local communication and identify Cx43 as the molecular mediator necessary for connectivity between local signaling neighborhoods. This work provides resolution in how stem cells at different stages of the cell cycle communicate and how that diversity of phases is essential for tissue wide communication and signaling flow during epidermal regeneration. Our approach provides a framework to investigate stem cell populations and their signaling dynamics, previously not possible.


title: G2 stem cells orchestrate time-directed, long-range coordination of calcium signaling during skin epidermal regeneration


title: Oncogenic KRAS Requires Complete Loss of BAP1 Function for Development of Murine Intrahepatic Cholangiocarcinoma

abstract: Intrahepatic cholangiocarcinoma (ICC) is a primary biliary malignancy that harbors a dismal prognosis. Oncogenic mutations of KRAS and loss of function mutations of BRCA1-associated protein 1 (BAP1) have been identified as recurrent somatic alterations in ICC. However, an autochthonous genetically engineered mouse model of ICC that genocopies the co-occurrence of these mutations has never been developed. By crossing Albumin-Cre mice bearing conditional alleles of mutant Kras and/or floxed Bap1, Cre-mediated recombination within the liver was induced. Mice with hepatic expression of mutant KrasG12D alone (KA), bi-allelic loss of hepatic Bap1 (BhomoA), and heterozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhetKA) developed primary hepatocellular carcinoma (HCC), but no discernible ICC. In contrast, mice with homozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhomoKA) developed discrete foci of HCC and ICC. Further, the median survival of BhomoKA mice was significantly shorter at 24 weeks, when compared to median survival of [≥]40 weeks in BhetKA mice and approximately 50 weeks in BhomoA and KA mice (p <0.001). Microarray analysis performed on liver tissue from KA and BhomoKA mice identified differentially expressed genes in the setting of BAP1 loss and suggests that deregulation of ferroptosis might be one mechanism by which loss of BAP1 cooperates with oncogenic Ras in hepato-biliary carcinogenesis. Our autochthonous model provides an in vivo platform to further study this lethal class of neoplasm.


title: RNAi screening of RING/U-box domain ubiquitin ligases identifies critical regulators of stem cells and tissue regeneration in planarians

abstract: Regenerative processes depend on the interpretation of signals to coordinate cell behaviors. The role of ubiquitin-mediated signaling is known to be important in many cellular and biological contexts, but its role in regeneration is not well understood. To investigate how ubiquitylation impacts tissue regeneration in vivo, we are studying planarians that are capable of regenerating after nearly any injury using a population of stem cells. Here we used RNAi to screen RING/U-box E3 ubiquitin ligases that are highly expressed in planarian stem cells and stem cell progeny. RNAi screening identified nine genes with functions in regeneration, including the spliceosomal factor prpf19 and histone modifier rnf2; based on their known roles in developmental processes, we further investigated these two genes. We found that prpf19 was required for animal survival but not for stem cell maintenance, suggesting a role in promoting cell differentiation. Because RNF2 is the catalytic subunit of the Polycomb Repressive Complex 1 (PRC1), we also examined other putative members of this complex (CBX and PHC). We observed a striking phenotype of regional tissue misspecification in cbx and phc RNAi planarians. To identify genes regulated by PRC1, we performed RNA-seq after knocking down rnf2 or phc. Although these proteins are predicted to function in the same complex, we found that the set of genes differentially expressed in rnf2 versus phc RNAi were largely non-overlapping. Using in situ hybridization, we showed that rnf2 regulates gene expression levels within a tissue type, whereas phc is necessary for the spatial restriction of gene expression, findings consistent with their respective in vivo phenotypes. This work not only uncovered roles for RING/U-box E3 ligases in stem cell regulation and regeneration, but also identified differential gene targets for two putative PRC1 factors required for maintaining cell-type-specific gene expression in planarians.

category: developmental biology


title: RNAi screen of RING/U-box domain ubiquitin ligases identifies critical regulators of tissue regeneration in planarians


title: Nephronectin-Integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development

abstract: During development, cells aggregate at tissue boundaries to form normal tissue architecture of organs. However, how cells are segregated into tissue precursors remains largely unknown. Cornea development is a perfect example of this process whereby neural crest cells aggregate in the periocular region prior to their migration and differentiation into corneal cells. Our recent RNA-Seq analysis identified upregulation of Nephronectin (Npnt) transcripts during early stages of corneal development where its function has not been investigated. We found that Npnt mRNA and protein are expressed by various ocular tissues including the migratory periocular neural crest (pNC), which also express the integrin alpha 8 (Itg8) receptor. Knockdown of either Npnt or Itg8 attenuated cornea development, whereas overexpression of Npnt resulted in cornea thickening. Moreover, overexpression of Npnt variants lacking RGD binding sites did not affect corneal thickness. Neither the knockdown or augmentation of Npnt caused significant changes in cell proliferation, suggesting that Npnt directs pNC migration into the cornea. In vitro analyses showed that Npnt promotes pNC migration from explanted periocular mesenchyme, which requires Itg8. Combined, these findings show that Npnt specifies and tunes cell migration into the presumptive cornea ECM by providing a substrate for Itg8-positive pNC cells.


title: Role of the reaction-structure coupling in temperature compensation of the KaiABC circadian rhythm

abstract: When the mixture solution of cyanobacterial proteins, KaiA, KaiB, and KaiC, is incubated with ATP in vitro, the phosphorylation level of KaiC shows stable oscillations with the temperature-compensated circadian period. We analyzed this temperature compensation by developing a theoretical model describing the feedback relations among reactions and structural transitions in the KaiC molecule. The model showed that the reduced structural cooperativity should weaken the negative feedback coupling among reactions and structural transitions, which enlarges the oscillation amplitude and period, explaining the observed significant period extension upon single amino-acid residue substitution. We propose that an increase in thermal fluctuations similarly attenuates the reaction-structure feedback, explaining the temperature compensation in the KaiABC clock. The model suggests that the ATPase reactions in the CI domain of KaiC affect the period depending on how the reaction rates are modulated. The KaiABC clock provides a unique opportunity to analyze how the reaction-structure coupling regulates the system-level synchronized oscillations of molecules.


