Miracle-dz/UniFAR-FTUs huggingface dataset:
Visualization of a Limonene Synthesis Metabolon Inside Living Bacteria by Hyperspectral SRS Microscopy

Metabolons consisting of cellular structure elements and sequential metabolic enzymes are expected to be involved in diverse biological processes. However, direct visualization of metabolons in prokaryotic cells is still challenging. In this study, we report direct visualization of concentrated subcellular regions of limonene synthesis inside single engineered Escherichia coli by using hyperspectral stimulated Raman scattering (hSRS) microscopy. Equipped with spectral unmixing, hSRS imaging provides a reliable method to quantify intracellular limonene content. In E. coli strains with a complete limonene synthesis pathway, intracellular limonene is found locally concentrated and colocalized with proteins. Furthermore, dual-modality SRS and two-photon fluorescence imaging showed colocalization of limonene and GFP-fused limonene synthase. Significance Statement Monitoring biosynthesis activity at the single-cell level is key to metabolic engineering but is still difficult to achieve in a label-free manner. Using hyperspectral stimulated Raman scattering imaging in the 670-900 cm−1 region, we visualized localized limonene synthesis inside engineered E. coli. The colocalization of limonene and GFP-fused limonene synthase was confirmed by co-registered stimulated Raman scattering and two-photon fluorescence images. Our finding suggests a limonene synthesis metabolon with a polar distribution inside the cells. This finding expands our knowledge of de novo limonene biosynthesis in engineered bacteria and highlights the potential of SRS chemical imaging in metabolic engineering research.
Silencing of ApoE with Divalent siRNAs Drives Activation of Immune Clearance Pathways and Improves Amyloid Pathology in Mouse Models of Alzheimer’s Disease

The most common genetic risk factor for late-onset Alzheimer’s disease (AD) is the APOE4 allele, with evidence for gain- and loss-of-function mechanisms. ApoE knockout in mice abrogates AD phenotypes but causes severe atherosclerosis due to the role of liver ApoE in cholesterol homeostasis. Previous attempts to inhibit brain-specific ApoE with anti-sense oligonucleotides only modestly reduced ApoE expression and had no effect on amyloid burden in adult AD mice. Here, we optimized a divalent small interfering RNA (di-siRNA) to selectively and potently silence ApoE in the brain. Silencing brain ApoE in AD mice significantly reduced amyloid plaque formation without affecting systemic cholesterol levels, confirming that brain and liver APOE pools are spatially and functionally distinct. Mechanistically, APOE appears to be a scaffold for beta-amyloid aggregation that limits clearance by microglia. Di-siRNAs from this study can be taken to pre-clinical and clinical trials to accelerate development of AD-modifying therapies and establish siRNA-based modulation of ApoE as a viable path towards therapeutic development.
Development of allosteric, selective cyclin-dependent kinase 2 (CDK2) inhibitors that are negatively cooperative with cyclin binding and show potential as contraceptive agents

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception.1 However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs.1-2 In this paper, we describe the development and mechanism of action of new type III inhibitors that bind CDK2 with nanomolar affinity, making them the highest affinity, structurally confirmed allosteric CDK inhibitors reported. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as efficacious contraceptive agents is seen by incubation with mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.
Redundancy in Citrate and cis-Aconitate Transport in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a bacterium that infects hospitalized patients and is often highly resistant to antibiotic treatment. It preferentially uses small organic acids called tricarboxylates rather than sugars as a source of carbon for growth. ABSTRACT Tricarboxylates such as citrate are the preferred carbon sources for Pseudomonas aeruginosa, an opportunistic pathogen that causes chronic human infections. However, the membrane transport process for the tricarboxylic acid cycle intermediates citrate and cis-aconitate is poorly characterized. Transport is thought to be controlled by the TctDE two-component system, which mediates transcription of the putative major transporter OpdH. Here, we search for previously unidentified transporters of citrate and cis-aconitate using both protein homology and RNA sequencing approaches. We uncover new transporters and show that OpdH is not the major citrate importer; instead, citrate transport primarily relies on the tripartite TctCBA system, which is encoded in the opdH operon. Deletion of tctA causes a growth lag on citrate and loss of growth on cis-aconitate. Combinatorial deletion of newly discovered transporters can fully block citrate utilization. We then characterize transcriptional control of the opdH operon in tctDE mutants and show that loss of tctD blocks citrate utilization due to an inability to express opdH-tctCBA. However, tctE and tctDE mutants evolve heritable adaptations that restore growth on citrate as the sole carbon source. IMPORTANCE Pseudomonas aeruginosa is a bacterium that infects hospitalized patients and is often highly resistant to antibiotic treatment. It preferentially uses small organic acids called tricarboxylates rather than sugars as a source of carbon for growth. The transport of many of these molecules from outside the cell to the interior occurs through unknown channels. Here, we examined how the tricarboxylates citrate and cis-aconitate are transported in P. aeruginosa. We then sought to understand how production of proteins that permit citrate and cis-aconitate transport is regulated by a signaling system called TctDE. We identified new transporters for these molecules, clarified the function of a known transport system, and directly tied transporter expression to the presence of an intact TctDE system.
Transcription factors regulating the fate and developmental potential of a multipotent progenitor in Caenorhabditis elegans

