Miracle-dz/UniFAR-FTUs huggingface dataset:
Secondary metabolites of Bacillus subtilis impact soil-derived semi-synthetic bacterial community assembly

Secondary metabolites provide Bacillus subtilis with increased competitiveness towards other microorganisms. In particular nonribosomal peptides (NRPs) have an enormous antimicrobial potential by causing cell lysis, perforation of fungal membranes, enzyme inhibition or disruption of bacterial protein synthesis. This knowledge was primarily acquired in vitro when B. subtilis was competing with other microbial monocultures. However, our understanding of the true ecological role of these small molecules is limited. In this study, we have established soil-derived semi-synthetic mock communities containing 13 main genera and supplemented them with B. subtilis P5_B1 WT, its NRP deficient strain sfp or single NRP mutants incapable of producing surfactin, plipastatin or bacillaene. 16S amplicon sequencing revealed that the invasion of NRP-producing B. subtilis strains had no major impact on the bacterial communities. Still, the abundances of the two genera Lysinibacillus and Viridibacillus were reduced. Interestingly, this effect was diminished in communities supplemented with the NRPs deficient strain. Growth profiling of Lysinibacillus fusiformis M5 exposed to either spent media of the B. subtilis strains or pure surfactin indicates the sensitivity of this strain towards the biosurfactant surfactin. Our study provides a more in-depth insight into the influence of B. subtilis NRPs on semi-synthetic bacterial communities and helps to understand their ecological role.
Structural basis for CDK7 activation by MAT1 and Cyclin H

Significance The fundamental processes of cell-cycle regulation and transcription are linked by the heterotrimeric CDK-activating kinase (CAK) complex. We solved the crystal structure of the active CAK complex and provide a molecular rationale for CAK activation, regulation, and substrate recognition. Our data thus highly advance our understanding of this essential factor which is also a proven target for cancer therapy. Cyclin-dependent kinase 7 (CDK7), Cyclin H, and the RING-finger protein MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex which is vital for transcription and cell-cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH the trimeric complex phosphorylates the T-loop of CDKs that control cell-cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on two proteins for activation. We solved the structure of the CAK complex from the model organism Chaetomium thermophilum at 2.6-Å resolution. Our structure reveals an intricate network of interactions between CDK7 and its two binding partners MAT1 and Cyclin H, providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements and functional mutagenesis show that CDK7 activation can occur independent of T-loop phosphorylation and is thus exclusively MAT1-dependent by positioning the CDK7 T-loop in its active conformation.
Increased and synchronous recruitment of release sites underlies hippocampal mossy fiber presynaptic potentiation

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of long-term potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed two-photon imaging of the genetically encoded glutamate sensor iGluu that revealed an increase in the surface area used for glutamate release at potentiated terminals. Moreover, time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between immunofluorescence signals from calcium channels and release sites. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements likely enable long-term increase in synaptic strength.
Tissue-specific transcription footprinting using RNA PoI DamID (RAPID) in C. elegans

Differential gene expression across cell types underlies the development and cell physiology in multicellular organisms. C. elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates, however, to the difficulty to obtain comprehensive tissue-specific gene transcription data, since available methods are still challenging to execute and/or require large worm populations. Here, we introduce the RNA Pol DamID (RAPID) approach, in which the Dam methyltransferase is fused to an ubiquitous RNA polymerase subunit in order to create transcriptional footprints via methyl marks on the DNA of transcribed genes. To validate the method, we determined the polymerase footprints in whole animals, sorted embryonic blastomeres and in different tissues from intact young adults by driving Dam fusion expression tissue-specifically. We obtained meaningful transcriptional footprints in line with RNA-seq studies in whole animals or specific tissues. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX neuroendocrine cells, representing 0.2% of the somatic cell content of the animals. We identified 2362 candidate genes with putatively active transcription in XXX cells, among which the few known markers for these cells. Using transcriptional reporters for a subset of new hits, we confirmed their XXX expression for a majority of them and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging. Article summary Gene expression is a major determinant of cell fate and physiology, yet it is notoriously difficult to characterize for the widely used model system C. elegans. By using a new method based on in vivo covalent modification of DNA by RNA polymerases, we determine genome-wide transcription patterns in single tissues of embryos or young adult animals. We show that the method is able to identify actively transcribed genes in tissues representing down to 0.2% of the somatic cells in adult animals. Additionally, this method can be fully performed in a single laboratory by using third generation sequencing methods (ONT).
Primary visual cortex injury produces loss of inhibitory neurons and long-term visual circuit dysfunction

