title: Multi-environment genome-wide association mapping of culm morphology traits in barley

abstract:
In cereals with hollow internodes, lodging resistance is influenced by morphological characteristics such as internode diameter and culm wall thickness. Despite their relevance, knowledge of the genetic control of these traits and their relationship with lodging is lacking in temperate cereals such as barley. To fill this gap, we developed an image-analysis based protocol to accurately phenotype culm diameter and culm wall thickness across 261 barley accessions. Analysis of culm trait data collected from field trials in 7 different environments revealed genetic control as supported by high heritability values, as well as genotype-by-environment interactions. The collection was structured mainly according to row-type, which had a confounding effect on culm traits as evidenced by phenotypic correlations. In addition, culm traits showed strong negative correlations with lodging but weak correlations with plant height across row-types, indicating the possibility of improving lodging resistance independent of plant height. Using 50k iSelect SNP genotyping data, we conducted multi-environment genome-wide association studies using mixed model approach across the whole panel and row-type subsets: we identified a total of 192 QTLs for the studied traits, including subpopulation-specific QTLs and several main effect loci for culm traits showing negative effects on lodging without impacting plant height. Providing first insights into the genetic architecture of culm morphology in barley and the possible role of candidate genes involved in hormone and cell wall related pathways, this work supports the potential of loci underpinning culm features to improve lodging resistance and increase barley yield stability under changing environments.

One-sentence summaryGenetic analysis of a diverse collection of European barleys reveals genomic regions underpinning stem morphological features associated with lodging resistance.


title: Active Anemosensing Hypothesis: How Flying Insects Could Estimate Ambient Wind Direction Through Sensory Integration & Active Movement

abstract: Estimating the direction of ambient fluid flow is a crucial step during chemical plume tracking for flying and swimming animals. How animals accomplish this remains an open area of investigation. Recent calcium imaging with tethered flying Drosophila has shown that flies encode the angular direction of multiple sensory modalities in their central complex: orientation, apparent wind (or airspeed) direction, and direction of motion. Here we describe a general framework for how these three sensory modalities can be integrated over time to provide a continuous estimate of ambient wind direction. After validating our framework using a flying drone, we use simulations to show that ambient wind direction can be most accurately estimated with trajectories characterized by frequent, large magnitude turns. Furthermore, sensory measurements and estimates of their derivatives must be integrated over a period of time that incorporates at least one of these turns. Finally, we discuss approaches that insects might use to simplify the required computations, and present a list of testable predictions. Together, our results suggest that ambient flow estimation may be an important driver underlying the zigzagging maneuvers characteristic of plume tracking animals trajectories.

category: animal behavior and cognition


title: Targeted Proteomics and Support Vector Classification Reveal Potential Biomarkers for the Early Detection of High-grade Serous Ovarian Cancer

abstract: The 5-year prognosis of late-stage epithelial ovarian cancer (EOC) remains poor, thus the discovery of early-stage EOC biomarkers is of paramount importance. Extracellular vesicles (EVs) circulating in blood are thought to contain proteomic cargo originating from an EOC microenvironment and are thus amenable for clinical biomarker discovery. We profiled the proteome of EVs purified from patient blood plasma, ascites and cell lines using strong cation exchange peptide fractionation and Orbitrap-based tandem mass spectrometry. To further increase sensitivity and specificity of the method, CD9-affinity purification and ultracentrifugation were used to purify EVs. Using parallel reaction monitoring we identified a compendum of 240 proteins that were differentially enrirched in EVs derived from EOC (n=10) patients versus women with non-cancerous gynecological conditions (n=9). Support vector machines were optimized using leave-one-out cross-validation and this methodology was implemented on a test set of malignant (n=4) and control (n=3) donors. Using the relative levels of >450 EV-associated peptides in a cohort of plasma-derived EVs, we identified several combinatorial peptides capable of discriminating high-grade serous EOC with up to 100% accuracy in Stage I, II, and III donors. This study demonstrates an adaptable biomarker discovery pipeline and provides pinoeering evidence of EV-associated biomarkers for the detection of early-stage EOC.

category: cancer biology


title: GPR183 mediates the capacity of the novel CD47-CD19 bispecific antibody TG-1801 to heighten ublituximab-umbralisib (U2) anti-lymphoma activity

abstract: Targeted therapies have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decade; however, most subtypes remain incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory B-NHL patients either as a single-agent or in combination with ublituximab, a CD20 antibody, which is also being combined with the PI3K{delta}/CK1e inhibitor, umbralisib ("U2"-regimen). In this study, we demonstrated that TG-1801 potentiates ublituximab-mediated antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP), leading to an additive anti-tumour effect of the TG-1801/U2 combination in B-NHL co-cultures. Accordingly, in a B-NHL xenotransplant model, the triplet achieved a 93% tumour growth inhibition, with 40% of the animals remaining tumour-free 35 days after the last dosing. Transcriptomic analysis further uncovered the upregulation of the G protein-coupled receptor, GPR183, as a crucial event associated with TG-1801/U2 synergism, while pharmacological blockade or genetic depletion of this factor impaired ADCP initiation, as well as cytoskeleton remodelling and cell migration, in B-NHL cultures exposed to the drug combination. Thus, our results set the preclinical rationale and support a combination strategy of TG-1801 with PI3K{delta}- and CD20-targeting agents in patients with B-NHL.


title: The mouse claustrum synaptically connects cortical network motifs

abstract: Spatially distant areas of cerebral cortex coordinate their activity into networks that are integral to cognitive processing. A common structural motif of cortical networks is co-activated frontal and posterior cortical regions. Knowledge of the neural circuit mechanisms underlying such widespread inter-areal cortical coordination is lacking. Using anesthetized mouse functional magnetic resonance imaging (fMRI) we discovered that mouse frontal cortical functional connectivity reflects the common cortical network motif in its functional connectivity to posterior cortices, but also demonstrates significant functional connectivity with the claustrum. Exploring whether the claustrum may synaptically support such network architecture, we used a channelrhodopsin-assisted electrophysiological circuit mapping approach to assess the strength of synaptic connectivity of 35 unique frontal cortico-claustral-cortical connections through 1,050 subtype-identified claustrum projection neurons. We observed significant trans-claustral synaptic connectivity from the anterior cingulate cortex and prelimbic prefrontal cortex back to originating frontal cortical regions as well as to posteriorly-lying visual and parietal association cortices contralaterally. The infralimbic prefrontal cortex possessed significant trans-claustral synaptic connectivity with the posteriorly-lying retrosplenial cortex, but to a far lesser degree with visual and parietal association cortices. These data reveal discrete extended cortical pathways through the claustrum that are positioned to support cortical network motifs central to cognitive control functions.

category: neuroscience


title: Follicular helper T cells expressing Blimp1 are specialized for plasma cell differentiation

abstract:
B cells differentiate into antibody-producing plasma cells (PC) and germinal center (GC) B cells under the guidance of specialized CD4+ follicular helper T (TFH) cells. Here, we demonstrate that CD4 T cells require Prdm1 expression for both early PC differentiation and post-GC PC formation. Using dual Blimp1/Foxp3 reporter mice and single cell-indexed analysis, we segregate persistent compartments and expressed transcriptional programs of Blimp1+ CXCR5+PD1hi TFH (referred to here as PC-TFH) from canonical Blimp1- Bcl6+ TFH (GC-TFH) and Blimp1+Foxp3+ TFR immune regulators. Antigen recall expands localized PC-TFH compartments with rapidly divergent antigen-specific memory PC-TFH and GC-TFH programs. Thus, Blimp1 is a central mediator of PC-TFH function producing specialized TFH subsets that co-ordinate with GC-TFH function to establish high-affinity long-lasting protective immunity to vaccines and infection.

One-Sentence SummaryBlimp1 expressing TFH cells express unique transcriptional programs to control PC formation

RESEARCH ARTICLE SUMMARYO_ST_ABSIntroductionC_ST_ABSAdaptive B cell immunity rapidly emerges to form plasma cells (PC) for antibody production and non-PC that enter germinal centers (GC) to evolve higher affinity B cell receptors. Both pathways are essential to long-term high-affinity immune protection. The early PC to GC cell fate division is driven by B cell expression of mutually antagonist transcriptional repressors Blimp1 and Bcl6. This dichotomous B cell outcome is orchestrated through antigen-specific contact by follicular helper T (TFH) cells that express Bcl6 to upregulate CXCR5, localize into B cell regions and express transcriptional programs that influence B cell fate and function. It remains unclear what TFH cell mechanisms differentially impact these divergent B cell pathways.

RationaleBlimp1 is found in Foxp3+ follicular regulatory T (TFR) cells known to impact GC B cell outcomes and play a role controlling antibody-mediated autoimmunity. In the context of infection, induced Blimp1 expression in CD4 T cells is expressed by conventional non-TFH effector cell compartments. Blimp1 segregates with emigrant CD4 T cells that leave the reactive lymphoid tissue to control innate immune function at the site of antigen entry. Conversely, Bcl6 is predominantly expressed in the GC regulating TFH pathway and is demonstrated to suppress Blimp1 expression. Germline ablation of Bcl6 exaggerates type 2 effector TH cell functions that promote excessive antibody production in the absence of the GC reaction. Similarly, loss of Bcl6 in CD4 T cells abrogates GC formation and post-GC PC responses, however multiple recent reports indicate continued support for antibody production without a Bcl6+ TFH compartment. To reconcile these findings, we propose a division of TFH function with separable pathways to regulate PC and GC differentiation. We hypothesize a central role for persistent CD4 T cell expressed Blimp1 that segregates early TFH transcriptional control to create an effector cell program that selectively targets PC differentiation.

