Miracle-dz/UniFAR-FTUs huggingface dataset:
Dissection of prostate tumour, stroma and immune transcriptional components reveals a key contribution of the microenvironment for disease progression

Prostate cancer is caused by genomic aberrations in normal epithelial cells, however clinical translation of findings from analyses of cancer cells alone has been very limited. A deeper understanding of the tumour microenvironment is needed to identify the key drivers of disease progression and reveal novel therapeutic opportunities. In this study, the experimental enrichment of selected cell-types and the development of a Bayesian inference model for continuous differential transcript abundance permitted us to define the transcriptional landscape of the prostate cancer microenvironment along the disease progression axis. An important role of monocytes and macrophages in prostate cancer progression and disease recurrence was uncovered, supported by both transcriptional landscape findings and by differential tissue composition analyses. These findings were corroborated and validated by spatial analyses at the single-cell level using multiplex immunohistochemistry. This study advances our knowledge concerning the role of monocyte-derived recruitment in primary prostate cancer, and supports their key role in disease progression, patient survival and prostate microenvironment immune modulation.
Evidence for non-methanogenic metabolisms in globally distributed archaeal clades basal to the Methanomassiliicoccales

Recent discoveries of mcr and mcr-like complexes in genomes from diverse archaeal lineages suggest that methane (and more broadly alkane) metabolism is an ancient pathway with complicated evolutionary histories. The conventional view is that methanogenesis is an ancestral metabolism of the archaeal class Thermoplasmata. Through comparative genomic analysis of 12 Thermoplasmata metagenome-assembled genomes (MAGs), we show that these microorganisms do not encode the genes required for methanogenesis, which suggests that this metabolism may have been laterally acquired by an ancestor of the order Methanomassiliicoccales. These MAGs include representatives from four orders basal to the Methanomassiliicoccales, including a high-quality MAG (95% complete) that likely represents a new order, Ca. Lunaplasma lacustris ord. nov. sp. nov. These MAGs are predicted to use diverse energy conservation pathways, such as heterotrophy, sulfur and hydrogen metabolism, denitrification, and fermentation. Two of these lineages are globally widespread among anoxic, sedimentary environments, with the exception of Ca. Lunaplasma lacustris, which has thus far only been detected in alpine caves and subarctic lake sediments. These findings advance our understanding of the metabolic potential, ecology, and global distribution of the Thermoplasmata and provide new insights into the evolutionary history of methanogenesis within the Thermoplasmata.
Interferon stimulated genes mark pathogenic cells of the myelopoietic response to sterile tissue injury in mice and humans

Sterile tissue injury recruits myeloid cells (neutrophils and monocytes) from the bone marrow by stimulating a process known as emergency myelopoiesis. It is commonly assumed that myeloid cells activate proinflammatory signaling pathways within the injured tissue after sensing innate immune-activating molecules in the local microenvironment. Here, we show that pathogenic innate immune signaling in myeloid cells begins far from the site of injury, at their origins in the bone marrow. Using myocardial infarction (MI) as a model of sterile injury, we profiled single cell transcriptomes of >50,000 myeloid cells and found evidence of type I interferon signaling characterized by expression of interferon stimulated genes (ISGs) in a subset of neutrophils and monocytes in the heart, blood, and bone marrow after MI. We validated the findings in humans by quantifying ISG-expression in myeloid cells of patients presenting with acute MI, which revealed ISG levels far exceed that of healthy subjects and are comparable to patients with the interferon-associated autoimmune disease, systemic lupus erythematous. The identification of remote innate immune education raises important questions about how distant tissue injury activates interferon signaling in the hematopoietic compartment. Clinically, our data provide a strategy for quantifying pathogenic subsets of emergency myelopoietic cells in the blood as a biomarker that may identify patients who have disproportionately worse outcomes or who may benefit from immune modulatory therapy after acute MI.
Integrating Multiplex SiMPull and Computational Modeling to Evaluate Combinatorial Aspects of EGFR Signaling

The Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1) plays an important role in both physiological and cancer-related processes. To study the factors that influence receptor phosphorylation, we have coupled Single Molecule Pull-down (SiMPull) measurements with rule-based modeling of EGFR signaling. Using SiMPull, we quantified the phosphorylation state of thousands of individual receptors. These measurements enabled the first direct detection of multisite phosphorylation on full-length EGFR and revealed that the extent of phosphorylation varies by tyrosine site and is dependent on the relative abundance of signaling partners that limit access by tyrosine phosphatases. We also evaluated the impact of oncogenic mutations and ligands with varying affinity on phosphorylation kinetics. Simulations highlight the importance of dimer lifetimes on EGFR phosphorylation and signaling output.
Targeted genome editing in Nicotiana tabacum using inducible CRISPR/Cas9 system

Targeted genome editing has been achieved in multiple plant species using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, in which the Cas9 gene is usually driven by constitutive promoters. However, constitutive expression of Cas9 is not necessary and can be harmful to plant development. In this study, we developed an estrogen-inducible CRISPR/Cas9 system by taking advantage of the chimeric transcription activator XVE and tested the efficacy of this inducible system in Nicotiana tabacum by targeting the phytoene desaturase (NtPDS) gene, whose mutation resulted in albino phenotypes. Treatment of four independent transgenic lines with exogenous estradiol successfully induced targeted mutagenesis in NtPDS. Sanger sequencing assay uncovered the presence of indel mutations (nucleotides insertions or deletions) at the target site as expected, and at least two types of mutations were identified for each line. Transgenic plants with mutated NtPDS gene after estradiol treatment exhibited pale green or incomplete albino leaves. Moreover, the expression of Cas9 in transgenic plants was strongly induced by estradiol treatment. Our results demonstrate the efficacy of XVE-based CRISPR/Cas9 system in N. tabacum, and the system reported here promises to be a useful approach for conditional genome editing, which would facilitate the study of genes of interest, especially those developmentally important genes.
Convolutional neural network, personalised, closed-loop Brain-Computer Interfaces for multi-way control mode switching in real-time

Exoskeletons and robotic devices are for many motor disabled people the only way to interact with their envi-ronment. Our lab previously developed a gaze guided assistive robotic system for grasping. It is well known that the same natural task can require different interactions described by different dynamical systems that would require different robotic controllers and their selection by the user in a self paced way. Therefore, we investigated different ways to achieve transitions between multiple states, finding that eye blinks were the most reliable to transition from ‘off’ to ‘control’ modes (binary classification) compared to voice and electromyography. In this paper be expanded on this work by investigating brain signals as sources for control mode switching. We developed a Brain Computer Interface (BCI) that allows users to switch between four control modes in self paced way in real time. Since the system is devised to be used in domestic environments in a user friendly way, we selected non-invasive electroencephalographic (EEG) signals and convolutional neural networks (ConvNets), known by their capability to find the optimal features for a classification task, which we hypothesised would add flexibility to the system in terms of which mental activities the user could perform to control it. We tested our system using the Cybathlon BrainRunners computer game, which represents all the challenges inherent to real time control. Our preliminary results show that an efficient architecture (SmallNet) composed by a convolutional layer, a fully connected layer and a sigmoid classification layer, is able to classify 4 mental activities that the user chose to perform. For his preferred mental activities, we run and validated the system online and retrained the system using online collected EEG data. We achieved 47, 6% accuracy in online operation in the 4-way classification task. In particular we found that models trained with online collected data predicted better the behaviour of the system in real time suggesting, as a side note, that similar (ConvNets based) offline classifying methods present in literature might find a decay in performance when applied online. To the best of our knowledge this is the first time such an architecture is tested in an online operation task. While compared to our previous method relying on blinks with this one we reduced in less than half (1.6 times) the accuracy but increased by 2 the amount of states among which we can transit, bringing the opportunity for finer control of specific subtasks composing natural grasping in a self paced way.
How to navigate the myosin-V motor through the actin network