title: Ex Vivo Culture Models of Hidradenitis Suppurativa for defining molecular pathogenesis and treatment efficacy of novel drugs

abstract: Hidradenitis suppurativa (HS) is a complex inflammatory and debilitating skin disease for which no effective treatment is available. This is partly because of the unavailability of suitable human or animal models with which exact pathobiology of the disease can be defined. Here, we describe the development of air-liquid (A-L) interface, liquid-liquid/liquid-submersion (L-S) and bioreactor (Bio) ex vivo skin culture models. All three ex vivo platforms were effective for culturing skin samples up to day-14, with the tissue architecture and integrity remaining intact for at least 3 days for healthy skin while for 14 days for HS skin. Up to day-3, no significant differences were observed in % early apoptotic cells among all three platforms. However, an increase was observed in late apoptotic/necrotic cells in HS skin at day-3 in A-L and Bio culture of HS skin. These cultures efficiently support the growth of various cells populations, including keratinocytes and immune cells. Profiling of the inflammatory genes using HS skin from these ex vivo cultures showed dynamic expression changes at day-3 and day-14. All of these cultures are necessary to represent the inflammatory gene status of HS skin at day-0 suggesting that not all gene clusters are identically altered in each culture method. Similarly, cytokine/chemokine profiling of the supernatant from vehicle- and drug-treated ex vivo HS cultures again showed better prediction of drug efficacy against HS. Overall, development of these three systems collectively provide a powerful tool to uncover the pathobiology of HS progression and screen various drugs against HS.

category: pathology


title: Speckle illumination microscopy enables slide-free and non-destructive pathology of human lung adenocarcinoma

abstract: Histopathology based on formalin-fixed and paraffin-embedded tissues remains the gold standard for surgical margin assessment (SMA). However, routine pathological practice is lengthy and laborious, failing to provide immediate feedback to surgeons and pathologists for intraoperative decision-making. In this report, we propose a cost-effective and easy-to-use histological imaging method with speckle illumination microscopy (i.e., HiLo). HiLo can achieve rapid and non-destructive imaging of large and fluorescently-labelled resection tissues at an acquisition speed of 5 cm2/min with 1.3-m lateral resolution and 5.8-m axial resolution, demonstrating a great potential as an intraoperative SMA tool that can be used by surgeons and pathologists to detect residual tumors at surgical margins. It is experimentally validated that HiLo enables rapid diagnosis of different subtypes of human lung adenocarcinoma and hepatocellular carcinoma, producing images with remarkably recognizable cellular features comparable to the gold-standard histology. This work will facilitate the clinical translations of HiLo microscopy to improve the current standard-of-care.


title: Dynamic metabolic network modeling of a mammalian cell cycle using time-course multi-omics data

abstract: Cell cycle is a fundamental process for cell growth and proliferation, and its dysregulation leads to many diseases. How metabolic networks are regulated and rewired during the cell cycle is unknown. Here we apply a dynamic genome-scale metabolic modeling framework (DFA) to simulate a cell cycle of cytokine-activated murine pro-B cells. Phase-specific reaction activity predicted by DFA using time-course metabolomics were validated using matched time-course proteomics and phospho-proteomics data. Our model correctly predicted changes in methionine metabolism at the G1/S transition and the activation of lysine metabolism, nucleotides synthesis, fatty acid elongation and heme biosynthesis at the critical G0/G1 transition into cell growth and proliferation. Metabolic fluxes predicted from proteomics and phosphoproteomics constrained metabolic models were highly consistent with DFA fluxes and revealed that most reaction fluxes are regulated indirectly. Our model can help predict the impact of changes in nutrients, enzymes, or regulators on this critical cellular process.


title: Individual alpha power predicts language comprehension

abstract:
Alpha power attenuation during cognitive task performing has been suggested to reflect a process of release of inhibition, increase of excitability, and thereby benefit the improvement of performance. Here, we hypothesized that changes in individual alpha power during the execution of a complex language comprehension task may correlate with the individual performance in that task. We tested this using magnetoencephalography (MEG) recorded during comprehension of German sentences of different syntactic complexity.

Results showed that neither the frequency nor the power of the spontaneous oscillatory activity at rest were associated with the individual performance. However, during the execution of a sentences processing task, the individual alpha power attenuation did correlate with individual language comprehension performance. Source reconstruction localized effects in temporal-parietal regions of both hemispheres. While the effect of increased task difficulty is localized in the right hemisphere, the difference in power attenuation between tasks of different complexity exhibiting a correlation with performance was localized in left temporal-parietal brain regions known to be associated with language processing.

From our results, we conclude that in-task attenuation of individual alpha power is related to the essential mechanisms of the underlying cognitive processes, rather than merely to general phenomena like attention or vigilance.