ResultsDirect intracellular staining for protein, confirmed with single Blimp1 and dual (Foxp3) reporter mice, identified Blimp1 expressing CXCR5+PD1hi TFH and TFR subsets within the spleen, bone marrow and other lymphoid tissues at steady-state. Conditional deletion of Prdm1 in CD4 T cells and adoptive transfer into immunodeficient hosts with splenic B cells, truncated both early pre-GC and late post-GC formation of PC providing a causal link to both pathways of differentiation in vivo. Across steady-state splenic T cells, in vitro activated Blimp1+CD25- CD4 T cells in T-B cell co-cultures correlated with significant levels of PC induction. Integrated single cell-indexed strategies segregate the transcriptional programs of Blimp1 expressing TFH cells (referred to here as PC-TFH) from canonical GC-inducing Bcl6+ TFH cells (GC-TFH), both distinct from Blimp1+ TFR cell programs in the steady-state. Immunization and recall produce follicular localized PC-TFH with pMHCII-tetramer binding memory response TFH cells that segregate across PC-TFH and GC-TFH compartments re-iterating the dichotomous transcriptome seen at steady-state.

ConclusionThis study identifies Blimp1 as a key mediator of PC-TFH cells that sub-specialize as inducers of PC differentiation and bifurcate from the Bcl6+ GC-TFH cell pathway and functions. Persistent PC-TFH compartments assort across multiple lymphoid tissues at steady-state and are distinct from Foxp3+Blimp1+ TFR immune regulators. While PC TFH cells alone are required for early and rapid antibody responses, both TFH sub-classes are essential to the generation of high-affinity long-lived and memory response PC compartments. Cellular organization and molecular components of the PC-TFH transcriptional program indicate functional sub-specialization that can be separately targeted for immunotherapeutic purposes and adjuvant design in future vaccines.

Sub-specialized Blimp1+ PC-TFH cells control PC differentiationAdaptive immune protection requires balancing the evolution of BCR affinity within germinal center (GC) B cells and the differentiation of plasma cells (PC) for production of antibodies. Both functional B cell pathways require the antigen-specific induction of specialized CD4+ follicular T (TFH) cells. Within GC-inducing TFH cells, Bcl6 is required to drive the formation and function of GC B cells. Here, we segregate PC-inducing TFH cells that require Blimp1 as a key mediator of antigen-specific PC differentiation. The Blimp1+ PC-TFH transcriptional program diverges from Bcl6+ GC-TFH compartment and Blimp1+Foxp3+ follicular regulatory T (TFR) compartments. Antigen-specific PC-TFH emerge and segregate rapidly from GC-TFH after priming and recall to co-operatively induce effective long-term adaptive immunity.

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category: immunology


title: MAIT cells activate dendritic cells to promote T follicular helper cell differentiation and humoral immunity

abstract: Protective immune responses against respiratory pathogens, including influenza virus are initiated by the mucosal immune system. However, most licensed vaccines are administered parenterally and are largely ineffective at inducing mucosal immunity. The development of safe and effective mucosal vaccines has largely been hampered by the lack of a suitable mucosal adjuvant. In this study we explore a novel class of adjuvant that harness mucosal-associated invariant T (MAIT) cells. We show evidence that intranasal immunisation of MAIT cell agonists co-administered with protein, including haemagglutinin from influenza A virus induced potent humoral immunity and immunoglobulin (Ig)A production, which protected mice against infection. MAIT cell adjuvant activity was mediated by CD40L-dependent activation of dendritic cells and subsequent priming of CD4+ T follicular helper cells. In summary, we show that MAIT cells are promising vaccine targets that can be utilised as cellular adjuvants in mucosal vaccines.


title: Rapid and accurate identification of Escherichia coli STEC O157:H7 by mass spectrometry, artificial intelligence and detection of specific biomarkers peaks.

abstract:
The different pathotypes of Escherichia can produce a large number of human diseases. Surveillance becomes complex since their differentiation are not easy.

Particularly, the detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 consists of stool culture of a diarrheal sample in enrichment and/or selective media, identification of presumptive colonies and confirmation by Multiplex PCR technique for the genotypic characterization of serogroup O157 and Shiga toxins (stx1 and stx2), in addition to the traditional biochemical identification.

All of these procedures are laborious, require a certain level of training, are time consuming and expensive. Among the currently most widely used methodologies, MALDI-TOF MS mass spectrometry (matrix-assisted laser desorption/ionization with time-of-flight mass detection), allows a quick and easy way to obtain a protein spectrum of a microorganism, not only in order to identify the genus and species, but also the discovery of potential biomarker peaks of a certain characteristic. In the present work, the information obtained from 60 clinical isolates was used to detect peptide fingerprints of STEC O157:H7 and other diarrheagenic E. coli. The differences found in the protein profiles of the different pathotypes established the foundations for the development and evaluation of classification models through automated training.

The application of the Biomarkers in combination with the predictive models on a new set of samples (n=142), achieved 99.3% of correct classifications, allowing the distinction between STEC O157:H7 isolates from the other diarrheal Escherichia coli.

Therefore, given that STEC O157:H7 is the main causal agent of haemolytic uremic syndrome and based on the performance values obtained in the present work (Sensitivity=98.5% and Specificity=100%), this development could be a useful tool for diagnosis of the disease in clinical microbiology laboratories.

category: microbiology


title: Development of a long-read NGS workflow for improved characterization of fastidious respiratory mycoplasmas

abstract: Mycoplasmas are respiratory pathogens in humans and animals and due to their fastidious nature, they have been historically underdiagnosed. Lack of standardised diagnostic, typing and antimicrobial susceptibility testing methods makes clinical management and epidemiological studies challenging. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of respiratory mycoplasmas using a rapid long-read sequencing platform. Critical aspects of bacterial whole genome sequencing were explored using fastidious respiratory Mycoplasma (M. felis and M. cynos) isolated from animals including: (i) four solid and liquid-based media based on a specialized formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for sequencing purposes, and (iii) two de novo assembly platforms as key components of a bioinformatic pipeline including Flye and Canu assemblers. DNA quality and quantity compatible with long-read sequencing requirements were obtained with culture volumes of 160ml in modified Hayflicks broth incubated for 96 hours. The other three culture approaches investigated did not meet the DNA quality criteria required for long-read sequencing. The use of bead-beating bacterial cell lysis in the extraction protocol resulted in smaller fragments and shorter reads compared to enzymatic lysis methods. Overall, Flye generated more contiguous assemblies than the Canu assembler. This novel study provides a step-by-step sequencing workflow including mycoplasma culture, DNA extraction and de novo assembly approaches for the characterization of highly fastidious respiratory mycoplasmas. This workflow will provide diagnosticians, epidemiologists, and researchers with a more comprehensive tool than the laborious conventional methods for a complete genomic characterization of respiratory mycoplasmas.


title: Cell type-specific Expression Quantitative Trait Loci

abstract: Genetic regulation of gene expression can vary across cell types. To map cell type-specific gene expression quantitative trait loci (eQTLs), a popular approach is to assess the interaction between genotype and cell type proportions in a linear model. A linear model requires transformation of RNA-seq count data, which distorts the relation between gene expression and cell type proportions and it could lead to reduced power and inflated false positive findings. We have developed a statistical method, CSeQTL, to perform cell type-specific eQTL mapping for bulk RNA-seq data while directly modeling RNA-seq count data. CSeQTL utilizes both total expression and allele-specific expression to improve the power of eQTL mapping. As demonstrated through simulations and applications on human whole blood and brain RNA-seq data, CSeQTL controls false positive probability and reports much more findings than the linear model approach. We have conducted a comprehensive evaluation of the overlap between cell type-specific eQTLs and the genetic loci associated with 21 categories of human traits and identified several interesting findings. For example, the genetic loci associated with substance addiction are enriched among the eQTLs in the excitatory neurons of schizophrenia subjects, but not in healthy controls.

category: genomics


title: PanGu Drug Model: Learn a Molecule Like a Human

abstract: Recent achievements in large-scale pre-trained models like GPT-3 and PanGu- showed amazing performances in many downstream tasks, which makes AI friendlier toward industrial users. Deep learning has been recognized as the most promising technology for pharmaceuticals, a powerful molecule pre-trained model could save researchers tons of time. In chemistry classes, the students learn two molecule representations, the molecular formula and the structure formula, and learn to translate them from one way to the other. Inspired by this, we developed a novel deep learning architecture using a graph-to-sequence asymmetric conditional variational autoencoders, called PanGu Drug Model, which can appropriately characterize the molecule from both representations and improve the performance of downstream drug discovery tasks. After pretrained with 1.7 billion small molecules, our proposed model achieved the state-of-the-art results in 20 drug discovery tasks, such as molecule property prediction (predict ADMET properties, compound-target interactions, drug-drug interactions and chemical reaction productivity), molecule generation and molecule optimization. A new drug screening library of 100 million drug-like small molecules with 99.68% novelty was generated by PanGu molecule generator, which could efficiently produce novel compounds with similar physiochemical properties to given distribution, this library could be used to supplement existing compound databases. In addition, PanGu molecule optimizer could optimize the chemical structures of starting molecule with improved molecular property of interest. An automatic multi-objective optimization web application implemented by PanGu Drug Model is available at http://www.pangu-drug.com/.

category: biochemistry


title: Prediction of brain functions from structural connectome using graph neural network

abstract: AO_SCPLOWBSTRACTC_SCPLOWThe relationship between brain structure and function remains elusive, amidst the tremendous advances in brain mapping techniques. In this work, we attempt to partially disentangle this relationship by connecting task-evoked functional MRI (fMRI) responses with the underlying structural connectome using graph neural network (GNN). MRI data (n = 1,063) were collected from the Human Connectome Project. We demonstrate that our GNN-based model predicts task-evoked fMRI responses with high fidelity. Using a graph attention mechanism, it is possible to infer the subsets of neighboring cortical regions whose structural connections are important for the prediction of the functional responses of individual cortical regions. Notably, for each cortical region, such subset of neighboring cortical regions is predominantly localized to the ipsilateral hemisphere and much smaller than that with direct structural connections. We found that the higher cognitive functions subserved by the cingulo-opercular, dorsal attention, frontoparietal and default mode clusters may depend on neighboring cortical regions across a wide range of functional brain clusters in the ipsilateral hemisphere, whilst the sensory functions subserved by the visual1 and auditory clusters on neighboring cortical regions across much fewer functional brain clusters.