Myosin-V (MyoV) is a ubiquitous motor protein that transports an astonishingly diverse set of cargos on the actin network in eukaryotes. Phosphorylation-dependent processes often regulate MyoV-mediated cargo transport, molecular details of which remain largely unknown. We previously showed that phosphorylation regulates MyoV’s switching from microtubules onto actin filaments, not its motor activity. Regulation of switching at reconstituted microtubule-actin-crossings in fact sufficed to recapitulate the MyoVa-driven redistribution of pigment-organelles in amphibian melanophores. However, in those cells, MyoVa also encounter many actin-actin crossings. Here, we show that isolated MyoVa motors switch with equal probabilities at reconstituted actin-actin-crossings. Under the control of its adaptor-protein melanophilin (Mlph), however, the motor differentiates between the actin filaments at crossing points in a phosphorylation-regulated manner. Whereas phosphorylation of Mlph forced about ∼2/3 of MyoVa to ignore the intersections, dephosphorylation completely reversed this behavior and forced ∼2/3 to switch. We show that the filament-binding domain (FBD) of Mlph controls this switching behavior. This property evolved in amphibians, but not in the early vertebrate zebrafish. By protein engineering, we demonstrate that changes of a few residues are sufficient to impart actin-binding capability onto the zebrafish Mlph. We thus unmask the molecular beginnings of dual filament binding in Mlph that allow it to control the switching behavior of MyoVa at cytoskeletal crossings. We therefore propose a direct link between intracellular phosphorylation activity and the adaptor-protein, not to regulate MyoVa activity, but to navigate the motor through the entire cytoskeletal maze for correct positioning of cargo. Significance statement In virtually all eukaryotic cells, numerous myosin motors have to navigate through an elaborate actin network for timely transport of intracellular cargo. Here, we unmask an unintuitive regulation of the myosin-Va motor that is involved in pigment organelle transport. We demonstrate that myosin-Va differentiates between the same actin filaments and displays regulated switching at reconstituted actin-actin crossings, an unexpected behavior that has been predicted from previous theoretical work. We trace this regulation back to the adaptor protein of the myosin-Va motor and show that this regulation was present in amphibian but had not evolved in the early vertebrate zebrafish. Notably, we demonstrate that the evolution of actin-binding capability is achieved by changing a few residues in the adaptor protein.
Effect of Interglomerular Inhibitory Networks on Olfactory Bulb Odor Representations

Lateral inhibition is a fundamental feature of circuits that process sensory information. In the mammalian olfactory system, inhibitory interneurons called short axon cells (SACs) comprise the first network mediating lateral inhibition between glomeruli, the functional units of early olfactory coding and processing. The connectivity of this network and its impact on odor representations is not well understood. To explore this question, we constructed a computational model of the interglomerular inhibitory network using detailed characterizations of SAC morphologies taken from mouse olfactory bulb (OB). We then examined how this network transformed glomerular patterns of odorant-evoked sensory input (taken from previously-published datasets) as a function of the selectivity of interglomerular inhibition. We examined three connectivity schemes: selective (each glomerulus connects to few others with heterogeneous strength), nonselective (glomeruli connect to most others with heterogenous strength), or global (glomeruli connect to all others with equal strength). We found that both selective and nonselective interglomerular networks could mediate heterogeneous patterns of inhibition across glomeruli when driven by realistic sensory input patterns, but that global inhibitory networks were unable to produce input-output transformations that matched experimental data and were poor mediators of intensity-dependent gain control. We further found that networks whose interglomerular connectivities were tuned by sensory input profile decorrelated odor representations moreeffectively. These results suggest that, despite their multiglomerular innervation patterns, SACs are capable of mediating odorant-specific patterns of inhibition between glomeruli that could, theoretically, be tuned by experience or evolution to optimize discrimination of particular odorants. SIGNIFICANCE STATEMENT Lateral inhibition is a key feature of circuitry in many sensory systems including vision, audition, and olfaction. We investigate how lateral inhibitory networks mediated by short axon cells (SACs) in the mouse olfactory bulb (OB) might shape odor representations as a function of their interglomerular connectivity. Using a computational model of interglomerular connectivity derived from experimental data, we find that SAC networks, despite their broad innervation patterns, can mediate heterogeneous patterns of inhibition across glomeruli, and that the canonical model of global inhibition does not generate experimentally observed responses to stimuli. In addition, inhibitory connections tuned by input statistics yield enhanced decorrelation of similar input patterns. These results elucidate how the organization of inhibition between neural elements may affect computations.
Platelet counts predict prognosis in IPF, but are not the main source of pulmonary TGFβ1