Primary sensory areas of the mammalian neocortex have a remarkable degree of plasticity, allowing neural circuits to adapt to dynamic environments. However, little is known about the effect of traumatic brain injury on visual system function. Here we applied a mild focal contusion injury to primary visual cortex (V1) in adult mice. We found that, although V1 was largely intact in brain-injured mice, there was a reduction in the number of inhibitory interneurons that extended into deep cortical layers. In general, we found a preferential reduction of interneurons located in superficial layers, near the impact site, while interneurons positioned in deeper layers were better preserved. Three months after injury, V1 neurons showed dramatically reduced responses to visual stimuli and weaker orientation selectivity and tuning, consistent with the loss of cortical inhibition. Our results demonstrate that V1 neurons no longer robustly and stably encode visual input following a mild traumatic injury. Highlights Inhibitory neurons are lost throughout brain injured visual cortex Visually-evoked potentials are severely degraded after injury Injured V1 neurons show weaker selectivity and tuning consistent with reduced interneurons
Simultaneous transcriptome and methylome profiles of single mouse oocytes provide novel insights on maturation and aging

Background Advanced maternal aging has become a worldwide public health issue that contributes to female fertility decline and significant risk to embryo development. Despite transcriptional and epigenetic alterations reported in oocyte maturation and development, the dynamics of gene expression and DNA dynamics associated with aging remain elusive. Here we generated simultaneous transcriptome and methylome profiles of mouse oocytes during aging and maturation at single-cell and single-base resolution to examine key biological processes and identify the key targets for novel treatment options. Results We report the dynamics in transcriptome and DNA methylome in mouse oocytes during maternal aging and oocyte maturation. Age-associated gene expression changes showed mitochondrial dysfunction in GV oocytes and defects of chromosome segregation and spindle assembly in MII oocytes. EIF2 signaling protein synthesis pathway was also impaired during aged oocyte maturation. Moreover, distinctive DNA methylation patterns were demonstrated during maternal aging in GV and MII oocytes. A positive correlation between gene expression and methylation in gene body was characterized. Furthermore, we identified several promising biomarkers, including IL-7, to assess oocyte quality, which are potential therapeutic targets for improve oocyte maturation. More importantly, we built the first mouse oocyte maturation and age prediction model using transcriptome data and validated its feasibility in published data. Conclusions This work provides a better understanding of molecular and cellular mechanisms during mouse oocyte aging, points a new direction of oocyte quality assessment, and paves the way for developing novel treatments to improve oocyte maturation and quality in the future.
Optogenetic activation of heterotrimeric Gi proteins by LOV2GIVe— a rationally engineered modular protein

Heterotrimeric G-proteins are signal transducers that mediate the action of many natural extracellular stimuli as well as of many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we engineered LOV2GIVe, a modular protein that allows the activation of Gi proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, i.e. metazoan opsins, which are light-activated GPCRs. To make LOV2GIVe, we fused a peptide derived from a non-GPCR protein that activates Gαi (but not Gαs, Gαq, or Gα12) to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity. GRAPHICAL SUMMARY
Functional validation of a finding from a mouse genome-wide association study demonstrates that a mutant allele of Azi2 alters sensitivity to methamphetamine

