Miracle-dz/UniFAR-FTUs huggingface dataset:
DNA methylation in Verticillium dahliae requires only one of three putative DNA methyltransferases, yet is dispensable for growth, development and virulence

DNA methylation is an important epigenetic control mechanism that in many fungi is restricted to genomic regions containing transposons. Two DNA methyltransferases, Dim2 and Dnmt5, are known to perform methylation at cytosines in fungi. While most ascomycete fungi encode both Dim2 and Dnmt5, only few functional studies have been performed in species containing both. In this study, we report functional analysis of both Dim2 and Dnmt5 in the plant pathogenic fungus Verticillium dahliae. Our results show that Dim2, but not Dnmt5 or the putative sexual-cycle related DNA methyltransferase Rid, is responsible for nearly all DNA methylation. Single or double DNA methyltransferase mutants did not show altered development, virulence, or transcription of genes or transposons. In contrast, Hp1 and Dim5 mutants that are impacted in chromatin-associated processes upstream of DNA methylation are severely affected in development and virulence and display extensive transcriptional reprogramming in specific hypervariable genomic regions (so-called lineage-specific (LS) regions) that contain genes associated with host colonization. As these LS regions are largely devoid of DNA methylation and of Hp1- and Dim5-associated heterochromatin, the differential transcription is likely caused by pleiotropic effects rather than by differential DNA methylation. Overall, our study suggests that Dim2 is the main DNA methyltransferase in V. dahliae and, in conjunction with work on other fungi, is likely the main active DNMT in ascomycetes, irrespective of Dnmt5 presence. We speculate that Dnmt5 acts under specific, presently enigmatic, conditions or, alternatively, acts in DNA-associated processes other than DNA methylation.
Highly redundant neuropeptide volume co-transmission underlying episodic activation of the GnRH neuron dendron

The necessity and functional significance of neurotransmitter co-transmission remains unclear. The glutamatergic “KNDy” neurons co-express kisspeptin, neurokinin B (NKB) and dynorphin and exhibit a highly stereotyped synchronized behavior that reads out to the gonadotropin-releasing hormone (GnRH) neuron dendrons to drive episodic hormone secretion. Using expansion microscopy, we show that KNDy neurons make abundant close but non-synaptic appositions with the GnRH neuron dendron. Confocal GCaMP6 calcium imaging demonstrated that, of the neurotransmitters co-expressed by KNDy neurons, only kisspeptin was able to activate the GnRH neuron dendron. The selective deletion of kisspeptin from KNDy neurons resulted in mice in which the synchronized behavior of the KNDy neurons was maintained but their ability to drive episodic hormone secretion was abolished. This indicates that KNDy neurons drive episodic hormone secretion through converse modes of highly redundant neuropeptide co-transmission orchestrated by differential postsynaptic neuropeptide receptor expression at their two target sites.
Facilitation and biodiversity jointly drive mutualistic networks

Facilitation by nurse plants increases understorey diversity and supports ecological communities. In turn, biodiversity shapes ecological networks and enhances ecosystem functioning. However, whether and how facilitation and increased biodiversity jointly influence community structure and ecosystem functioning remains unclear. We performed a field experiment disentangling the relative contribution of nurse plants and increasing understorey plant diversity in driving pollination interactions. Both the presence of nurse shrubs as well as increased understorey plant diversity increased pollinator diversity and visitation rates. While nurse and understorey diversity effects on pollinator visitation rates did not interact, the effects of increasing understorey plant diversity on pollinator diversity were stronger in the absence than in the presence of shrubs, meaning that nurse shrubs attenuated the effects of high understorey diversity and buffered the effects of low understorey diversity. We also found positive complementarity effects among understorey species as well as complementarity between nurse plants and understorey species at high diversity. Results also indicate negative selection effects, suggesting that species with generally few pollinators benefit the most in the polyculture, while a species (possibly the nurse plant) with generally lots of pollinators does not. The corresponding changes in pollination networks with the experimental treatments were due to both changes in the frequency of visits and turnover in pollinator community composition. Synthesis Plant–plant facilitative systems, where a nurse plant increases understorey plant diversity, are common in stressful environments. Here, we show that these facilitative systems positively influence mutualistic interactions with pollinators via both direct nurse effects and indirect positive effects of increasing plant diversity. Conserving and supporting nurse plant systems is crucial not only for maintaining plant diversity but also for supporting ecosystem functions and services.
Global identification of RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq

Posttranscriptional regulation of gene expression in bacteria is performed by a complex and hierarchical signaling cascade. Pseudomonas aeruginosa harbors two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, in vivo binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied in vivo UV crosslinking immunoprecipitation followed by RNA-sequencing (UV CLIP-seq) to detect RsmA/N binding sites at single-nucleotide resolution and mapped more than 500 peaks to approximately 400 genes directly bound by RsmA/N in P. aeruginosa. This also demonstrated the ANGGA sequence in apical loops skewed towards 5’UTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results identified previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the small non-coding RNAs RsmY/RsmZ, which sequester RsmA/N away from target mRNAs, are positively regulated by the RsmA/N-mediated translational repression of hptB, encoding a histidine phosphotransfer protein, and cafA, encoding a cytoplasmic axial filament protein, thus providing a possible mechanistic explanation for homeostasis of the Rsm system. Our findings present the global RsmA/N-RNA interaction network that exerts pleiotropic effects on gene expression in P. aeruginosa. IMPORTANCE The ubiquitous bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen causing life-threatening acute and chronic infections in immunocompromised patients. P. aeruginosa infection processes are governed by two major gene regulatory systems, namely, the GacA/GacS (GacAS) two-component system and the RNA-binding proteins RsmA/RsmN (RsmA/N). RsmA/N basically function as a translational repressor or activator directly by competing with the ribosome. In this study, we identified hundreds of RsmA/N regulatory target RNAs and the consensus motifs for RsmA/N bindings by UV crosslinking in vivo. Moreover, our CLIP-seq revealed that RsmA/N posttranscriptionally regulate cell wall organization and exert feedback control on GacAS-RsmA/N systems. Many genes including small regulatory RNAs identified in this study are attractive targets for further elucidating the regulatory mechanisms of RsmA/N in P. aeruginosa.
Quantification of iPSC-derived vascular networks in novel phototunable angiogenic hydrogels

Vascularization of engineered scaffolds remains a critical obstacle hindering the translation of tissue engineering from the bench to the clinic. Previously, we demonstrated the robust micro-vascularization of collagen hydrogels with induced pluripotent stem cell (iPSC)-derived endothelial progenitors; however, physically cross-linked collagen hydrogels compact rapidly and exhibit limited strength. To address these challenges, we synthesized an interpenetrating polymer network (IPN) hydrogel comprised of collagen and norbornene-modified hyaluronic acid (NorHA). This dual-network hydrogel combines the natural cues presented by collagen’s binding sites and extracellular matrix (ECM)-mimicking fibrous architecture with the in situ modularity and chemical cross-linking of NorHA. We modulated the stiffness and degradability of this novel IPN hydrogel by varying the concentration and sequence, respectively, of the NorHA peptide cross-linker. Rheological characterization of the photo-mediated gelation process revealed that the stiffness of the IPN hydrogel increased with cross-linker concentration and was decoupled from the bulk NorHA content. Conversely, the swelling of the IPN hydrogel decreased linearly with increasing cross-linker concentration. Collagen microarchitecture remained relatively unchanged across cross-linking conditions, although the mere addition of NorHA delayed collagen fibrillogenesis. Upon iPSC-derived endothelial progenitor encapsulation, robust, lumenized microvascular networks developed in IPN hydrogels over two weeks. Subsequent computational analysis showed that an initial rise in stiffness increased the number of branch points and vessels, but vascular growth was suppressed in high stiffness IPN hydrogels. These results suggest that an IPN hydrogel consisting of collagen and NorHA is highly tunable, compaction resistant, and capable of stimulating angiogenesis. STATEMENT OF SIGNIFICANCE We have synthesized the first tunable collagen and norbornene functionalized hyaluronic acid (NorHA) interpenetrating polymer network hydrogel. This unique biomaterial allows for control over hydrogel stiffness, independent of the total polymer concentration, by varying the concentration of a peptide cross-linker and was specifically designed to produce a biomimetic vasculogenic microenvironment. Using the system, we performed a detailed study of the vasculogenesis of induced pluripotent stem cell-derived (iPSC) endothelial progenitors, a poorly studied cell source with considerable therapeutic potential. Our results show that vascular growth can be tuned by altering the stiffness and degradability of the scaffolds independently. Finally, we improved upon our open-source computational pipeline programmed in ImageJ and MATLAB to further quantify vascular topologies in three dimensions. Graphical abstract
The medaka alg2 mutant is a model for hypo-N-glycosylation-associated retinitis pigmentosa