category: bioengineering


title: Informational Masking Can Constrain Acoustically Guided Mating Behavior

abstract: Noisy social environments constrain human speech intelligibility in two primary ways: spectro-temporal overlap between signals and noise reduces speech audibility ("energetic masking") and noise interferes with processing the informative features of otherwise audible speech ("informational masking"). To date, informational masking has not been investigated in studies of vocal communication in nonhuman animals, even though their behavioral decisions frequently depend on extracting information from vocalizations in noisy aggregations. In this study of a treefrog, in which females chose mates in noisy breeding choruses, we investigated whether informational masking can disrupt the processing of signal information in the contexts of species recognition and sexual selection. We observed significant reductions in response to the vocalizations of a potential mate when they were broadcast concurrently with other biologically relevant sounds designed to reduce or eliminate energetic masking. These effects were more pronounced when competing sounds were present in a particularly salient frequency range for processing vocalizations. Among responsive subjects, however, performance in signal recognition and discrimination tasks remained largely unchanged. These results confirm that informational masking is a general communication problem among humans and other animals and suggest it may be a crucial yet understudied source of selection shaping the evolution of animal communication.


title: History of previous midlife estradiol treatment permanently alters interactions of brain insulin-like growth factor-1 signaling and hippocampal estrogen synthesis to enhance cognitive aging in a rat model of menopause

abstract:
Across species, including humans, elevated levels of brain estrogen receptor (ER)  are associated with enhanced cognitive aging even in the absence of circulating estrogens. In rodents, short-term estrogen treatment--such as that commonly used in the menopausal transition--results in long-term increases in levels of ER in the hippocampus, leading to enhanced memory long after termination of estrogen treatment. However, mechanisms by which increased levels of brain ER enhances cognitive aging remain unclear. Here we show that in the hippocampus, insulin-like growth factor-1 (IGF-1)--which can activate ER via ligand-independent mechanisms--requires concomitant synthesis of brain-derived neuroestrogens to phosphorylate ER via MAPK signaling, ultimately resulting in enhanced memory. In a rat model of menopause involving long-term ovarian hormone deprivation, hippocampal neuroestrogen activity decreases, altering IGF-1 activity and resulting in impaired memory. However, this process is reversed by short-term estradiol treatment. Forty-days of estradiol exposure following ovariectomy results in maintenance of neuroestrogen levels that persist beyond the period of hormone treatment, allowing for continued interactions between IGF-1 and neuroestrogen signaling, elevated levels of hippocampal ER, and ultimately enhanced memory. Collectively, results demonstrate that short-term estradiol use following loss of ovarian function has long-lasting effects on hippocampal function and memory by dynamically regulating cellular mechanisms that promote activity of ER in the absence of circulating estrogens. Translational impacts of these findings suggest lasting cognitive benefits of short-term estrogen use near menopause and highlight the importance of hippocampal ER--independent from the role of circulating estrogens--in regulating memory in aging females.

Significance statementDeclines in ovarian hormones following menopause coincide with increased risk of cognitive decline. Due to potential health risks, current recommendations are that menopausal estrogen therapy be limited to a few years. Long-term consequences for the brain and memory of this short-term midlife estrogen therapy are unclear. Here, in a rodent model of menopause, we determined mechanisms by which short-term midlife estrogen exposure can enhance hippocampal function and memory with cognitive benefits and molecular changes enduring long after termination of estrogen exposure. Our model indicates long-lasting benefits of maintaining hippocampal estrogen receptor function in the absence of ongoing estrogen exposure and suggests potential strategies for combating age-related cognitive decline.


title: Hippocampal CA1 represents action and reward events instantly compared to the superficial and deep layers of the lateral entorhinal cortex

abstract: The entorhinal cortex (EC) is the main interface between the hippocampus and the neocortex. The EC plays a critical role in learning and memory. We investigated the neuronal representation of behavioral events during operant learning in the hippocampal-entorhinal circuit of head-fixed rats. Both CA1 and lateral entorhinal cortex (LEC) neurons develop task-related activities after learning. Among diverse task-related activities, we compared the transient peak activities that represent action and reward and found a distinct difference in the timing of behavioral event representation between CA1 and LEC. CA1 represents action and reward events in close to real-time, whereas both the superficial and deep layers of the LEC showed delayed representation of those events. Our results suggest that subpopulations exist within which CA1 and LEC neurons process the information in a different order from the anatomically defined hippocampal-entorhinal circuit.


title: Different olfactory neuron classes use distinct temporal and molecular programs to complete synaptic development

abstract: Developing neurons must meet core molecular, cellular, and temporal requirements to ensure the correct formation of synapses, resulting in functional circuits. However, because of the vast diversity in neuronal class and function, it is unclear whether or not all neurons use the same organizational mechanisms to form synaptic connections and achieve functional and morphological maturation. Moreover, it remains unknown if neurons united in a common goal and comprising the same sensory circuit develop on similar timescales and using identical molecular approaches to ensure the formation of the correct number of synapses. To begin to answer these questions, we took advantage of the Drosophila antennal lobe, a model olfactory circuit with remarkable genetic access and synapse-level resolution. Using tissue-specific genetic labeling of active zones, we performed a quantitative analysis of synapse formation in multiple classes of neurons throughout development and adulthood. We found that olfactory receptor neurons (ORNs), projection neurons (PNs), and local interneurons (LNs) each have unique time-courses of synaptic development, addition, and refinement, demonstrating that each class follows a distinct developmental program. This raised the possibility that these classes may also have distinct molecular requirements for synapse formation. We genetically altered neuronal activity in each neuronal subtype and observed differing effects on synapse number based on the neuronal class examined. Silencing neuronal activity in ORNs, PNs, and LNs impaired synaptic development but only in ORNs did enhancing neuronal activity influence synapse formation. ORNs and LNs demonstrated similar impairment of synaptic development with enhanced activity of a master kinase, GSK-3{beta}, suggesting that neuronal activity and GSK-3{beta} kinase activity function in a common pathway. ORNs also, however, demonstrated impaired synaptic development with GSK-3{beta} loss-of-function, suggesting additional activity-independent roles in development. Ultimately, our results suggest that the requirements for synaptic development are not uniform across all neuronal classes with considerable diversity existing in both their developmental timeframes and molecular requirements. These findings provide novel insights into the mechanisms of synaptic development and lay the foundation for future work determining their underlying etiologies.


title: Irrational choices via a curvilinear representational geometry for value

abstract: We make decisions by comparing values, but how is value represented in the brain? Many models assume, if only implicitly, that the representational geometry of value is linear. However, in part due to a historical focus on noisy single neurons, rather than neuronal populations, this hypothesis has not been rigorously tested. Here, we examined the representational geometry of value in the ventromedial prefrontal cortex (vmPFC), a part of the brain linked to economic decision-making. We found that values were encoded along a curved manifold in vmPFC. This curvilinear geometry predicted a specific pattern of irrational decision-making: that decision-makers will make worse choices when an irrelevant, decoy option is worse in value, compared to when it is better. Indeed, we observed these irrational choices in behavior. Together, these results not only suggest that the representational geometry of value is nonlinear, but that this nonlinearity could impose bounds on rational decision-making.


title: An Open and Continuously Updated Fern Tree of Life (FTOL)

abstract:
1Ferns, with about 12,000 species, are the second most diverse clade of vascular plants after angiosperms. They have been the subject of numerous molecular phylogenetic studies, resulting in the publication of trees for every major clade and DNA sequences from nearly half of all species. Global fern phylogenies have been published periodically, but as molecular systematics research continues at a rapid pace, these become quickly outdated.

Here, we develop a mostly automated, reproducible, open pipeline to generate a continuously updated fern tree of life (FTOL) from DNA sequence data available in GenBank. Our tailored sampling strategy combines whole plastomes (few taxa, many loci) with commonly sequenced plastid regions (many taxa, few loci) to obtain a global, species-level fern phylogeny with high resolution along the backbone and maximal sampling across the tips. We use an curated reference taxonomy to resolve synonyms in general compliance with the community-driven Pteridophyte Phylogeny Group I classification.

The current FTOL includes 5,563 species, an increase of ca. 40% relative to the most recently published global fern phylogeny. Using an updated and expanded list of 65 fern fossil constraints, we find estimated ages for most families and deeper clades to be considerably older than earlier studies.

FTOL and its accompanying datasets, including the fossil list and taxonomic database, will be updated on a regular basis and are available via a web portal (https://fernphy.github.io) and R packages, enabling immediate access to the most up-to-date, comprehensively sampled fern phylogeny. FTOL will be useful for anybody studying this important group of plants over a wide range of taxonomic scales, from smaller clades to the entire tree. We anticipate FTOL will be particularly relevant for macroecological studies at regional to global scales and will inform future taxonomic systems with the most recent hypothesis of fern phylogeny.

category: evolutionary biology


title: Direct and indirect impacts of positive selection on genomic variation in Drosophila serrata

abstract: Understanding the extent to which microevolutionary adaptation relies on novel beneficial mutations, as opposed to previously neutral standing genetic variation, is an important goal of evolutionary genetics. Progress towards this goal has been enhanced during the genomic era through the study of selective sweeps. Selective sweeps fall into two categories: hard sweeps via new mutations and soft sweeps via pre-existing mutations. However, data are currently lacking on the relative frequency of these two types of selective sweep. In this study, we examined 110 whole genome sequences from Drosophila serrata sampled from eastern Australia and searched for hard and soft sweeps using a deep learning algorithm (diploS/HIC). Analyses revealed that approximately 15% of the D. serrata genome was directly impacted by soft sweeps, and that 46% of the genome was indirectly influenced via linkage to these soft sweeps. In contrast, hard sweep signatures were very rare, only accounting for 0.1% of the scanned genome. Gene ontology enrichment analysis further supported our confidence in the accuracy of sweep detection as several traits expected to be under frequent selection due to evolutionary arms races (e.g. immunity and sperm competition) were detected. Within soft sweep regions and those flanking them, there was an over-representation of SNPs with predicted deleterious effects, suggesting positive selection drags deleterious variants to higher frequency due to their linkage with beneficial loci. This study provides insight into the direct and indirect contributions of positive selection in shaping genomic variation in natural populations.