Multipotent stem and progenitor cells have the capacity to generate a limited array of related cell types. The two C. elegans somatic gonadal precursors (SGPs) are multipotent progenitors that generate all 143 cells of the somatic gonad, including complex tissues and specialized signaling cells. To screen for candidate regulators of cell fate and multipotency, we identified transcription factor genes with higher expression in SGPs than in their differentiated sister, the head mesodermal cell. We used RNA interference or genetic mutants to reduce the function of 183 of these genes and examined the worms at two developmental stages. Eight genes were identified that regulate the SGP fate, including the Ci/GLI homolog tra-1, which is the terminal regulator of sex determination. We show that tra-1 regulates SGP fate independently of its role in sex determination, supporting the idea that tra-1 retains ancestral functions. Four genes were necessary for SGPs to generate the correct number and type of descendant cells. We show that the E2F homolog, efl-3, regulates the cell fate decision between distal tip cells and the sheath/spermathecal precursor. We find that the FACT complex gene hmg-4 is required for the generation of the correct number of SGP descendants, and we define an earlier role for the nhr-25 nuclear hormone receptor-encoding gene, in addition to its previously described role in regulating the asymmetric division of SGPs. Overall, our data show that genes regulating cell fate are largely different from genes regulating developmental potential, demonstrating that these processes are genetically separable.
Bronchus-associated macrophages efficiently capture and present soluble inhaled antigens and are capable of local Th2 cell activation

In allergic asthma, allergen inhalation leads to local Th2 cell activation and peribronchial inflammation. However, the mechanisms for local antigen capture and presentation remain unclear. By two-photon microscopy, we established that soluble antigens in the bronchial airway lumen were efficiently captured and presented by a population of CD11c+ interstitial macrophages with high CX3CR1-GFP and MHC class II expression. We refer to these cells as Bronchus-Associated Macrophages (BAMs) based on their localization underneath the bronchial epithelium. BAMs were enriched in collagen-rich regions near some airway branchpoints, where inhaled antigens are likely to deposit. BAMs engaged in extended interactions with effector Th2 cells and promoted Th2 cytokine production. BAMs were also often in contact with dendritic cells (DCs). After exposure to inflammatory stimuli, DCs migrated to draining lymph nodes, whereas BAMs remained lung resident. We propose that BAMs act as local antigen presenting cells in the lung and also transfer antigen to DCs.
RootSlice – a novel functional-structural model for root anatomical phenotypes

Root anatomy is an important determinant of root metabolic costs, soil exploration, and soil resource capture. Root anatomy varies substantially within and among plant species. RootSlice is a multicellular functional-structural model of root anatomy developed to facilitate the analysis and understanding of root anatomical phenotypes. RootSlice can capture phenotypically accurate root anatomy in three dimensions of different root classes and developmental zones, of both monocotyledonous and dicotyledonous species. Several case studies are presented illustrating the capabilities of the model. For maize nodal roots, the model illustrated the role of vacuole expansion in cell elongation; and confirmed the individual and synergistic role of increasing root cortical aerenchyma and reducing the number of cortical cell files in reducing root metabolic costs. Integration of RootSlice for different root zones as the temporal properties of the nodal roots in the whole-plant and soil model OpenSimRoot/maize enabled the multiscale evaluation of root anatomical phenotypes, highlighting the role of aerenchyma formation in enhancing the utility of cortical cell files for improving plant performance over varying soil nitrogen supply. Such integrative in silico approaches present avenues for exploring the fitness landscape of root anatomical phenotypes. Summary statement Root anatomy remains an underutilized target for crop breeding. RootSlice, a multicellular functional-structural model of root anatomy, simulates the costs and benefits of diverse root anatomical phenotypes to estimate their utility for plant fitness in unfavorable soil environments.
HT-SIP: A semi-automated Stable Isotope Probing pipeline identifies interactions in the hyphosphere of arbuscular mycorrhizal fungi