Transforming growth factor-β1 (TGFβ1) is the key pro-fibrotic cytokine implicated in the interstitial lung diseases (ILD), including idiopathic pulmonary fibrosis (IPF), but the primary source of TGFβ1 in these diseases is unknown. Platelets have abundant TGFβ1 stores, however their role in IPF is ill-defined. We sought to investigate whether platelets or platelet-derived TGFβ1 mediate IPF disease progression. ILD/IPF and non-ILD patients were recruited to determine platelet reactivity and followed for mortality. To study whether platelet-derived TGFβ1 modulates pulmonary fibrosis, mice with a targeted deletion of TGFβ1 in megakaryocytes and platelets (TGFβ1fl/fl.PF4-Cre) were used in the bleomycin-induced PF model. We found a significantly higher mortality in IPF patients with elevated platelet counts, along with significantly increased platelets, neutrophils, TGFβ1 and CCL5 in the lung and bronchoalveolar lavage (BAL) of ILD patients. Despite platelets being readily detected within the lungs of bleomycin-treated mice, neither the degree of pulmonary inflammation or fibrosis were significantly different between TGFβ1fl/fl.PF4-Cre and control mice. Our results demonstrate for the first-time that platelet-derived TGFβ1 is redundant in driving pulmonary fibrosis in an animal model. However, platelets can predict mortality in IPF implicating other platelet-derived mediators, such as CCL5, in promoting human IPF disease.
Harnessing Expressed Single Nucleotide Variation and Single Cell RNA Sequencing to Define Immune Cell Chimerism in the Rejecting Kidney Transplant

In solid organ transplantation, donor derived immune cells are assumed to decline with time after surgery. Whether donor leukocytes persist within kidney transplants or play any role in rejection is unknown, however, in part because of limited techniques for distinguishing recipient and donor cells. To address this question, we performed paired whole exome sequencing of donor and recipient DNA and single cell RNA sequencing (scRNA-seq) of 5 human kidney transplant biopsy cores. Exome sequences were used to define single nucleotide variations (SNV) across all samples. By analyzing expressed SNVs in the scRNA-seq dataset we could define recipient vs. donor cell origin for all 81,139 cells. The leukocyte donor to recipient ratio varied with rejection status for macrophages and with time post-transplant for lymphocytes. Recipient macrophages were characterized by inflammatory activation and donor macrophages by antigen presentation and complement signaling. Recipient origin T cells expressed cytotoxic and pro-inflammatory genes consistent with an effector cell phenotype whereas donor origin T cells are likely quiescent expressing oxidative phosphorylation genes relative to recipient T cells. Finally, both donor and recipient T cell clones were present within the rejecting kidney, suggesting lymphoid aggregation. Our results indicate that donor origin macrophages and T cells have distinct transcriptional profiles compared to their recipient counterparts and donor macrophages can persist for years post transplantation. This study demonstrates the power of this approach to accurately define leukocyte chimerism in a complex tissue such as the kidney transplant coupled with the ability to examine transcriptional profiles at single cell resolution.
Marine snow as a habitat for microbial mercury methylators in the Baltic Sea