category: genetics


title: De novo sequencing, diploid assembly, and annotation of the black carpenter ant, Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing.

abstract: The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America east of the Rocky Mountains. Its frequent infestation in human buildings justifies close genetic examination and its large size and small genome make it ideal for individual sequencing. From a single individual I used long-read nanopore sequencing to assemble a genome of 306 Mb, with an N50 of 565 Kb, and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon related ant assemblies. The reads provided secondary information in the form of parasitic and symbiont genomes, as well as epigenetic information. I assembled a complete Wolbachia bacterial isolate with a genome size of 1.2 Mb and 76X coverage, as well as a commensal bacterium of the carpenter ant tribe, the species-specific symbiont Blochmannia pennsylvanicus, at 791 Kb, 2400X coverage, which matched to within 200 bp of its previously published reference. I also produced a complete mitochondrial genome with over 5000X coverage, revealing minor rearrangements and the first assembly for this species. DNA methylation and hydroxymethylation was measured simultaneously at whole genome, base-pair resolution level from the same nanopore reads and confirmed extremely low levels seen in the Formicidae family of Hymenoptera. A phased diploid assembly was built, revealing a moderate level of heterozygosity, with 0.16% of bases having biallelic SNPs from the two parental haplotypes. Protein prediction yielded 20,209 putative amino acid sequences and annotation identified 86% matched to previously known proteins. All assemblies were derived from a single Minion flow cell generating 20 Gb of sequence for a cost of $1047 including all consumable reagents. Adding fixed costs for required equipment and batch costs for reagents brings the cost to assemble an ant-sized genome to less than $5000. Complete analysis was performed in under 1 week on a commodity computer desktop with 64 Gb memory.


title: Drug combination prediction for cancer treatment using disease-specific drug response profiles and single-cell transcriptional signatures

abstract: Developing novel cancer treatments is a challenging task that can benefit from computational techniques matching transcriptional signatures to large-scale drug response data. Here, we present  retriever, a tool that extracts robust disease-specific transcriptional drug response profiles based on cellular response profiles to hundreds of compounds from the LINCS-L1000 project. We used retriever to extract transcriptional drug response signatures of triple-negative breast cancer (TNBC) cell lines and combined these with a single-cell RNA-seq breast cancer atlas to predict drug combinations that antagonize TNBC-specific disease signatures. After systematically testing 152 drug response profiles and 11,476 drug combinations, we identified the combination of kinase inhibitors QL-XII-47 and GSK-690693 as the topmost promising candidate for TNBC treatment. Our new computational approach allows the identification of drugs and drug combinations targeting specific tumor cell types and subpopulations in individual patients. It is, therefore, highly suitable for the development of new personalized cancer treatment strategies.

category: bioinformatics


title: Size matters - the impact of nucleus size on results from spatial transcriptomics

abstract: Visium Spatial Gene Expression (ST) is a method combining histological spatial information with transcriptomics profiles directly from tissue sections. The use of spatial information has made it possible to discover novel modes of gene expression regulations. However, in the ST experiment, the nucleus size of cells may exceed the thickness of a tissue slice. This may, in turn, negatively affect comprehensive capturing the transcriptomics profile in a single slice, especially for tissues having large differences in the size of nuclei. Here, we defined the effect of Consecutive Slices Data Integration (CSDI) on results from spatial transcriptomic study of human postmortem brains. CSDI can be applied to investigate consecutive sections studied with ST in the human cerebral cortex, avoiding misinterpretation of spot clustering and annotation, increasing accuracy of cell recognition as well as improvement in uncovering the layers of grey matter in the human brain.


title: Beneficial and Detrimental Effects of Nonspecific Binding During DNA Hybridization

abstract: DNA strands have to sample numerous states to find the alignment that maximizes Watson-Crick-Franklin base pairing. This process depends strongly on sequence, which affects the stability of the native duplex as well as the prevalence of non-native inter- and intra-molecular helices. We present a theory which describes DNA hybridization as a three stage process: diffusion, registry search, and zipping. We find that non-specific binding affects each of these stages in different ways. Mis-registered intermolecular binding in the registry search stage helps DNA strands sample different alignments and accelerates the hybridization rate. Non-native intramolecular structure affects all three stages by rendering portions of the molecule inert to intermolecular association, limiting mis-registered alignments to be sampled, and impeding the zipping process. Once inregister base-pairs are formed, the stability of the native structure is important to hold the molecules together long enough for non-native contacts to break.

category: biophysics


title: Antibody evolution to SARS-CoV-2 after single-dose Ad26.COV2.S vaccine

abstract:
The single dose Ad.26.COV.2 (Janssen) vaccine elicits lower levels of neutralizing antibodies and shows more limited efficacy in protection against infection than either of the available mRNA vaccines. In addition, the Ad.26.COV.2 has been less effective in protection against severe disease during the Omicron surge. Here, we examined the memory B cell response to single dose Ad.26.COV.2 vaccination. Compared to mRNA vaccines, Ad.26.COV.2 recipients had significantly lower numbers of RBD-specific memory B cells 1.5 or 6 months after vaccination. Memory antibodies elicited by both vaccine types show comparable neutralizing potency against SARS-CoV-2 and Delta. However, the number of memory cells producing Omicron neutralizing antibodies was somewhat lower after Ad.26.COV.2 than mRNA vaccination. The data help explain why boosting Ad.26.COV.2 vaccine recipients with mRNA vaccines is effective, and why the Janssen vaccine appears to have been less protective against severe disease during the Omicron surge than the mRNA vaccine.

One-Sentence SummaryAd.26.COV.2 vaccine results in lower quantity but comparable quality of protective memory B cells compared to mRNA vaccines.


title: In vivo prenylomic profiling in the brain of a transgenic mouse model of Alzheimer's disease reveals increased prenylation of a key set of proteins

abstract: Dysregulation of protein prenylation has been implicated in many diseases, including Alzheimers disease (AD). Prenylomic analysis, the combination of metabolic incorporation of an isoprenoid analogue (C15AlkOPP) into prenylated proteins with a bottom-up proteomic analysis, has allowed identification of prenylated proteins in various cellular models. Here, transgenic AD mice were administered with C15AlkOPP through intracerebroventricular (ICV) infusion over 13 days. Using prenylomic analysis, 36 prenylated proteins were enriched in the brains of AD mice. Importantly, the prenylated forms of 15 proteins were consistently upregulated in AD mice compared to non-transgenic wild-type controls. These results highlight the power of this in vivo metabolic labeling approach to identify multiple post-translationally modified proteins that may serve as potential therapeutic targets for a disease that has proved refractory to treatment thus far. Moreover, this method should be applicable to many other types of protein modifications, significantly broadening its scope.


title: A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy

abstract: Reconstitution of membrane proteins into model membranes is an essential approach for their functional analysis under chemically defined conditions. Established model-membrane systems used in ensemble average measurements are limited by sample heterogeneity and insufficient knowledge of lipid and protein content at the single vesicle level, which limits quantitative analysis of vesicle properties and prevents their correlation with protein activity. Here, we describe a versatile total internal reflection fluorescence microscopy-based bleaching protocol that permits parallel analyses of multiple parameters (physical size, tightness, unilamellarity, membrane protein content and orientation) of individual proteoliposomes prepared with fluorescently tagged membrane proteins and lipid markers. The approach makes use of commercially available fluorophores including the commonly used nitrobenzoxadiazole (NBD) dye and may be applied to deduce functional molecular characteristics of many types of reconstituted fluorescently tagged membrane proteins.


title: miRNA upregulate protein and glycan expression via direct activation in proliferating cells

abstract:
The dominant paradigm is that miRNA binding to mRNA represses protein expression. Activation by miRNA has been observed in select circumstances (quiescent cells, mitochondria), but is not thought a feature of miRNA action in actively dividing cells. Herein, we comprehensively map the miRNA regulation of -2,6-sialyltransferases ST6GAL1 and ST6GAL2 using a high-throughput assay (miRFluR). We find the majority of miRNA targeting ST6GAL1, the main enzyme controlling -2,6-sialylation, upregulate protein expression. In contrast, those that regulate ST6GAL2 are predominantly downregulatory. We provide evidence that miRNA-mediated upregulation occurs in proliferating cells and is a direct effect. Further, we show that AGO2 and FXR1 are required. Our data expands current understanding of miRNA, providing strong evidence of both upregulatory and downregulatory roles for these non-coding RNA.

One-Sentence SummarymiRNA directly activate expression of -2,6-sialyltransferases and sialylation, expanding miRNA actions in dividing cells.


title: Decision Tree Ensembles Utilizing Multivariate Splits Are Effective at Investigating Beta-Diversity in Medically Relevant 16S Amplicon Sequencing Data

abstract:
Canonical distance and dissimilarity measures can fail to capture important relationships in high-throughput sequencing datasets since these measurements are unable to represent feature interactions. By learning a dissimilarity using decision tree ensembles, we can avoid this important pitfall. We used 16S rRNA data from the lumen and mucosa of the distal and proximal human colon and the stool of patients suffering from immune-mediated inflammatory diseases and compared how well the Jaccard and Aitchison metrics preserve the pairwise relationships between samples to dissimilarities learned using Random Forests, Extremely Randomized Trees, and LANDMark. We found that dissimilarities learned by unsupervised LANDMark models were better at capturing differences between communities in each set dataset. For example, differences in the microbial communities of colons distal lumen and mucosa were better reflected using LANDMark dissimilarity (p [≤] 0.001, R2 = 0.476) than using the Jaccard distance (p [≤] 0.001, R2 = 0.313) or Random Forest dissimilarity (p [≤] 0.001, R2 = 0.237). In addition, applying Uniform Manifold Approximation and Projection to dissimilarity matrices and transforming the result using principal components analysis created two-dimensional projections that captured the main axes of variation while also preserving the pairwise distances between samples (eg: {rho} = 0.8804, p [≤] 0.001 for the distal colon dissimilarities). Finally, supervised LANDMark models tend to outperform both Random Forest and Extremely Randomized Tree classifiers. Models employing multivariate splits can improve the analysis of complex high-throughput sequencing datasets. The improvements observed in this work likely result from the ability of these models to reduce noise from uninformative features. In an unsupervised setting, LANDMark models can preserve pairwise relationships between samples. When used in a supervised manner, these methods tend to learn a decision boundary that is more reflective of the biological variation within the dataset.