HighlightsO_LIComprehension of structurally complexed embedded sentences is correlated with individual alpha power attenuation during task but not with alpha power at rest.
C_LIO_LIThese effects were localized in temporal-parietal brain regions known to be associated with language processing.
C_LI

Data availability statementAnonymized raw data will be made available upon request via email to the corresponding author provided the requesting researchers sign a formal data sharing agreement and cite this paper as origin of the data.


title: Predicting Individual Traits from Unperformed Tasks

abstract: Relating individual differences in cognitive traits to brain functional organization is a long-lasting challenge for the neuroscience community. Individual intelligence scores were previously predicted from whole-brain connectivity patterns, extracted from functional magnetic resonance imaging (fMRI) data acquired at rest. Recently, it was shown that task-induced brain activation maps outperform these resting-state connectivity patterns in predicting individual intelligence, suggesting that a cognitively demanding environment improves prediction of cognitive abilities. Here, we use data from the Human Connectome Project to predict task-induced brain activation maps from resting-state fMRI, and proceed to use these predicted activity maps to further predict individual differences in a variety of traits. While models based on original task activation maps remain the most accurate, models based on predicted maps significantly outperformed those based on the resting-state connectome. Thus, we provide a promising approach for the evaluation of measures of human behavior from brain activation maps, that could be used without having participants actually perform the tasks.


title: Predictive regulation of cardiovascular system on emergence of auditory-motor interaction in young infants

abstract: Humans develop auditory-motor interaction to produce a variety of rhythmic sounds using body movements, which are often produced and amplified with tools, such as drumming. The extended production of sounds allows us to express a wide range of emotions, accompanied by physiological changes. According to previous studies, even young infants enhance movements in response to auditory feedback. However, their exhibition of physiological adaptation on emergence of auditory-motor interaction is unclear. We investigated the movement and cardiac changes associated with auditory feedback to spontaneous limb movements in 3-month-old infants. The results showed that infants increased the frequency of limb movements inducing auditory feedback, while they exhibited the more regular rhythm of the limb movements. Furthermore, heart rate increase associated with the limb movement was first inhibited immediately after the timing of the auditory feedback, which may reflect sustained attention to the auditory stimuli. Then, through auditory-motor experience, the heart rate increase was inhibited even prior to the auditory feedback, leading to suppression on the peak intensity of the heart rate increase. These findings suggest that young infants regulate the cardiovascular system as well as limb movements in anticipation of the auditory feedback. The anticipatory regulation associated with movement and attentional changes may contribute to reduced cardiovascular stress in auditory-motor interaction, and provide a developmental basis for more sophisticated goal-directed behavior of producing rhythmic sounds.


title: Anticipatory regulation of cardiovascular system on emergence of auditory-motor interaction in young infants


title: Neural dynamics of illusory tactile pulling sensations

abstract:
The sensation of directional forces and their associated sensorimotor commands are inextricably intertwined, complicating the identification of brain circuits responsible for tactile pulling sensations. One hypothesis is that, like tactile frequency discrimination, pulling sensations are generated by early sensory-frontal activity. Alternatively, they may be generated later in the somatosensory association cortex. To dissociate these accounts and uncouple the pulling sensation from unrelated but correlated sensory and motor processing, we combined high-density EEG with an oddball paradigm and asymmetric vibration, which creates an illusory sensation of the hand being directionally pulled. Oddballs that created a pulling sensation in the opposite direction to common stimuli were compared to the same oddballs in the context of neutral common stimuli (symmetric vibration) and to neutral oddballs. Brain responses to having directional pulling expectations violated by directional stimuli were therefore isolated. Contrary to the sensory-frontal account, frontal N140 brain activity was actually larger for neutral than pulling oddballs. Instead, pulling sensations were associated with amplitude and latency modulations of midline P200 and P3b potentials, and specifically, to contralateral parietal lobe activity 280ms post-stimulus. The timing of this activity suggested pulling sensations involve spatial processing, such as tactile remapping between coordinate frames. Source localization showed this activity to be centered on the postcentral sulcus, superior parietal lobule and intraparietal sulcus, suggesting that pulling sensations arise via the processing of body position, tactile orientation and peripersonal space. Our results demonstrate how tactile illusions can uniquely disambiguate parietal contributions to somatosensation by removing unrelated sensory processing.

Significance statementThe neural mechanisms of tactile pulling sensations are poorly understood. Competing early sensory-frontal and later somatosensory association cortex accounts are hard to dissociate due to confounding sensory and motor signals present when forces are applied to the skin. Here, we used EEG and a novel asymmetric vibration approach to induce an illusory pulling sensation, which circumvents these issues. We found that pulling sensations were associated with parietal lobe activity 280ms post-stimulus and modulations of the P200. The timing and location of this activity suggested that pulling sensations necessitate spatial processing and supported a somatosensory association cortex account of the pulling sensation.


title: Intermanual transfer of visuomotor learning is facilitated by a cognitive strategy

abstract:
Humans continuously adapt their movement to a novel environment by recalibrating their sensorimotor system. Recent evidence, however, shows that explicit planning to compensate for external changes, i.e. a cognitive strategy, can also aid performance. If such a strategy is indeed planned in external space, it should improve performance in an effector independent manner. We tested this hypothesis by examining whether promoting a cognitive strategy during a visual-force adaptation task performed in one hand can facilitate learning for the opposite hand. Participants rapidly adjusted the height of visual bar on screen to a target level by isometrically exerting force on a handle using their right hand. Visuomotor gain increased during the task and participants learned the increased gain. Visual feedback was continuously provided for one group, while for another group only the endpoint of the force trajectory was presented. The latter has been reported to promote cognitive strategy use. We found that endpoint feedback produced stronger intermanual transfer of learning and slower response times than continuous feedback. In a separate experiment, we confirmed that the aftereffect is indeed reduced when only endpoint feedback is provided, a finding that has been consistently observed when cognitive strategies are used. The results suggest that intermanual transfer can be facilitated by a cognitive strategy. This indicates that the behavioral observation of intermanual transfer can be achieved either by forming an effector-independent motor representation, or by sharing an effector-independent cognitive strategy between the hands.