Methylmercury (MeHg), a neurotoxic compound biomagnifying in aquatic food webs, can be a threat to human health via fish consumption. However, the composition and distribution of the microbial communities mediating the methylation of mercury (Hg) to MeHg in marine systems remain largely unknown. In order to fill this gap of knowledge, we used the Baltic Sea Reference Metagenome (BARM) dataset to study the distribution of the genes involved in Hg methylation (the hgcAB gene cluster). We determined the relative abundance of the hgcAB genes and their taxonomic identity in 81 brackish metagenomes that cover spatial, seasonal and redox variability in the Baltic Sea water. The hgcAB genes were predominantly detected in anoxic water, but some hgcAB genes were also detected in hypoxic and normoxic waters. Higher relative quantities of hgcAB genes were found in metagenomes from marine snow compared to free-living communities in anoxic water, suggesting that marine snow are hotspot habitats for Hg methylators in oxygen-depleted seawater. Phylogenetic analysis identified well-characterized Hg methylators such as Deltaproteobacteria in oxygen-depleted water, but also uncovered Hg methylators within the Spirochaetes and Lentisphaerae phyla. Altogether, our work unveils the diversity of the microorganisms mediating MeHg production in the Baltic Sea and pinpoint the ecological niches of these microorganisms within the marine water column.
P4HA2 is associated with prognosis, promotes proliferation, invasion, migration and EMT in glioma

Prolyl-4-hydroxylase subunit 2 (P4HA2), as a member of collagen modification enzymes, is induced under hypoxic conditions with essential roles in the collagen maturation, deposition as well as the remodeling of extracellular matrix(ECM). Mounting evidence has suggested that deregulation of P4HA2 is common in cancer. However, the expression pattern and molecular mechanisms of P4HA2 in glioma remain unknown. Here, we demonstrate that P4HA2 is overexpressed in glioma and inversely correlates with patient survival. Knockdown of P4HA2 inhibits proliferation, migration, invasion, and epithelial-to-mesenchymal transition (EMT)-like phenotype of glioma cells in vitro and suppressed tumor xenograft growth in vivo. Mechanistically, bioinformatics analysis shows that ECM-receptor interaction and PI3K/AKT pathway are the most enriched pathways of the co-expressed genes with P4HA2. Furthermore, P4HA2 mRNA was positively correlated with mRNA expressions of a series of collagen genes, but not mRNA of PI3K or AKT1/2. Conversely, both the protein expressions of collagens and phosphorylated PI3K/AKT could be downregulated either by silencing of P4HA2 expression or inhibition of its prolyl hydroxylase. Moreover, the inhibitory effects on the migration, invasion and the EMT-related molecules by P4HA2 knockdown can be recapitulated by the Akt phosphorylation activator. Taken together, our findings for the first time reveal an oncogenic role of P4HA2 in the glioma malignancy. By regulating the expression of fibrillar collagens and the downstream PI3K/AKT signaling pathway, it may serve as a potential anti-cancer target for the treatment of glioma. Highlights P4HA2 is overexpressed and correlated with poor prognosis in glioma. P4HA2 depletion inhibits glioma proliferation, migration, invasion and EMT-like phenotype in vitro and tumorigenesis in vivo. P4HA2 depletion attenuates the PI3K/AKT signaling pathway in a collagen-dependent manner.
Efficacy of Tulathramycin in the Treatment of Respiratory Pasteurollosis in Rabbits

| Pasteurella multocida is the most critical respiratory bacterial infection of rabbits, around the globe. Beside passive immunization, anti-bacterial treatment is the primary choice of disease control. Since tulathromycin may be retained in the lung for many days after single administration, it can be applied for treatment of respiratory diseases. Hence, the purpose of current study was to evaluate the efficacy of tulathromycin in treating experimentally infected rabbits with P. multocida. In the sensitivity test, tulathromycin showed a potent inhibitory effect on P. multocida compared to several well-established antimicrobial agents. In vivo, treatment with tulathromycin improved clinical signs, mortality rate, lesion scores and growth performance parameters in infected rabbits. Furthermore, treatment with tulathromycin ameliorated the hematological picture, lowered the level of biochemical parameters which were significantly increased due to infection as liver enzymes, blood urea, creatinine and creatine kinase (CK-MB) and elevated the levels of total protein and albumin. Biochemical findings were supported by histopathological picture. It can be concluded that tulathromycin is an ideal and safe antibiotic for treating infection with Pasteurella multocida in rabbits.