Author SummaryDistance and dissimilarity measures are often used to investigate the structure of biological communities. However, our investigation into two commonly used distance measures, the Jaccard and Aitchison distances, demonstrates that these measures can fail to capture important relationships in microbiome communities. This is likely due to their inability to identify dependencies between features. For example, both the Jaccard and Aitchison metrics are unable to identify subsets of samples where the presence of one feature depends on another. Previous research has found that Random Forest embeddings can be used to create an alternative dissimilarity measure for dimensionality reduction in genomic datasets. We show that dissimilarities learned by decision tree ensembles, especially those using base-estimators capable of partitioning data using oblique and non-linear cuts, can be superior since these approaches naturally model these interactions.


title: Identification and receptor mechanism of TIR-catalyzed small molecules in plant immunity

abstract: Plant nucleotide-binding leucine-rich-repeat receptors (NLRs) with an N-terminal toll/interleukin-1 receptor (TIR) domain sense pathogen effectors to enable TIR-encoded NADase activity for immune signaling. TIR-NLR (TNL) signaling requires conserved helper NLRs NRG1 and ADR1 and the lipase-like protein EDS1 that functions as a heterodimer with each of its paralogs PAD4 and SAG101. We show that TIR-containing proteins catalyze production of 2-(5-phosphoribosyl)-5-adenosine mono-/di-phosphate (pRib-AMP/ADP) in vitro and in planta. Biochemical and structural data demonstrate that EDS1-PAD4 is a receptor complex for pRib-AMP/ADP. pRib-ADP binding triggers a conformational change in the PAD4 C-terminal domain to allosterically promote EDS1-PAD4 interaction with ADR1-L1 but not NRG1A. Our study identifies TIR-catalyzed pRib-AMP/ADP as a missing link in TIR signaling via EDS1-PAD4 and as likely second messengers for plant immunity.

category: plant biology


title: New broad-spectrum anti-Flavivirus human antibodies with Zika virus-neutralizing potential

abstract:
Flavivirus infections show recurrent outbreaks and can be responsible for disease complications such as Hemorrhagic Dengue Fever and Congenital Zika Virus Syndrome. Effective therapeutic interventions are still a challenge. Antibodies can provide significant protection, although antibody response may fail due to ADE (Antibody-Dependent Enhancement) reactions or immune escape mutations. To generate effective neutralizing antibodies, the choice of the target antigen is a crucial part of the process. Human anti-Flavivirus antibodies were selected from a combinatorial library displayed on a phage surface. The antibodies were selected against a mimetic peptide based on the fusion loop region in Domain II of the protein E, which is highly conserved among different Flavivirus. Four rounds of selection were performed using the synthetic peptide in two strategies: the first was using acidic elution of bound phages, and the second was applying a competing procedure. After panning, the selected VH and VL domains were determined by combining NGS and bioinformatic approaches. Three different human monoclonal antibodies were expressed as scFvs and further characterized. All showed binding capacity to Zika (ZIKV), Yellow Fever (YFV), and Dengue (DENV) viruses. Two of these antibodies, AZ1p and AZ6m, could neutralize the ZIKV infection in a PRNT assay. These new antibodies have the potential to be used in therapeutic intervention against different Flavivirus illnesses and, due to the conservation of the fusion loop region, they may be resistant to scape mutations.

Author summaryThe central idea of this work is to present a possible unique therapeutic approach to combat different diseases that cause health problems annually, such as Dengue and yellow fever infections and Zika congenital syndromes. The viruses that cause these diseases, of the Flavivirus genus, typically have disease amplification reactions and evasion mechanisms of the immune response that hinder the success of specific therapies and vaccines and require new forms of effective and safe treatments. We have developed new neutralizing human antibodies so that they bind to a highly conserved region of Flavivirus, called the fusion loop, and in such a way as to avoid adverse effects associated with anti-Flavivirus antibodies. We showed that the antibodies have high cross-reactivity against different Flaviviruses and we exemplified their neutralizing capacity for Zika virus infection in an ex vivo assay. New monoclonal antibodies such as those presented here may contribute to the control of important tropical diseases in a safer and more efficient way.


title: Recall of pre-existing cross-reactive B cell memory following Omicron breakthrough infection

abstract:
Understanding immune responses following SARS-CoV-2 breakthrough infection will facilitate the development of next-generation vaccines. Here, we profiled spike (S)-specific B cell responses following Omicron/BA.1 infection in mRNA-vaccinated donors. The acute antibody response was characterized by high levels of somatic hypermutation (SHM) and a bias toward recognition of ancestral SARS-CoV-2 strains, suggesting the early activation of vaccine-induced memory B cells (MBCs). BA.1 breakthrough infection induced a shift in B cell immunodominance hierarchy from the S2 subunit toward the receptor binding domain (RBD). A large proportion of RBD-directed neutralizing antibodies isolated from BA.1 breakthrough infection donors displayed convergent sequence features and broadly recognized SARS-CoV-2 variants of concern (VOCs). Together, these findings provide fundamental insights into the role of pre-existing immunity in shaping the B cell response to heterologous SARS-CoV-2 variant exposure.

One sentence summaryBA.1 breakthrough infection activates pre-existing memory B cells with broad activity against SARS-CoV-2 variants.


title: Potent Human Broadly SARS-CoV-2 Neutralizing IgA and IgG Antibodies Effective Against Omicron BA.1 and BA.2

abstract: Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in COVID-19 convalescents combining serological, cellular and monoclonal antibody explorations, revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell, demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.


title: Environment and the Evolutionary Trajectory of Horizontal Gene Transfer.

abstract: Horizontal Gene Transfer (HGT) is a powerful evolutionary force facilitating bacterial adaptation and emergence of novel phenotypes. Several factors, including environmental ones, are predicted to restrict HGT, but we lack data supporting these predictions. Here, we address this gap by measuring the relative fitness of 44 genes horizontally transferred from E. coli to S. Typhimurium strain 4/74 in four infection relevant environments and estimated the distribution of fitness effects (DFE) in each environment. Roughly half of the genes, 40-50%, had significant fitness effects in at least one environment, with most genes showing significant gene by environment (G x E) interactions. We further identified 12 genes with dosage-dependent effects across the four environments, indicating that intolerance of the recipient strain to high protein dosage is a significant barrier to HGT. We also found longer genes had stronger fitness detriments than shorter ones, showing that gene length can significantly hinder HGT. In summary, a substantial fraction of transferred genes had a significant fitness cost on the recipient, with both gene characteristics and the environment providing barriers to HGT.


title: Frequency of change determines effectiveness of microbial response strategies in sulfidic stream microbiomes

abstract: Nature challenges microbes with change at different frequencies and demands an effective response for survival. Here, we used controlled laboratory experiments to investigate the ecological success of different response strategies, such as post-translational modification, transcriptional regulation, and specialized versus adaptable metabolisms. For this, we inoculated replicated chemostats with an enrichment culture obtained from sulfidic stream microbiomes 16 weeks prior. The chemostats were submitted to alternatingly oxic and anoxic conditions at three frequencies, with periods of 1 day, 4 days and 16 days. The microbial response was recorded with 16S rRNA gene amplicon sequencing, shotgun metagenomics, transcriptomics and proteomics. Metagenomics resolved 26 nearly complete genomes of bacterial populations, mainly affiliated with Proteobacteria and Bacteroidetes. Almost all these populations maintained a steady growth rate under both redox conditions at all three frequencies of change. Apparently, oscillating oxic/anoxic conditions selected for generalistic species, rather than species specializing in only a single condition. Rapid (1-day) dynamics yielded more stochasticity, both in community dynamics and gene expression, indicating that bet-hedging might be an effective coping strategy for relatively rapid environmental change. Codon-usage bias, previously associated with copiotrophic and oligotrophic lifestyles, was found to be a powerful predictor of ecological success at different frequencies, with copiotrophs and oligotrophs more successful at a rapid and a slow pace of change, respectively, independent of growth rate.


title: Functional Biomarkers of Ex-vivo Dental Caries Onset

abstract:
BackgroundThe etiology of dental caries remains poorly understood. With the advent of next generation sequencing, a number of studies have focused on the microbial ecology of the disease. However, taxonomic associations with caries have not been consistent. Researchers have also pursued function-centric studies of the caries microbial communities aiming to identify consistently conserved functional pathways. A major question is whether changes in microbiome are a cause or a consequence of the disease. Thus, there is a critical need to define conserved functional biomarkers at the onset of dental caries.

MethodsSince it is unethical to induce carious lesions clinically, we developed an innovative longitudinal ex-vivo model integrated with the advanced non-invasive multiphoton second harmonic generation bioimaging to spot the very early signs of dental caries, combined with 16S rRNA short amplicon sequencing and liquid chromatography-mass spectrometry-based targeted metabolomics.

FindingsFor the first time, we induced longitudinally-monitored caries lesions validated with the scanning electron microscope. Consequently, we spotted the caries onset and, associated to it, distinguished five differentiating metabolites - Lactate, Pyruvate, Dihydroxyacetone phosphate, Glyceraldehyde 3-phosphate (upregulated) and Fumarate (downregulated). Those metabolites co-occurred with certain bacterial taxa; Streptococcus, Veillonella, Actinomyces, Porphyromonas, Fusobacterium, and Granulicatella, regardless of the abundance of other taxa.

InterpretationThese findings are crucial for understanding the etiology and dynamics of dental caries, and devising targeted interventions to prevent disease progression.

FundingThe study was funded by the National Institute for Dental and Craniofacial Research of the National Institutes of Health and the University of Minnesota.

Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSStudies have shown that dental caries, tooth decay, occurs as a result of disruptive imbalance in the oral ecosystem. Excessive dietary intake of fermentable carbohydrates is a critical contributor to disease progression by promoting bacterial production of acids, which shifts the microbial community to an imbalanced and a less diverse one. Studies have also shown that microbial associations with caries have not been consistent while their functions are relatively conserved across individuals. Still, the specific microbial functions associated with the dental caries onset is still unknown due to its infeasible clinical diagnosis.

Added value of this studyThis study applied a novel longitudinal ex-vivo model, integrated with advanced non-invasive bioimaging, for experimental dental caries induction. This model enabled the detection of the exact onset of the disease, which is undetected clinically. Then, the microbial communities accompanying the caries onset were analyzed for their microbial composition and metabolic functions in comparison to normal conditions. Our study identified five metabolites differentiating the caries onset. Further, we investigated the co-occurrence of these metabolic biomarkers with certain oral bacteria.

Implications of all the available evidenceOur study provides carefully validated evidence for biomarkers of the dental caries onset. These data are critical for early diagnostics and development of timely intervention strategies to interfere with the disease progression that otherwise requires invasive and costly health care expenses. Moreover, our data open new avenues for developing therapeutics to neutralize the identified metabolic biomarkers or target the accountable bacteria for caries prevention.


title: Surface microbiota of Mediterranean loggerhead sea turtles unravelled by 16S and 18S amplicon sequencing

abstract: Loggerhead sea turtle is considered a keystone species with major ecological role in Mediterranean marine environment. As with other wild reptiles, their outer microbiome is rarely studied. Although there are several studies on sea turtles macro-epibionts and endo-microbiota, there has been little research on epibiotic microbiota associated with sea turtles skin and carapace. Therefore, we provide the first identification of combined epibiotic eukaryotic and prokaryotic microbiota on Mediterranean loggerhead sea turtles. In this study, we sampled skin and carapace of 26 loggerheads from the Mediterranean Sea during 2018 and 2019. To investigate the overall microbial diversity and composition, amplicon sequencing of 16S and 18S rRNA genes was performed. We found that the Mediterranean loggerhead sea turtle epibiotic microbiota is a reservoir of a vast variety of microbial species. Microbial communities between samples mostly varied by different locations and seas, while within prokaryotic communities significant difference was observed between sampled body sites (carapace vs. skin). In terms of relative abundance, Proteobacteria and Bacteroidota were the most represented phyla within prokaryotes, while Alveolata and Stramenopiles thrived among eukaryotes. This study, besides providing a first survey of microbial eukaryotes on loggerheads via metabarcoding, identifies fine differences within both prokaryotic and eukaryotic microbial communities that seem to reflect the host anatomy and habitat. Multi-domain epi-microbiome surveys provide additional layers of information that are complementary with previous morphological studies and enable better understanding of the biology and ecology of these vulnerable marine reptiles.


title: GTP-bound E. coli FtsZ filaments are composed of Tense monomers: A DNP NMR study using interface detection.

abstract: FtsZ filaments are the major structural component of the bacterial Z-ring and are drivers of bacterial division. While crystal structures for FtsZ from some gram positive bacteria in the presence of GTP-analog like compounds suggest the possibility of a high energy "Tense" conformation, to date it remains an important question to elucidate whether this Tense form is the dominant form in filaments. Using dynamic nuclear polarization (DNP) solid-state NMR and differential isotopic labelling, we directly detect residues located at the inter-monomer interface of GTP-bound WT Escherichia coli FtsZ filaments. We combine chemical shift prediction, homology modelling, and heteronuclear dipolar recoupling techniques to characterize the E. coli FtsZ filament interface and demonstrate that the monomers in active filaments assume a Tense conformation.


title: Feedback linking cell envelope stiffness, curvature, and synthesis enables robust rod-shaped bacterial growth

abstract:
Bacterial growth is remarkably robust to environmental fluctuations, yet the mechanisms of growth-rate homeostasis are poorly understood. Here, we combine theory and experiment to infer mechanisms by which Escherichia coli adapts its growth rate in response to changes in osmolarity, a fundamental physicochemical property of the environment. The central tenet of our theoretical model is that cell-envelope expansion is only sensitive to local information such as enzyme concentrations, cell-envelope curvature, and mechanical strain in the envelope. We constrained this model with quantitative measurements of the dynamics of E. coli elongation rate and cell width after hyperosmotic shock. Our analysis demonstrated that adaptive cell-envelope softening is a key process underlying growth-rate homeostasis. Furthermore, our model correctly predicted that softening does not occur above a critical hyperosmotic shock magnitude and precisely recapitulated the elongation-rate dynamics in response to shocks with magnitude larger than this threshold. Finally, we found that to coordinately achieve growth-rate and cell-width homeostasis, cells employ direct feedback between cell-envelope curvature and envelope expansion. In sum, our analysis points to new cellular mechanisms of bacterial growth-rate homeostasis and provides a practical theoretical framework for understanding this process.

Significance StatementThe bacterial cell envelope is the critical structure that defines cell size and shape, and its expansion therefore defines cell growth. Although size, shape, and growth rate are important cellular variables that are robust to environmental fluctuations, the feedback mechanisms by which these variables influence cell-envelope expansion are unknown. Here, we explore how E. coli cells achieve growth-rate and cell-width homeostasis during fluctuations in osmolarity, a key environmental property. A biophysical model in which the cell envelope softens after an osmotic shock and envelope expansion depends directly on local curvature quantitatively recapitulated all experimental observations. Our study elucidates new mechanisms of bacterial cell morphogenesis and highlights the deep interplay between global cellular variables and the mechanisms of cell-envelope expansion.


title: The temporal transcriptomic signature of cartilage formation

abstract:
Chondrogenesis is a multistep process whereby cartilage progenitor cells generate a tissue with distinct structural and functional properties. Although several approaches to cartilage regeneration rely on the differentiation of implanted progenitor cells, the temporal transcriptomic landscape of in vitro chondrogenesis in different models has not been reported. Using RNA sequencing, we examined the differences between gene expression patterns during early cartilage formation in micromass cultures of embryonic limb bud-derived progenitors. Principal component analysis indicated a progressively different and distinct transcriptome during chondrogenesis. Differentially expressed genes (DEGs) based on pairwise comparisons of samples from consecutive days were classified into clusters and analysed. We confirmed the involvement of the top DEGs in chondrogenic differentiation with pathway analysis. We discovered several chondrogenesis-associated transcription factors and new collagen subtypes not previously linked to cartilage formation. These results provide, for the first time, a detailed insight into the molecular mechanism of in vitro chondrogenesis and provide a new gold standard for the temporal transcriptomic landscape of chondrogenic differentiation that may serve as a platform for new approaches in cartilage tissue engineering.

Highlights O_LIChondroprogenitors derived from chicken embryonic limb buds were differentiated into chondrocytes.
C_LIO_LIRNA-sequencing revealed expression levels of 24,145 transcripts.
C_LIO_LIWe examined the global expression patterns during chondrogenesis and defined 6 clusters.
C_LIO_LIUsing WGCNA analysis, we created a module of genes that had similar patterns to the chondrogenic transcription factor SOX9.
C_LIO_LIWe created a list of transcription factors with a hitherto unexplored role in chondrogenesis.
C_LI

category: molecular biology


title: Convergence of immune escape strategies highlights plasticity of SARS-CoV-2 spike

abstract: The SARS-CoV-2 spike protein is the target of neutralizing antibodies and the immunogen used in all currently approved vaccines. The global spread of the virus has resulted in emergence of lineages which are of concern for the effectiveness of immunotherapies and vaccines based on the early Wuhan isolate. Here we describe two SARS-CoV-2 isolates with large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis showed that the deletions result in complete reshaping of the antigenic surface of the NTD supersite. The remodeling of the NTD affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite for both spike variants. A unique escape mechanism with high antigenic impact observed in the {Delta}N135 variant was based on the loss of the Cys15-Cys136 disulfide due to the P9L-mediated shift of the signal peptide cleavage site and deletion of residues 136-144. Although the observed large loop and disulfide deletions are rare, similar modifications became independently established in several other lineages, highlighting the possibility of a general escape mechanism via the NTD supersite. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.


title: Evidence that Nek1 does not phosphorylate Rad54-S572 during recovery from IR

abstract: A main focus of the work in our lab is on the activity of Tousled Like Kinase 1 (TLK1) in the area of DNA Damage and Repair. As one of its key interactor, TLK1 phosphorylates NIMA related kinase 1 (Nek1), and Nek1 was reported by Spies et al. to phosphorylate and regulate the activity of the key HRR protein Rad541, which suggested an intriguing signal transduction pathway: TLK1>Nek1>Rad54. In an effort to confirm such relations, we now report that we have not been able to reproduce key findings from that study. Specifically, we found that Nek1 does not phosphorylate RAD54-S572 as was reported. We generated Nek1-KO mouse NT1 cells2 and Nek1-Knock-down in Hek293 (same cells as in Spies et al.), and the pRAD54-S572 signal does not change with our custom Ab, with or w/o IR. When we used an Ab from Lobrichs lab, it detected an immunoreactive band of wrong size for RAD54, which also did not change after IR even in synchronized G2 cells, contrary to their report. We also note that their P-assignment was based on guessing a weak consensus Nek1 sequence, and that site-directed mutagenesis of RAD54-S572 failed to yield biological effects in their in in vitro studies1. To conclusively establish that S572 is not a site of phosphorylation of Nek1, we carried out a IVK with purified Nek1 and RAD54 followed by MS analysis of the phosphatides, which revealed several but not S572. We also could not reproduce their copurification of Nek1-RAD54 by coIP, calling into question this interaction.