New and noteworthyThe causes and consequences of cognitive strategy use for motor learning are poorly understood. We tested whether a visuomotor task learned using a strategy generalizes across effectors. Visual feedback was manipulated to enhance the use of a cognitive strategy. Learning using a cognitive strategy for one hand transferred to the task performed by the un-learned hand. Our result suggests that intermanual transfer can also result from a common cognitive strategy used to control both hands.


title: Multi-parameter optical imaging of immune cell activity in chimeric antigen receptor T-cell and checkpoint blockade therapies

abstract: Longitudinal multimodal imaging presents unique opportunities for noninvasive surveillance and prediction of treatment response to cancer immunotherapy. In this work we first designed a novel granzyme B activated self-assembly small molecule, G-SNAT, for quantitative assessment of cytotoxic T lymphocyte mediated cancer cell killing in vivo. In lymphoma tumor bearing mice, the retention of cyanine 5 labeled G-SNAT-Cy5 was shown to be highly correlated to CAR T-cell mediated granzyme B release and tumor eradication. In colorectal tumor-bearing transgenic mice, expressing firefly luciferase in hematopoietic cells, and which received combination treatment of anti-PD-1 and anti-CTLA-4, longitudinal bioluminescence and fluorescence imaging revealed the dynamics of immune cell expansion, trafficking, tumor infiltration, and cytotoxic activity which predicted therapeutic outcome before tumor shrinkage was evident. These results support further development of G-SNAT for imaging early immune response to checkpoint blockade and CAR T-cell therapy in patients and highlight the utility of multimodality imaging for improved mechanistic insights into cancer immunotherapy.

category: immunology


title: Membrane permeability differentiation at the lipid divide

abstract: One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and is assumed to bestow different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes are more permeable, yet systematic analysis based on direct measurements is absent. Here we develop a new approach for assessing the membrane permeability of cell-sized unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of twenty metabolites demonstrates that archaeal-type membranes are permeable to a range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Surprisingly, permeability is much lower in bacterial-type membranes, in contradiction to current orthodoxy. We then show that archaeal permeability traits are specifically linked to both the methyl branches present on the archaeal phospholipid tails and the ether link between the tails and the head group. To explore this result further, we compare the abundance of transporter-encoding families present on genomes sampled from across the prokaryotic tree of life. Interestingly, archaea have a reduced repertoire of transporter gene families, consistent with an increased dependency on membrane permeation for a subset of metabolites. Taken together, these results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell and protocell evolution.

category: evolutionary biology


title: Chromosome-level assembly of the common vetch reference genome (Vicia sativa)

abstract:
BackgroundVicia sativa L. (Common Vetch, n = 6) is an annual, herbaceous, climbing legume that is distributed in tropical, sub-tropical and temperate climates. Originating in the Fertile Crescent of the Middle East, V. sativa is now widespread and grows in the Mediterranean basin, West, Central and Eastern Asia, North and South America. V. sativa is of economic importance as a forage legume in countries such as Australia, China, and the USA and contributes valuable nitrogen to agricultural rotation cropping systems. To accelerate precision genome breeding and genomics-based selection of this legume, we here present a chromosome-level reference genome sequence for V. sativa.

ResultsWe applied a combination of long-read Oxford Nanopore sequencing, short-read Illumina sequencing, and high-throughput chromosome conformation data (CHiCAGO and Hi-C) analysis to construct a chromosome-level genome of V. sativa. The chromosome-level assembly of six pseudo-chromosomes has a total genome length of 1.9 gigabases (Gb) with a median contig length of 684 kb. Benchmarking Universal Single-Copy Orthologs (BUSCO) of the assembly demonstrated a very high completeness of 98 % of the dicotyledonous orthologs. RNA-seq analysis and gene modelling enabled the annotation of 58,415 protein-coding genes.

ConclusionsThe high-quality chromosome-level genome assembly of V. sativa will provide novel insights into vetch genome evolution and be a valuable resource for genomic breeding, genetic diversity and for understanding adaption to diverse arid environments.

category: genomics


title: Social and asocial learning in zebrafish are encoded by a shared brain network that is differentially modulated by local activation

abstract: Group living animals can use social and asocial cues to predict the presence of a reward or a punishment in the environment through associative learning. The degree to which social and asocial learning share the same mechanisms is still a matter of debate, and, so far, studies investigating the neuronal basis of these two types of learning are scarce and have been restricted to primates, including humans, and rodents. Here we have used a Pavlovian fear conditioning paradigm in which a social (fish image) or an asocial (circle image) conditioned stimulus (CS) have been paired with an unconditioned stimulus (US=food), and we have used the expression of the immediate early gene c-fos to map the neural circuits associated with social and asocial learning. Our results show that the learning performance is similar with social (fish image) and asocial (circle image) CSs. However, the brain regions involved in each learning type are distinct. Social learning is associated with an increased expression of c-fos in olfactory bulbs, ventral zone of ventral telencephalic area, ventral habenula and ventromedial thalamus, whereas asocial learning is associated with a decreased expression of c-fos in dorsal habenula and anterior tubercular nucleus. Using egonetworks, we further show that each learning type has an associated pattern of functional connectivity across brain regions. Moreover, a community analysis of the network data reveals four segregated functional submodules, which seem to be associated with different cognitive functions involved in the learning tasks: a generalized attention module, a visual response module, a social stimulus integration module and a learning module. Together, these results suggest that, although there are localized differences in brain activity between social and asocial learning, the two learning types share a common learning module and social learning also recruits a specific social stimulus integration module. Therefore, our results support the occurrence of a common general-purpose learning module, that is differentially modulated by localized activation in social and asocial learning.

category: animal behavior and cognition


title: Functional single cell selection and annotated profiling of dynamically changing cancer cells

abstract: A method connecting single cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, would be transformative for single cell and cancer biology. Here, we present fSCS: functional single cell selection. This technology combines a custom-built ultrawide field-of-view optical screening microscope, fast automated image analysis and a new photolabeling method, phototagging, using a newly synthesized visible-light-photoactivatable dye. Using fSCS, we screen, selectively photolabel and isolate cells of interest from large heterogeneous populations based on functional dynamics like fast migration, morphological variation, small molecule uptake or cell division. We combined fSCS with single cell RNA sequencing for functionally annotated transcriptomic profiling of fast migrating and spindle-shaped MCF10A cells with or without TGF{beta} induction. We identified critical genes and pathways driving aggressive migration as well as mesenchymal-like morphology that could not be detected with state-of-the-art single cell transcriptomic analysis. fSCS provides a crucial upstream selection paradigm for single cell sequencing independent of biomarkers, allows enrichment of rare cells and can facilitate the identification and understanding of molecular mechanisms underlying functional phenotypes.