Methamphetamine is a widely abused psychostimulant. In a previous genome-wide association study (GWAS), we identified a locus that influenced the stimulant response to methamphetamine. That locus was also an eQTL for the gene Azi2. Based on those findings, we hypothesized that heritable differences in the expression of Azi2 were causally related to the differential response to methamphetamine. In this study, we created a mutant Azi2 allele that caused lower Azi2 expression and enhanced the locomotor response to methamphetamine; however, based on the GWAS findings, we had expected lower Azi2 to decrease rather than increase the stimulant response to methamphetamine. We then sought to explore the mechanism by which Azi2 influenced methamphetamine sensitivity. A recent publication had reported that the 3’UTR of Azi2 mRNA downregulates the expression of Slc6a3, which encodes the dopamine transporter (DAT), which is a key target of methamphetamine. We evaluated the relationship between Azi2/Azi2 3’UTR and Slc6a3 expression in the VTA in the mutant Azi2 mice and in a new cohort of CFW mice. We did not observe any correlation between Azi2 and Slc6a3 in the VTA in either cohort. However, RNA sequencing confirmed that the Azi2 mutation altered Azi2 expression and also revealed a number of potentially important genes and pathways that were regulated by Azi2, including the metabotropic glutamate receptor group III pathway and nicotinic acetylcholine receptor signaling pathway. Our results support a role for Azi2 in methamphetamine sensitivity; however, the exact mechanism does not appear to involve regulation of Slc6a3 and thus remains unknown.
Exposing distinct subcortical components of the auditory brainstem response evoked by continuous naturalistic speech

The auditory brainstem is important for processing speech, yet we have much to learn regarding the contributions of different subcortical structures. These deep neural generators respond quickly, making them difficult to study during dynamic, ongoing speech. Recently developed techniques have paved the way to use natural speech stimuli, but the auditory brainstem responses (ABRs) they provide are temporally broad and thus have ambiguous neural sources. Here we describe a new method that uses re-synthesized “peaky” speech stimuli and deconvolution analysis of EEG data to measure canonical ABRs to continuous naturalistic speech of male and female narrators. We show that in adults with normal hearing, peaky speech quickly yields robust ABRs that can be used to investigate speech processing at distinct subcortical structures from auditory nerve to rostral brainstem. We further demonstrate the versatility of peaky speech by simultaneously measuring bilateral and ear-specific responses across different frequency bands. Thus, the peaky speech method holds promise as a powerful tool for investigating speech processing and for clinical applications.
Evaluating an e-nose ability to detect biogas plant efficiency: a case study

Abstract The demand for online monitoring and control of biogas process is increasing, since better monitoring and control system can improve process plants stability and economy. A number of parameters in both the liquid and the gas phase have been suggested as process indicators (pH, alkalinity, VFA and H2, redox potential, biogas production rate, biogas composition, FOS/TAC ratio, COD and/or VS reduction). The present study proposes the use of complex sensors as a possible solution to engineer a consistent control system. Tests were performed to analyze the biogas coming from a biogas plant in which conditions of pre-overloading and overloading were artificially inducted. The different inducted functioning conditions were recognised using a FOS/TAC tester, or the ratio volatile organic acids/alkaline buffer capacity. The FOS/TAC ratio has long been recognized extending as far as the imminent inversion of the digester biology to be detected at an early stage. Data coming from the e-nose were sorted and classified according to FOS/TAC ratio as a reference method. Not all the sensors of the e-nose were reactive to manure digestates, but four of them, sensitive to aromatic compounds, ammonia alkanes and methane, resulted crucial in the samples identification. Results confirmed that the e-nose can discriminate different digestion conditions, demonstrating the possibility to reduce the number of sensors in this innovative tool for biogas control systems. However, this instrument cannot be considered a complete alternative to traditional analysis systems, as, for example, the FOS/TAC titration, but a supporting tool for a quick analysis of the system.
Estimating inbreeding and its effects in a long-term study of snapdragons (Antirrhinum majus)