title: USP18 is an essential regulator of muscle cell differentiation and maturation

abstract: USP18 negatively regulates the IFN-1 signalling pathway by removing ISG15, a diubiquitin analogue, from substrate proteins and plays a role in immune related pathologies. Dermatomyositis (DM), a rare muscle degeneration disease, is characterised by an excessive immune response and IFN-1 signaling. In DM muscles we found USP18 expression within myofibers at regenerating regions, suggesting a role in muscle cell biology. Surprisingly, we observed that USP18 depletion induced muscle cell differentiation under nutrient-rich conditions. USP18 expression and function was independent of ISG15 and IFN-1 signalling, but concomitant with reduced cell cycle and sarcomeric gene networks. USP18 depletion let to altered expression of myogenic (co-)transcription factors, indicating that USP18 tightly regulates the muscle cell differentiation program. Moreover, USP18-depletion impacted physiological features in differentiated muscle cells, suggesting a role in differentiation maintenance. Our results revealed USP18 as a critical regulator of multiple steps during muscle cell differentiation independent of the IFN-1 pathway.

category: cell biology


title: HSD3B1 is an Oxysterol 3β-Hydroxysteroid Dehydrogenase in Human Placenta

abstract: Most biologically active oxysterols have a 3{beta}-hydroxy-5-ene function in the ring system with an additional site of oxidation at C-7 or on the side-chain. In blood plasma oxysterols with a 7-hydroxy group are also observed with the alternative 3-oxo-4-ene function in the ring system formed by ubiquitously expressed 3{beta}-hydroxy-{Delta}5-C27-steroid oxidoreductase {Delta}5-isomerase, HSD3B7. However, oxysterols without a 7-hydroxy group are not substrates for HSD3B7 and are not usually observed with the 3-oxo-4-ene function. Here we report the unexpected identification of oxysterols in plasma derived from umbilical cord blood and blood from pregnant women taken before delivery at 37+ weeks of gestation, of side-chain oxysterols with a 3-oxo-4-ene function but no 7-hydroxy group. These 3-oxo-4-ene oxysterols were also identified in placenta, leading to the hypothesis that they may be formed by a previously unrecognised 3{beta}-hydroxy-{Delta}5-C27-steroid oxidoreductase {Delta}5-isomerase activity of HSD3B1, an enzyme which is highly expressed in placenta. Proof of principle experiments confirmed that HSD3B1 has this activity. We speculate that HSD3B1 in placenta is the source of the unexpected 3-oxo-4-ene oxysterols in cord and pregnant womens plasma and may have a role in controlling the abundance of biologically active oxysterols delivered to the fetus.


title: Actin turnover required for adhesion-independent bleb migration

abstract: Cell migration is critical for many vital processes, such as wound healing, as well as harmful processes, like cancer metastasis. Experiments have highlighted the diversity in migration strategies employed by cells in physiologically relevant environments. In 3D fibrous matrices and confinement between two surfaces, some cells migrate using round membrane protrusions, called blebs. In bleb-based migration, the role of substrate adhesion is thought to be minimal, and it remains unclear if a cell can migrate without any adhesion complexes. We present a 2D computational fluid-structure model of a cell using cycles of bleb expansion and retraction in a channel with several geometries. The cell model consists of a plasma membrane, an underlying actin cortex, and viscous cytoplasm. Cellular structures are immersed in viscous fluid which permeates them, and the fluid equations are solved using the method of regularized Stokeslets. Simulations show that the cell cannot effectively migrate when the actin cortex is modeled as a purely elastic material. We find that cells do migrate in rigid channels if actin turnover is included with a viscoelastic description for the cortex. Our study highlights the non-trivial relationship between cell rheology and its external environment during migration with cytoplasmic streaming.


title: Integrative conformational ensembles of Sic1 using different initial pools and optimization methods

abstract: Intrinsically disordered proteins play key roles in regulatory protein interactions, but their detailed structural characterization remains challenging. Here we calculate and compare conformational ensembles for the disordered protein Sic1 from yeast, starting from initial ensembles that were generated either by statistical sampling of the conformational landscape, or by molecular dynamics simulations. Two popular, yet contrasting optimization methods were used, ENSEMBLE and Bayesian Maximum Entropy, to achieve agreement with experimental data from nuclear magnetic resonance, small-angle X-ray scattering and single-molecule Forster resonance energy transfer. The comparative analysis of the optimized ensembles, including secondary structure propensity, inter-residue contact maps, and the distributions of hydrogen bond and pi interactions, revealed the importance of the physics-based generation of initial ensembles. The analysis also provides insights into designing new experiments that can maximally discriminate among the optimized ensembles. Overall, differences between ensembles optimized from different priors were greater than when using the same prior with different optimization methods. Generating increasingly accurate, reliable and experimentally validated ensembles for disordered proteins is an important step towards a mechanistic understanding of their biological function and involvement in various diseases.


title: Head-to-head comparison of CCN4, DNMT3A, PTPN11, and SPARC as suppressors of anti-tumor immunity

abstract:
BackgroundEffective communication between innate and adaptive immunity is essential for mounting an effective antitumor immune response. Emergent cancer cells likely secrete factors that inhibit this communication. To identify such factors, we applied an in vitro workflow that coupled a functional assay with proteomics to a syngeneic mouse model of melanoma known to be resistant to immunotherapies. Collectively, these in vitro results suggested CCN4, DNMT3A, PTPN11, and SPARC as secreted factors that potentially mediate immunosuppression and could potentially become easily accessible targets for novel immunotherapies. The objective of this study was to test for consistent clinical correlates in existing human data and to verify in vivo whether knocking out tumor cell production of these secreted factors improved immune-mediated control of tumor growth.

ResultsAnalyses of available human data indicate that high CCN4 expression is associated with reduced survival in primary melanoma patients. High DNMT3A, PTPN11, and SPARC expression showed no association with overall survival. In addition, scRNAseq data from patients with melanoma confirmed CCN4 is expressed by the tumor cells, as opposed to other cells in the tumor microenvironment. An experimental system was created using a CRISPR/Cas9 approach to generate knock out cell lines for each of the four genes of interest using the B16F0 mouse melanoma cell line. In a immunocompetent C57BL/6, DNMT3A, PTPN11, and SPARC knockouts had no effect on overall survival compared to mice challenged with wildtype B16F0 cells while the CCN4 knockout significantly increased survival. This increase in overall survival was lost when the CCN4 knockout cells were injected into severely immunocompromised NSG hosts, indicating that CCN4s effect on the tumor microenvironment is immune mediated. A kinetic analysis leveraging a Markov Chain Monte Carlo approach quantified the various knockouts effect on cells intrinsic growth rate. The analysis shows CCN4 is the only knockout tested that decreased the net proliferation rate in immunocompetent mice compared to wildtype cells.

ConclusionsThe results suggest that CCN4 is a mediator of immunosuppression in the melanoma tumor microenvironment and a potential collateral immunotherapy target.


title: Characterization of cell-fate decision landscapes by estimating transcription factor dynamics.

abstract:
Modulation of gene expression during differentiation by transcription factors promotes cell diversity. Despite their role in cell fate decisions, no experimental assays estimate their regulatory activity in a high-throughput manner and at the single-cell resolution. We present FateCompass for identifying lineage-specific transcription factors across differentiation. It uses single-cell transcriptomics data to infer differentiation trajectories and transcription factor activities. We combined a probabilistic framework with RNA velocities or a differentiation potential to estimate transition probabilities and perform stochastic simulations. Also, we learned transcription factor activities using a linear model of gene regulation. Considering dynamic changes and correlations, we identified lineage-specific regulators. We applied FateCompass to an islet cell formation dataset from the mouse embryo, and we found known and novel potential cell-type drivers. Also, when applied to a differentiation protocol dataset towards beta-like cells, we pinpointed undescribed regulators of an off-target population, which were experimentally validated. Thus, as a framework for identifying lineage-specific transcription factors, FateCompass could have implications on hypothesis generation to increase the understanding of the gene regulatory networks driving cell fate choices.

HighlightsO_LIWe developed FateCompass, a flexible pipeline to estimate transcription factor activities during cell-fate decision using single-cell RNA seq data.
C_LIO_LIFateCompass outlines gene expression stochastic trajectories by infusing the direction of differentiation using RNA velocity or a differentiation potential when RNA velocity fails.
C_LIO_LITranscription factor dynamics allow the identification of time-specific regulatory interactions.
C_LIO_LIFateCompass predictions revealed known and novel cell-subtype-specific regulators of mouse pancreatic islet cell development.
C_LIO_LIDifferential motif analysis predicts lineage-specific regulators of stem cell-derived human {beta}- cells and sheds light on the cellular heterogeneity of {beta}-cell differentiation protocols.
C_LIO_LIExperimental validation supports the proposed GRN controlling SC-EC differentiation predicted by FateCompass.
C_LI

category: systems biology


title: Physiopathological changes of ferritin mRNA density and distribution in hippocampal astrocytes in the mouse brain

abstract: Astrocytes are thought to play a crucial role in brain iron homeostasis. How they accomplish this regulation in vivo remains unclear. In a recent transcriptomic analysis, we showed that polysomal Ftl1 and Fth1 mRNAs, encoding the ferritin light (Ftl) and heavy (Fth) chains that assemble into ferritin, a critical complex for iron storage and reduction, are enriched in perisynaptic astrocytic processes as compared to astrocytic soma. These data suggested that ferritin translation plays a specific role at the perisynaptic astrocytic interface and is tighly regulated by local translation. Here, we used our recently described AstroDot 3D in situ methodology to study the density and localization of ferritin mRNAs in astrocytes in the hippocampus in three different contexts in which local or systemic iron overload has been documented: ageing, the hepcidin knock-out mouse model of hemochromatosis and the APP/PS1dE9 mouse model of Alzheimers disease (AD). Our results showed that in wild type mice, Fth1 mRNA density was higher than Ftl1 and that both mRNAs were mostly distributed in astrocyte fine processes. Ageing and absence of hepcidin caused an increased Fth1/Ftl1 ratio in astrocytes and in the case of ageing, led to a redistribution of Fth1 mRNAs in astrocytic fine processes. In contrast, in AD mice, we observed a lower Fth1/Ftl1 ratio and Fth1 mRNAs became more somatic. Hence, we propose that regulation of ferritin mRNA density and distribution in astrocytes regulates iron homeostasis in physiology and pathophysiology.


title: Fasciola hepatica Fatty Acid Binding Protein 1 modulates T cell polarization by promoting dendritic cell thrombospondin-1 secretion without affecting metabolic homeostasis in obese mice

abstract:
BackgroundThe parasitic trematode Fasciola hepatica evades host immune defenses through secretion of various immunomodulatory molecules. Fatty Acid Binding Proteins (fhFABPs) are among the main excreted/secreted proteins and have been shown to display anti-inflammatory properties. However, little is currently known regarding their impact on dendritic cells (DCs) and their subsequent capacity to prime specific CD4+ T cell subsets.