category: bioengineering


title: Ubiquitin Proteasome Pathway annotation in Diaphorina citri can reveal potential targets for RNAi based pest management

abstract: Ubiquitination is an ATP-dependent process that targets proteins for degradation by the proteasome. In this study, we annotated 15 genes from the ubiquitin-proteasome pathway in the Asian citrus psyllid, Diaphorina citri. This psyllid vector has come to prominence in the last decade due to its role in the transmission of the devastating bacterial pathogen, Candidatus Liberibacter asiaticus (CLas). Infection of citrus crops by this pathogen causes Huanglongbing (HLB or citrus greening disease) and results in the eventual death of citrus trees. The identification and correct annotation of these genes in D. citri will be useful for functional genomic studies that aid in the development of RNAi-based management strategies aimed at reducing the spread of HLB. Investigating the effects of CLas infection on the expression of ubiquitin-proteasome pathway genes may provide new information regarding the role that these genes play in the acquisition and transmission of CLas by D. citri.


title: Mapping tumor spheroid mechanics in dependence of 3D microenvironment stiffness and degradability by Brillouin microscopy

abstract: Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression.


title: Fast adaptation of cooperative channels engendersHopf bifurcations in auditory hair cells

abstract:
Since the pioneering work of Thomas Gold published in 1948, it has been known that we owe our sensitive sense of hearing to a process in the inner ear that can amplify incident sounds on a cycle-by-cycle basis. Termed the active process, it uses energy to counteract the viscous dissipation associated with sound-evoked vibrations of the ears mechanotransduction apparatus. Despite its importance, the mechanism of the active process and the proximate source of energy that powers it have remained elusive--especially at the high frequencies characteristic of mammalian hearing. This is partly due to our insufficient understanding of the mechanotransduction process in hair cells, the sensory receptors and amplifiers of the inner ear. It has previously been proposed that a cyclical binding of Ca2+ ions to individual mechanotransduction channels could power the active process. That model, however, relied on tailored reaction rates that structurally forced the direction of the cycle. Here, we ground our study on our previous model of hair-cell mechanotransduction, which relied on the cooperative gating of pairs of channels, and incorporate into it the cyclical binding of Ca2+ ions. With a single binding site per channel and reaction rates drawn from thermodynamic principles, our model shows that hair cells behave as nonlinear oscillators that exhibit Hopf bifurcations, dynamical instabilities long understood to be signatures of the active process. Using realistic parameter values, we find bifurcations at frequencies in the kilohertz range with physiological Ca2+ concentrations. In contrast to the myosin-based mechanism, responsible for low-frequency relaxation oscillations in the vestibular hair cells of amphibians, the current model relies on the electrochemical gradient of Ca2+ as the only energy source for the active process and on the relative motion of cooperative channels within the stereociliary membrane as the single mechanical driver. Equipped with these two mechanisms, a hair bundle proves capable of operating at frequencies in the kilohertz range, characteristic of mammalian hearing.

SIGNIFICANCEHow the inner ear amplifies incident sounds at audible frequencies of several kilohertz is a key question that has remained unanswered despite decades of research into several candidate mechanisms. Here, we model the behavior of hair cells, the sensory receptors of the inner ear, and show that they can undergo oscillatory instabilities called Hopf bifurcations due to the effect of Ca2+ on the cooperative opening and closing of mechanotransduction ion channels. Close to the bifurcation point, a hair cell behaves as a nonlinear oscillator that can amplify its input on a cycle-by-cycle basis. We find that our proposed mechanism can operate in the kilohertz range.


title: Early evolution of beetles regulated by the end-Permian deforestation

abstract: The end-Permian mass extinction (EPME) led to a severe terrestrial ecosystem collapse. However, the ecological response of insects--the most diverse group of organisms on Earth--to the EPME remains poorly understood. Here, we analyse beetle evolutionary history based on taxonomic diversity, morphological disparity, phylogeny, and ecological shifts from the Early Permian to Middle Triassic, using a comprehensive new data set. Permian beetles were dominated by xylophagous stem groups with a high diversity and disparity, which probably played an underappreciated role in the Permian carbon cycle. Our suite of analyses shows that Permian xylophagous beetles suffered a severe extinction during the EPME largely due to the collapse of forest ecosystems, resulting in an Early Triassic gap of xylophagous beetles. New xylophagous beetles appeared widely in the early Middle Triassic, which is consistent with the restoration of forest ecosystems. Our results highlight the ecological significance of insects in deep-time terrestrial ecosystems.

category: paleontology


title: AMPK mediates early activation of the unfolded protein response through a positive feedback loop in palmitate-treated muscle cells

abstract: Activation of the unfolded protein response (UPR) is closely associated with the pathogenesis of many metabolic diseases including obesity and type 2 diabetes. There is increasing evidence for the interdependence of the UPR and metabolic signaling pathways. The AMP-activated protein kinase (AMPK) signaling pathway controls energy balance in eukaryotes. The aim of this study was to investigate the possible interaction between AMPK signaling and the UPR in muscle cells exposed to a saturated fatty acid, as well as the underlying mechanism. The UPR was induced in C2C12 myotubes by treatment with palmitate along with activation of AMPK signaling. Inhibiting the AMPK pathway with compound C attenuated palmitate-induced UPR activation, while inhibiting the UPR with taurourdodeoxycholic acid alleviated palmitate-induced AMPK activation, suggesting a positive feedback loop between the UPR and AMPK. Additionally, 5-amino-1-{beta}-D-ribofuranosylimidazole-4-carboxamide, an AMPK agonist, caused a dose- and time-dependent upregulation of genes related to the UPR, including activating transcription factor (ATF)4, binding immunoglobulin protein (BIP), and growth arrest and DNA damage-inducible protein (GADD)34. These results provide the first evidence for the involvement of AMPK signaling in the early activation of the UPR induced by saturated fatty acid in skeletal muscle, and suggest that physiologic or pharmacologic activation of the AMPK pathway (ie, by exercise or metformin, respectively) can promote skeletal muscle health and function and thus improve quality of life for individuals with metabolic disorder due to a high-fat diet or obesity.