Background Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive to meet this goal, Stable Isotope Probing—SIP—remains the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, active microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated DNA-SIP pipeline to support well-replicated, temporally-resolved amplicon or metagenomics experiments that enable studies of dynamic microbial communities over space and time. To test this pipeline, we assembled SIP-metagenome assembled genomes (MAGs) from the hyphosphere zone surrounding arbuscular mycorrhizal fungi (AMF), in combination with a 13CO2 plant labelling study. Results Our semi-automated pipeline for DNA fractionation, cleanup, and nucleic acid quantification of SIP density gradients requires six times less hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients and reduced variation compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We then tested this pipeline on samples from a highly-constrained soil microhabitat with significant ecological importance, the AMF fungal hyphosphere. Processing via our quantitative SIP pipeline confirmed the AMF Rhizophagus intraradices and its associated microbiome were highly 13C enriched, even though the soils’ overall enrichment was only 1.8 atom% 13C. We assembled 212 13C-enriched hyphosphere MAGs, and the hyphosphere taxa that assimilated the most AMF-derived 13C (range 10-33 atom%) were from the phlya Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia oxidizing archaeon genus Nitrososphaeara. Conclusions Our semi-automated SIP approach decreases operator time and errors and improves reproducibility by targeting the most labor-intensive steps of SIP—fraction collection and cleanup. Here, we illustrate this approach in a unique and understudied soil microhabitat—generating MAGs of active microbes living in the AMF hyphosphere (without plant roots). Their phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.
A modular dCas9-based recruitment platform for combinatorial epigenome editing

CRISPR-dCas9 based targeted epigenome editing tools allow precise manipulation and functional investigation of various genome modifications. However, these tools often display substantial context dependency, with highly variable efficacy between target genes and cell types, potentially due to underlying variation in the chromatin modifications present. While simultaneous recruitment of multiple distinct ‘effector’ chromatin regulators has improved efficacy, these systems typically lack control over which effectors bind and their spatial organisation. To overcome this we have created a new modular combinatorial epigenome editing platform, called SSSavi. This system acts as an interchangeable and reconfigurable docking platform fused to dCas9 to enable simultaneous recruitment of up to four different effectors, allowing precise control and reconfiguration of the effector composition and spatial ordering of their binding. We demonstrate the activity and specificity of the SSSavi system and compare it to existing multi-effector targeting systems, establishing its efficacy. Furthermore, by altering the spatial ordering of effector recruitment, across multiple target genes and cell lines, we demonstrate the importance of effector recruitment order for effective transcriptional regulation. Together, this system offers the capacity to explore effector co-recruitment to specific loci to potentially enhance the manipulation of chromatin contexts previously resistant to targeted epigenomic editing.
MINI-EX: Integrative inference of single-cell gene regulatory networks in plants

Multicellular organisms, such as plants, are characterized by highly specialized and tightly regulated cell populations, establishing specific morphological structures and executing distinct functions. Gene regulatory networks (GRNs) describe condition-specific interactions of transcription factor (TF) regulating the expression of target genes, underpinning these specific functions. As efficient and validated methods to identify cell-type specific GRNs from single-cell data in plants are lacking, limiting our understanding of the organization of specific cell-types in both model species and crops, we developed MINI-EX (Motif-Informed Network Inference based on single-cell EXpression data), an integrative approach to infer cell-type specific networks in plants. MINI-EX uses single-cell transcriptomic data to define expression-based networks and integrates TF motif information to filter the inferred regulons, resulting in networks with increased accuracy. Next, regulons are assigned to different cell-types, leveraging cell-specific expression, and candidate regulators are prioritized using network centrality measures, functional annotations, and expression specificity. This embedded prioritization strategy offers a unique and efficient means to unravel signaling cascades in specific cell-types controlling a biological process of interest. We demonstrate MINI-EX’s stability towards input data sets with low number of cells and its robustness towards missing data, and we show it infers state-of-the-art networks with a better performance compared to related single-cell network tools. MINI-EX successfully identifies key regulators controlling root development in Arabidopsis and rice, and governing ear development in maize, enhancing our understanding of cell-type specific regulation and unraveling the role of different regulators controlling the development of specific cell-types in plants.
Enrichment of breast cancer stem cells following cytotoxic chemotherapy is mediated by hypoxia-inducible factors