Methodology/Principal FindingsThe immunomodulatory effects of both native F. hepatica extracts and recombinant fhFABPs were assessed on monocyte-derived human DCs (moDCs) and the underlying mechanism was next investigated using various approaches, including DC-allogenic T cell co-culture and DC phenotyping through transcriptomic, proteomic and FACS analyses. We mainly showed that fhFABP1 induced a tolerogenic-like phenotype in LPS-stimulated moDCs characterized by a dose-dependent increase in the cell-surface tolerogenic marker CD103 and IL-10 secretion, while DC co-stimulatory markers were not affected. A significant decrease in secretion of the pro-inflammatory cytokines IL-12p70 and IL-6 was also observed. In addition, these effects were associated with an increase in both Th2-on-Th1 ratio and IL-10 secretion by CD4+ T cells following DC-T cell co-culture. RNA sequencing and targeted proteomic analyses identified thrombospondin-1 (TSP-1) as a non-canonical factor highly expressed and secreted by fhFABP1-primed moDCs. The effect of fhFABP1 on T cell skewing was abolished when using a TSP-1 blocking antibody during DC-T cell co-culture. Immunomodulation by helminth molecules has been linked to improved metabolic homeostasis during obesity. Although fhFABP1 injection in high-fat diet-fed obese mice induced a potent Th2 immune response in adipose tissue, it did not improved insulin sensitivity or glucose homeostasis.

Conclusions/SignificanceWe show that fhFABP1 modulates T cell polarization, notably by promoting DC TSP-1 secretion in vitro, without affecting metabolic homeostasis in a mouse model of type 2 diabetes.


title: Omicron breakthrough infection drives cross-variant neutralization and memory B cell formation

abstract:
Omicron is the evolutionarily most distinct SARS-CoV-2 variant (VOC) to date and displays multiple amino acid alterations located in neutralizing antibody sites of the spike (S) protein. We report here that Omicron breakthrough infection in BNT162b2 vaccinated individuals results in strong neutralizing activity not only against Omicron, but also broadly against previous SARS-CoV-2 VOCs and against SARS-CoV-1. We found that Omicron breakthrough infection mediates a robust B cell recall response, and primarily expands preformed memory B cells that recognize epitopes shared broadly by different variants, rather than inducing new B cells against strictly Omicron-specific epitopes. Our data suggest that, despite imprinting of the immune response by previous vaccination, the preformed B cell memory pool has sufficient plasticity for being refocused and quantitatively remodeled by exposure to heterologous S protein, thus allowing effective neutralization of variants that evade a previously established neutralizing antibody response.

One Sentence SummaryBreakthrough infection in individuals double- and triple-vaccinated with BNT162b2 drives cross-variant neutralization and memory B cell formation.


title: A 3' UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria

abstract: Increasing numbers of small, regulatory RNAs (sRNAs) corresponding to 3 untranslated regions (UTR) are being discovered in bacteria. One such sRNA, denoted GlnZ, corresponds to the 3 UTR of the Escherichia coli glnA mRNA encoding glutamine synthetase. Several forms of GlnZ, processed from the glnA mRNA, are detected in cells growing with limiting ammonium. GlnZ levels are regulated transcriptionally by the NtrC transcription factor and post-transcriptionally by RNase III. Consistent with the expression, E. coli cells lacking glnZ show delayed outgrowth from nitrogen starvation compared to wild type cells. Transcriptome-wide RNA-RNA interactome datasets indicated that GlnZ binds to multiple target RNAs. Immunoblot and assays of fusions confirmed GlnZ-mediated repression of glnP and sucA, encoding proteins that contribute to glutamine transport and the citric acid cycle, respectively. Although the overall sequences of GlnZ from E. coli K-12, Enterohemorrhagic E. coli and Salmonella enterica have significant differences due to various sequence insertions, all forms of the sRNA were able to regulate the two targets characterized. Together our data show that GlnZ promotes survival of E. coli under low nitrogen conditions by modulating genes that affect carbon and nitrogen flux.


title: Periplasmic oxidized-protein repair during copper stress in E. coli: a focus on the metallochaperone CusF.

abstract:
Methionine residues are particularly sensitive to oxidation by reactive oxygen or chlorine species (ROS/RCS), leading to the appearance of methionine sulfoxide in proteins. This post-translational oxidation can be reversed by omnipresent protein repair pathways involving methionine sulfoxide reductases (Msr). In the periplasm of Escherichia coli, the enzymatic system MsrPQ, whose expression is triggered by the RCS, controls the redox status of methionine residues. Here we report that MsrPQ synthesis is also induced by copper stress via the CusSR two-component system, and that MsrPQ plays a role in copper homeostasis by maintaining the activity of the copper efflux pump, CusCFBA. Genetic and biochemical evidence suggest the metallochaperone CusF is the substrate of MsrPQ and our study reveal that CusF methionines are redox sensitive and can be restored by MsrPQ. Thus, the evolution of a CusSR-dependent synthesis of MsrPQ allows keeping copper homeostasis under aerobic conditions by maintenance of the reduced state of Met residues in copper-trafficking proteins.

AUTHOR SUMMARYOur study investigates the interconnection between the copper stress response and the methionine redox homeostasis in the Gram-negative bacterium Escherichia coli. We report that the copper-activation of the CusSR two-component system induces the expression of the periplasmic oxidized-protein repair system, MsrPQ. We demonstrate that MsrPQ is crucial for CusCFBA copper efflux pump activity under aerobic conditions as it maintains the periplasmic component CusF in its functional reduced form. Methionine emerges as a critical residue in copper trafficking proteins: however this naturally-selected advantage must be balanced by methionines high susceptibility to oxidation. Therefore the induction of MsrPQ by copper allows copper homeostasis under aerobic conditions, illustrating that E. coli has developed an integrated and dynamic circuit for sensing and counteracting stress caused by copper and oxidants, thus allowing bacteria to adapt to host cellular defences.


title: Comparative metagenomic analysis of biosynthetic diversity across sponge microbiomes highlights metabolic novelty, conservation and diversification

abstract: Marine sponges and their microbial symbiotic communities are rich sources of diverse natural products (NPs) that often display biological activity, yet little is known about their global distribution landscape and the symbionts that produce them. As the majority of sponge symbionts remain uncultured, it is a challenge to characterize their NP biosynthetic pathways, assess their prevalence within the holobiont and measure their diversity across sponge taxa and environments. Here, we explore the microbial biosynthetic landscapes of three sponge species from the Atlantic Ocean and the Mediterranean Sea. This dataset reveals striking novelty in its encoded biosynthetic potential, with less than 1% of the recovered gene cluster families (GCF) showing similarity to any characterized biosynthetic gene cluster (BGC). When zooming in on the microbial communities of each sponge, we observed higher variability of both secondary metabolic and taxonomic profiles between sponge species than within species. Nonetheless, we also identified conservation of GCFs, with 20% of sponge GCFs being shared between at least two sponge species, and a true GCF core comprised of 6% of GCFs shared across all species. Within this functional core, we identified a set of widespread and diverse GCFs encoding nonribosomal peptide synthetases (NRPS) that are potentially involved in the production of diversified ether lipids, as well as GCFs putatively encoding the production of highly modified proteusins. The present work contributes to the small, yet growing body of data characterizing NP landscapes of marine sponge symbionts, and to the cryptic biosynthetic potential contained in this environmental niche.


abstract: Single-molecule characterization of protein-DNA dynamics provides unprecedented mechanistic details about numerous nuclear processes. Here, we describe a new method that rapidly generates single-molecule information for fluorescently tagged proteins isolated from nuclear extracts of human cells. This approach determines binding lifetimes (koff), events per second ([~]kon), positional dependence (specificity), and characterizes 1D diffusion along DNA. We demonstrated the wide applicability of this approach on three forms of DNA damage using seven native DNA repair proteins and two structural variants, including: poly(ADP-ribose) polymerase (PARP1), heterodimeric ultraviolet-damaged DNA-binding protein (UV-DDB), xeroderma pigmentosum complementation group C protein (XPC), and 8-oxoguanine glycosylase 1 (OGG1). By measuring multiple fluorescent colors simultaneously, we additionally characterized the assembly and disassembly kinetics of multi-protein complexes on DNA. Thus, Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE) provides new insights about damage recognition and represents a universal technique that can be used to rapidly characterize numerous protein-DNA interactions.


title: Topographically distinct adaptive landscapes for skulls, skeletons, and size explain the dietary adaptive radiation of Carnivora (Mammalia)

abstract: AO_SCPLOWBSTRACTC_SCPLOWModels of adaptive radiation were originally developed to explain the early, rapid appearance of distinct modes of life within diversifying clades. Phylogenetic tests of this hypothesis have yielded limited support for temporally declining rates of phenotypic evolution across diverse clades, but the concept of an adaptive landscape that links form to fitness, while also crucial to these models, has received more limited attention. Using methods that assess the temporal accumulation of morphological variation and estimate the topography of the underlying adaptive landscape, I found evidence of an early partitioning of craniodental morphological variation in Carnivora (Mammalia) that occurs on an adaptive landscape with multiple peaks, consistent with classic ideas about adaptive radiation. Although strong support for this mode of adaptive radiation is present in traits related to diet, its signal is not present in body mass data or for traits related to locomotor behavior and substrate use. These findings suggest that adaptive radiations may occur along some axes of ecomorphological variation without leaving a signal in others and that their dynamics are more complex than simple univariate tests might suggest.