category: physiology


title: Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons

abstract:
In neurons, endoplasmic reticulum forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance. However, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high resolution microscopy and live cell imaging to investigate rapid movements of endoplasmic reticulum and ribosomes in axons of cultured motoneurons after stimulation with Brain-derived neurotrophic factor. Our results indicate that the endoplasmic reticulum extends into axonal growth cone filopodia where its integrity and dynamic remodeling are regulated mainly by actin and its motor protein myosin VI. Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with ER in response to extracellular stimuli which describes a novel function of axonal ER in dynamic regulation of local translation.

Summary statementIn axonal growth cones of motoneurons, dynamic remodeling of ER is coordinated through drebrin A-mediated actin and microtubule crosstalk and contributes to local translation as response to BDNF stimulation.

category: neuroscience


title: Brain endothelial antigen presentation detains CD8+ T cells at the blood-brain barrier leading to its breakdown

abstract: Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of CD8+ T cells are found in MS lesions and antigen (Ag)-presentation at the BBB was proposed to promote CD8+ T-cell entry into the CNS. Employing live cell imaging and primary mouse brain microvascular endothelial cells (pMBMECs) as in vitro model of the BBB and a mouse model of CNS autoimmunity, we here show that pMBMECs process and present antigens leading to effector CD8+ T-cell differentiation. Under physiological flow, endothelial Ag-presentation prohibited CD8+ T-cell crawling and diapedesis leading to pMBMEC apoptosis. Reduced motility of Ag-specific CD8+ T cells was also observed in CNS microvessels in neuroinflammation in vivo. Luminal MHC class I Ag-presentation at the BBB thus prohibits CD8+ T-cell entry into the CNS and rather triggers CD8+ T cell mediated focal BBB breakdown.


title: A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy

abstract: Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the "class 3" epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.

category: microbiology


title: An arsenic stress signaling code in yeast

abstract:
Arsenic is an environmental toxin that exists mainly as pentavalent arsenate and trivalent arsenite. Both forms activate the yeast SAPK Hog1, but with different consequences. We describe a mechanism by which cells distinguish between these arsenicals through one-step metabolism to differentially regulate the bidirectional glycerol channel Fps1, an adventitious port for arsenite. Cells exposed to arsenate reduce it to thiol-reactive arsenite, which modifies a set of cysteine residues in target proteins; whereas cells exposed to arsenite metabolize it to methylarsenite, which modifies an additional set of cysteine residues. Hog1 becomes arsenylated, which prevents it from closing Fps1. However, this block is overcome in cells exposed to arsenite through methylarsenylation of Acr3, an arsenite efflux pump that we found also regulates Fps1 directly. This adaptation allows cells to restrict arsenite entry through Fps1, but also allows its exit when produced from arsenate exposure. These results have broad implications for understanding how SAPKs activated by diverse stressors can drive stress-specific outputs.

Summary (for JCB table of contents)

Jongmin Lee, David E. Levin

Lee and Levin investigate the question of how different stressors can drive divergent outputs from an active SAPK. The work describes the mechanism by which two forms of arsenic that both activate the yeast SAPK Hog1 differentially regulate the glycerol channel Fps1.

category: molecular biology


title: Differential metabolism of arsenicals regulates Fps1-mediated arsenite transport


title: Frugivore gut passage increases seed germination: an updated meta-analysis

abstract: Many plants rely on animal mutualists for reproduction. Quantifying how animal mutualists impact plant performance provides a foundation for modelling how change in animal communities affects the composition and functioning of plant communities. We performed a meta-analysis of 2539 experiments, 6 times more than the last comprehensive meta-analysis, examining how gut passage by frugivores influences seed germination. We simultaneously analyzed multiple predictor variables related to study methodology, location, and frugivore identity to disentangle methodological from ecological impacts on effect sizes. We found that gut passage by birds, fish, reptiles, bats, primates, and other mammals on average increased seed germination, but that the magnitude varied across vertebrate groups. The positive effects of gut passage were largely explained by the de-inhibitory effects of pulp removal rather than by the scarification of seed tissues. Some previous studies and meta-analyses that found no effect of gut passage only tested scarification or did not distinguish between these tests of scarification and pulp removal. We found that, for a typical fleshy-fruited plant species, the lack of gut passage reduces germination by 60%. From an evolutionary perspective, this indicates a large risk associated with reliance on animal mutualists that is balanced against the benefits of animal-mediated seed dispersal. From a conservation perspective, this highlights the potential for large demographic consequences of frugivore declines on plant populations. Our database and findings advance quantitative predictions for the role of fruit-frugivore interactions in shaping plant communities in the Anthropocene.

category: ecology


title: Codiversification of gut microbiota with humans

abstract:
Some gut microbes have cospeciated with hominids, but whether they further codiversified with human populations is unclear. Here, we identify predominant gut microbial species sharing a parallel evolutionary history with human populations. Patterns of strain transfer between populations are generally consistent with an African origin, and suggest long-term vertical transmission over thousands of generations. We show the same strains also faithfully transmit between mothers and their children. Consistent with the development of intimate symbiosis, species with strongest patterns of codiversification have the smallest genomes. This study reveals long-term fidelity of gut microbiota with human populations through transmission among individuals living in close proximity. Dominance of specific strains in different populations is based in part on vertical transmission and they may provide population-specific health benefits.