Breast cancers (BCs) that do not express the estrogen or progesterone receptor or human epidermal growth factor receptor 2 are known as triple negative breast cancers (TNBCs). Women with TNBC receive non-targeted chemotherapy with a durable response rate of less than 20%. BC stem cells (BCSCs) are a small subpopulation of BC cells that are characterized by the capacity for infinite self-renewal; are the only BC cells capable of forming a secondary (recurrent or metastatic) BC; and must be eliminated in order to eradicate BC. Hypoxia-inducible factors (HIFs) activate hundreds of genes in TNBCs and HIF-1α expression in the diagnostic tumor biopsy is associated with patient mortality. In this paper, we report that treatment of TNBC cells with cytotoxic chemotherapy increased HIF-1α and HIF-2α protein levels and HIF target gene expression. Chemotherapy also increased the percentage of BCSCs through pathways involving interleukin-6 (IL-6), IL-8, and multidrug resistance 1. HIF inhibitors blocked increased BCSC specification in response to cytotoxic chemotherapy and combination therapy led to tumor eradication. Increased HIF target gene expression in BC biopsies was correlated with increased mortality, especially in those patients treated with chemotherapy alone. Our results suggest that HIF-dependent BCSC enrichment provides a molecular and cellular basis for the high incidence of relapse in women with TNBC.
Impaired neuromodulator crosstalk delays vigilance-dependent astroglia Ca2+ activation in mouse models of Alzheimer’s disease

Degeneration in neuronal nuclei producing the neuromodulators acetylcholine and norepinephrine is a hallmark of Alzheimer’s disease (AD). Therapeutic interventions that increase acetylcholine in brain ameliorate AD symptoms in human patients, and augmenting norepinephrine restores cognitive function in mouse models of AD as well as Down Syndrome, the most frequent cause of early onset AD. A prominent cellular target of noradrenergic and potentially cholinergic signaling during states of heightened vigilance are astroglia and recent studies indicate that astroglia Ca2+ dynamics in awake mice contribute to optimal cognitive performance. Here we tested the hypothesis that vigilance-dependent Ca2+ signaling in mouse primary visual cortex astrocytes is altered in mouse models of AD and provide mechanistic insight into upstream neuromodulator signaling that shapes astrocyte Ca2+ dynamics in healthy and AD conditions. In two mouse models of AD (APPswe/PSEN1dE9 and AppNL-F KI), we consistently observed delayed and less coordinated astrocyte Ca2+ elevations in response to locomotion, a well-controlled behavioral paradigm triggering widespread Ca2+ activation in astroglia throughout the brain. Combining pharmacological and genetic manipulations, we found that noradrenergic signaling to astrocytes was facilitated by cholinergic signaling, but this neuromodulator crosstalk was impaired in AppNL-F mice. Pharmacological facilitation of norepinephrine release rescued delayed and less coordinated astrocyte Ca2+ activation in AppNL-F mice and suggests that astrocytes preserve a functional reserve that can be recruited even during late-stage disease. Our findings of delayed and less coordinated astroglia Ca2+ activation predict impaired noradrenergic signaling and may contribute to the cognitive decline in AD.
A robustness analysis of Deep Q Networks

Deep Q Networks are a type of deep reinforcement learning algorithm that have been shown to be particularly adept at learning a variety of tasks with minimal priors. Specifically, DQN agents have been shown to learn a variety of Atari 2600 video games using only raw images of the game screen and the game score. To leverage DQNs in real world robotics applications, we must first understand how robust these networks are to the perceptual noise common to all robotics domains. In this pa- per, we present an analysis of the robustness of Deep Q Networks to various types of perceptual noise (changing brightness, Gaussian blur, salt and pepper, distractors). We present a benchmark example that involves playing the game Breakout though a webcam and screen environment, like humans do. We present a simple training approach to improve the performance maintained when transferring a DQN agent trained in simulation to the real world (36% vs. 1% maintained performance - see Table 1). We also evaluate DQN agents trained under a variety of simulation environments to report for the first time how DQNs cope with perceptual noise, common to real world robotic applications.