Inbreeding depression can be estimated by correlating heterozygosity with fitness components, but such heterozygosity-fitness correlations are typically weak. For over ten years, we studied a population of the self-incompatible plant, Antirrhinum majus, measuring heterozygosity and fitness proxies from 22,353 plants. Using a panel of 91 SNPs, we find that relatedness declines rapidly over short spatial scales. Individual heterozygosity varies more between individuals than expected, reflecting identity disequilibrium (g2) due to variation in inbreeding – a prerequisite for detecting inbreeding depression. We use two types of simulations to ask whether the heterozygosity distribution is consistent with spatially structured mating. First, we simulate offspring from matings with fathers at different distances and find that the distribution of heterozygosity in the field data is consistent with the measured pollen dispersal kernel. Second, we simulate a 1000-generation pedigree using the known spatial distribution, and find that identity disequilibrium, though highly variable between simulations, is consistent with that observed. Finally, we estimate inbreeding depression through the relationships between heterozygosity and six fitness proxies. Only the number of flowering stems is predicted by heterozygosity. Our approach provides a novel example of how long-term studies can elucidate population structure and fitness variation in the wild.
Cell-Cell Adhesion During Nephron Development Is Driven by Wnt/PCP Formin Daam1

E-cadherin junctions facilitate the assembly and disassembly of cell-cell contacts that drive development and homeostasis of epithelial tissues. The stability of E-cadherin-based junctions highly depends on their attachment to the actin cytoskeleton, but little is known about how the assembly of junctional actin filaments is regulated. Formins are a conserved group of proteins responsible for the formation and elongation of filamentous actin (F-actin). In this study, using Xenopus embryonic kidney and Madin-Darby canine kidney (MDCK) cells, we investigate the role of the Wnt/ planar cell polarity (PCP) formin protein Daam1 (Dishevelled-associated activator of morphogenesis 1) in regulating E-cadherin based intercellular adhesion. Using live imaging we show that Daam1 localizes to newly formed cell-cell contacts in the developing nephron. Furthermore, analyses of junctional F-actin upon Daam1 depletion indicate a decrease in microfilament localization and their slowed turnover. We also show that Daam1 is necessary for efficient and timely localization of junctional E-cadherin, which is mediated by Daam1’s formin homology domain 2 (FH2). Finally, we establish that Daam1 signaling is essential for promoting organized movement of renal cells. This study demonstrates that Daam1 formin junctional activity is critical for epithelial tissue organization.
Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease

Objective The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. Design We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. Results In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. Conclusion In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.
Extreme Y chromosome polymorphism corresponds to five male reproductive morphs

Sex chromosomes form once recombination is halted between the X and Y chromosomes. This loss of recombination quickly depletes Y chromosomes of functional content and genetic variation, which is thought to severely limit their potential to generate adaptive diversity. We examined Y diversity in Poecilia parae, where males occur as one of five discrete morphs, all of which shoal together in natural populations where morph frequency has been stable for over 50 years. Each morph utilizes different complex reproductive strategies, and differ dramatically from each other in color, body size, and mating behavior. Remarkably, morph phenotype is passed perfectly from father to son, indicating there are five Y haplotypes segregating in the species, each of which encodes the complex male morph characteristics. Using linked-read sequencing on multiple P. parae females and males of all five morphs from natural populations, we found that the genetic architecture of the male morphs evolved on the Y chromosome long after recombination suppression had occurred with the X. Comparing Y chromosomes between each of the morphs revealed that although the Ys of the three minor morphs that differ predominantly in color are highly similar, there are substantial amounts of unique genetic material and divergence between the Ys of the three major morphs that differ in reproductive strategy, body size and mating behavior. Taken together, our results reveal the extraordinary ability of evolution to overcome the constraints of recombination loss to generate extreme diversity resulting in five discrete Y chromosomes that control complex reproductive strategies. Significance Statement The loss of recombination on the Y chromosome is thought to limit the adaptive potential of this unique genomic region. Despite this, we describe an extraordinary case of Y chromosome adaptation in Poecilia parae. This species contains five co-occurring male morphs, all of which are Y-linked, and which differ in reproductive strategy, body size, coloration, and mating behavior. The five Y-linked male morphs of P. parae evolved after recombination was halted on the Y, resulting in five unique Y chromosomes within one species. Our results reveal the surprising magnitude to which non-recombining regions can generate adaptive diversity and have important implications for the evolution of sex chromosomes and the genetic control of sex-linked diversity.