One-sentence summaryIdentification of gut microbes that codiversified with human populations.


title: Modeling homing suppression gene drive in haplodiploid organisms

abstract: Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids and suffer more from functional resistance alleles. They are substantially more vulnerable to high resistance allele formation in the embryo due to maternally deposited Cas9 and gRNA and also to somatic cleavage activity. Examining spatial models where organisms move over a continuous landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly due to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations.


title: Modelling homing suppression gene drive in haplodiploid organisms


title: A dual endosymbiosis drives nutritional adaptation to hematophagy in the invasive tick Hyalomma marginatum

abstract: Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions: Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.


title: AUG-3387, a Human-Derived Monoclonal Antibody Neutralizes SARS-CoV-2 Variants and Reduces Viral Load from Therapeutic Treatment of Hamsters In Vivo

abstract: Infections from the SARS-CoV-2 virus have killed over 4.6 million people since it began spreading through human populations in late 2019. In order to develop a therapeutic or prophylactic antibody to help mitigate the effects of the pandemic, a human monoclonal antibody (mAb) that binds to the SARS-CoV-2 spike protein was isolated from a convalescent patient following recovery from COVID-19 disease. This mAb, designated AUG-3387, demonstrates a high affinity for the spike protein of the original viral strains and all variants tested to date. In vitro pseudovirus neutralization and SARS-CoV-2 neutralization activity has been demonstrated in vitro. In addition, a dry powder formulation has been prepared using a Thin-Film Freezing (TFF) process that exhibited a fine particle fraction (FPF) of 50.95 {+/-} 7.69% and a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 3.74 {+/-} 0.73 {micro}m and 2.73 {+/-} 0.20, respectively. The dry powder is suitable for delivery directly to the lungs of infected patients using a dry powder inhaler device. Importantly, AUG-3387, administered as a liquid by intraperitoneal injection or the dry powder formulation delivered intratracheally into Syrian hamsters 24 hours after intranasal SARS-CoV-2 infection, demonstrated a dose-dependent reduction in the lung viral load of the virus. These data suggest that AUG-3387 formulated as a dry powder demonstrates potential to treat COVID-19.

category: pharmacology and toxicology


title: Low antigen abundance limits efficient T-cell recognition of highly conserved regions of SARS-CoV-2

abstract: Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8+ T-cell epitopes across the nucleocapsid (N), spike (S) and ORF3a proteins of SARS-CoV-2 and five CD8+ T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8+ T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad pre-existing immunity in the community.


title: SARS-CoV-2 spike protein induces abnormal inflammatory blood clots neutralized by fibrin immunotherapy

abstract:
Blood clots are a central feature of coronavirus disease-2019 (COVID-19) and can culminate in pulmonary embolism, stroke, and sudden death. However, it is not known how abnormal blood clots form in COVID-19 or why they occur even in asymptomatic and convalescent patients. Here we report that the Spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to the blood coagulation factor fibrinogen and induces structurally abnormal blood clots with heightened proinflammatory activity. SARS-CoV-2 Spike virions enhanced fibrin-mediated microglia activation and induced fibrinogen-dependent lung pathology. COVID-19 patients had fibrin autoantibodies that persisted long after acute infection. Monoclonal antibody 5B8, targeting the cryptic inflammatory fibrin epitope, inhibited thromboinflammation. Our results reveal a procoagulant role for the SARS-CoV-2 Spike and propose fibrin-targeting interventions as a treatment for thromboinflammation in COVID-19.

One-Sentence SummarySARS-CoV-2 spike induces structurally abnormal blood clots and thromboinflammation neutralized by a fibrin-targeting antibody.


title: Rates of primary production in groundwater rival those in oligotrophic marine systems

abstract: The terrestrial subsurface contains nearly all of Earths freshwater reserves1 and harbors upwards of 60% of our planets total prokaryotic biomass2,3. While genetic surveys suggest these organisms rely on in situ carbon fixation, rather than the translocation of photosynthetically derived organic carbon4-6, corroborating measurements of carbon fixation in the subsurface are absent. Using a novel ultra-low level 14C-labeling technique, we show that in situ carbon fixation rates in a carbonate aquifer reached 10% of the median rates measured in oligotrophic marine surface waters, and were up to six-fold greater than those observed in lower euphotic zone waters where deep chlorophyll levels peak. Empirical carbon fixation rates were substantiated by both nitrification and anammox rate data. Metagenomic analyses revealed a remarkable abundance of putative chemolithoautotrophic members of an uncharacterized order of Nitrospiria - the first representatives of this class expected to fix carbon via the Wood-Ljungdahl pathway. Based on these fixation rates, we extrapolate global primary production in carbonate groundwaters to be 0.11 Pg of carbon per year.


title: Vht hydrogenase is required for hydrogen cycling during nitrogen fixation by the non-hydrogenotrophic methanogen Methanosarcina acetivorans.

abstract:
Methanosarcina acetivorans is the primary model to understand the physiology of methanogens that do not use hydrogenase to consume or produce hydrogen (H2) during methanogenesis. The genome of M. acetivorans encodes putative methanophenazine-reducing hydrogenases (Vht and Vhx), F420-reducing hydrogenase (Frh), and hydrogenase maturation machinery (Hyp), yet cells lack significant hydrogenase activity under all growth conditions tested to date. Thus, the importance of hydrogenase to the physiology of M. acetivorans has remained a mystery. M. acetivorans can fix dinitrogen (N2) using nitrogenase that is documented in bacteria to produce H2 during the reduction of N2 to ammonia. Therefore, we hypothesized that M. acetivorans uses hydrogenase to recycle H2 produced by nitrogenase during N2 fixation. Results demonstrate that hydrogenase expression and activity is higher in N2-grown cells compared to cells grown with fixed nitrogen (NH4Cl). To test the importance of each hydrogenase and the maturation machinery, the CRISPRi-dCas9 system was used to generate separate M. acetivorans strains where transcription of the vht, frh, vhx, or hyp operons is repressed. Repression of vhx and frh does not alter growth with either NH4Cl or N2 and has no effect on H2 metabolism. However, repression of vht or hyp results in impaired growth with N2 but not NH4Cl. Importantly, H2 produced endogenously by nitrogenase is detected in the headspace of culture tubes containing the vht or hyp repression strains. Overall, the results reveal that Vht hydrogenase recycles H2 produced by nitrogenase that is required for optimal growth of M. acetivorans during N2 fixation.

IMPORTANCEThe metabolism of M. acetivorans and closely related Methanosarcinales is thought to not involve H2. Here we show for the first time M. acetivorans is capable of H2 cycling like hydrogenotrophic Methanosarcinales (e.g., Methanosarcina barkeri). However, unlike M. barkeri hydrogenase activity and H2 cycling is tightly regulated in M. acetivorans and is only utilized during N2 fixation to consume H2 production endogenously by nitrogenase. The in vivo production of H2 by nitrogenase during N2 reduction is also demonstrated for the first time in a methanogen. Overall, the results provide new insight into the evolution and diversity of methanogen metabolism and new details about methanogen nitrogenase that could be leveraged for practical applications, such as nitrogenase-dependent production of H2 as a biofuel.


title: Development of a metatranscriptomic analysis method for multiple intestinal sites and its application to the common marmoset intestine

abstract:
BackgroundThe intestinal microbiome is closely related to host health, and metatranscriptomic analysis can assess the functional activity of microbiomes by quantifying the bacterial gene expression level, which helps to elucidate the interaction between the microbiome and the environment. However, functional changes in the microbiome along the host intestinal tract remain unknown, and previous analytical methods have limitations, such as potentially overlooking unknown genes due to dependence on existing databases and being unable to take full advantage of metatranscriptome to reveal the functional change among multiple environments.

ResultTo close these gaps, we develop a novel method that integrates metagenome and metatranscriptome to analyze the functional activity of microbiomes between intestinal sites. This method reconstructs a reference metagenomic sequence across multiple intestinal sites, allowing the gene expression levels of microbiome including unknown bacterial genes to be compared between multiple sites. As a result of applying this method to metatranscriptomic analysis in the intestinal tract of common marmoset, the reconstructed metagenome covered most of the expressed genes and it revealed that the changes in bacterial gene expressions among the caecum, transverse colon, and faeces were more dynamic and sensitive to environmental shifts than its abundance. In typical, the coenzyme synthesis gene and antibacterial resistance gene were more highly expressed in the caecum and transverse colon than in faeces, while there was no significant change in abundance of these genes.

ConclusionOur findings demonstrate that an analytical method that integrates metagenome and metatranscriptome in multiple intestinal sites captures functional changes in the microbiomes at the gene resolution level.


title: Transkingdom Analysis of the Female Reproductive Tract Reveals Bacteriophages Form Communities

abstract: The female reproductive tract (FRT) microbiome plays an important role in maintaining vaginal health. Viruses play a key role in regulating other microbial ecosystems, but little is known about how the FRT viruses (virome), particularly bacteriophages, impacts FRT health and dysbiosis. We hypothesize that bacterial vaginosis is associated with alterations in the FRT virome, and these changes correlate with bacteriome shifts. We conducted a retrospective, longitudinal analysis of vaginal swabs collected from 54 bacterial vaginosis (BV)-positive and 46 BV-negative South African women. Bacteriome analysis revealed samples clustered into five distinct bacterial community groups (CG). Bacterial alpha diversity was significantly associated with BV. Virome analysis on a subset of baseline samples showed FRT bacteriophages clustering into novel viral state types (VSTs), a viral community clustering system based on virome composition and abundance. Distinct BV bacteriophage signatures included increased alpha diversity along with Bacillus, Burkholderia and Escherichia bacteriophages. Discriminate bacteriophage-bacteria transkingdom associations were also identified between Bacillus and Burkholderia viruses and BV-associated bacteria, providing key insight for future studies elucidating transkingdom interactions driving BV-associated microbiome perturbations. In this cohort, bacteriophage-bacterial associations suggest complex interactions which may play a role in the establishment and maintenance of BV.


title: The cnf1 gene is associated to an expanding Escherichia coli ST131 H30Rx/C2 sublineage and confers a competitive advantage for host colonization

abstract: Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract acting as a source of urinary tract pathogens. We performed a high-throughput genetic screening of predominantly clinical E. coli isolates from wide geographical origins. This revealed a preferential distribution of the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, in four sequence types encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment and has known enhanced capacities to colonize the gastrointestinal tract (GIT). Statistical modeling uncovered a dominant global expansion of cnf1-positive strains within multidrug-resistant ST131 subclade H30Rx/C2. Despite the absence of phylogeographical signals, cnf1-positive isolates adopted a clonal distribution into clusters on the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. Functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2, established that a cnf1-deleted EC131GY is outcompeted by the wildtype strain in a mouse model of competitive infection of the bladder while both strains behave similarly during monoinfections. This points for positive selection of cnf1 during UTI rather than urovirulence. Wildtype EC131GY also outcompeted the mutant when concurrently inoculated into the gastrointestinal tract, arguing for selection within the gut. Whatever the site of selection, these findings support that the benefit of cnf1 enhancing host colonization by ST131-H30Rx/C2 in turn drives a worldwide dissemination of the cnf1 gene together with extended spectrum of antibiotic resistance genes.