Patients suffering from Congenital Disorders of Glycosylation (CDG) carry mutations in components of the evolutionarily highly conserved protein-glycosylation-machinery. Employing targeted genome editing, we modeled alleles in medaka fish based on a mutation described in an ALG2-index patient. The multisystemic phenotypes in our alg2 model closely resembled the patient’s syndromes. Molecularly, the mutation results in a reduction of the abundance of N-glycans without altering the profile of glycan structures in fish as well as in patient fibroblasts. This hypo-N-glycosylation impacted on protein abundance in two directions. We discovered a putative compensatory upregulation of the basic glycosylation and glycoprotein processing machinery highlighting the regulatory topology of the network. Conversely, proteins of the retinal phototransduction machinery were massively downregulated in the alg2 model. Those relate to the specific loss of rod photoreceptors that fail to be maintained in the alg2 mutants, a condition known as retinitis pigmentosa. Transient supply of human or medaka alg2 mRNA efficiently rescued the phenotypic spectrum and restored viability demonstrating that our alg2 model delivers key traits for the potential treatment of the disorder.
αKG inhibits Regulatory T cell differentiation by coupling lipidome remodelling to mitochondrial metabolism

The differentiation of CD4 T cells to a specific effector fate is metabolically regulated, integrating glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) with transcriptional and epigenetic changes. OXPHOS is tightly coordinated with the tricarboxylic acid (TCA) cycle but the precise role of TCA intermediates in CD4 T cell differentiation remain unclear. Here we demonstrate that α-ketoglutarate (αKG) inhibited regulatory T cell (Treg) generation while conversely, increasing Th1 polarization. In accord with these data, αKG promoted the effector profile of Treg-polarized chimeric antigen receptor-engineered T cells against the ErbB2 tumor antigen. Mechanistically, αKG significantly altered transcripts of genes involved in lipid-related processes, inducing a robust lipidome-wide remodelling and decreased membrane fluidity. A massive increase in storage and mitochondria lipids was associated with expression of mitochondrial genes and a significantly augmented OXPHOS. Notably, inhibition of succinate dehydrogenase activity, the bridge between the TCA cycle and the electron transport chain, enforced Treg generation. Thus, our study identifies novel connections between αKG, lipidome remodelling and OXPHOS in CD4 T cell fate decisions.
Dynamics of the N-terminal domain of SARS-CoV-2 nucleocapsid protein drives dsRNA melting in a counterintuitive tweezer-like mechanism

The N protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. Its N-terminal domain (NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRS), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of SARS-CoV-2 N-NTD to non-specific (NS) and TRS dsRNAs. We probed dsRNAs’ Watson and Crick (WC) base-pairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, initiating melting. N-NTD dsRNA destabilizing activity was more efficient for dsTRS than dsNS. N-NTD dynamics, especially a tweezer-like motion of β2-β3 and 2-β5 loops, played a key role in WC base-pairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD:dsRNA), matching MD simulations and raising different possibilities for N-NTD action: (i) two N-NTDs of dimeric N would act independently, increasing efficiency; (ii) two N-NTDs of dimeric N would bind to two different RNA sites, bridging distant regions of the genome; and (iii) monomeric N would be active, opening up the possibility of a regulatory dissociation event. IMPORTANCE Coronaviruses are among the largest positive-sense RNA viruses. They display a unique discontinous transcription mechanism, involving N protein as a major player. The N-NTD promote the dsRNA melting releasing the nascent sense negative strand via a poorly known mechanism of action. It specifically recognizes the body TRS conserved RNA motif located at the 5’ end of each ORF. N protein has the ability to transfer the nascent RNA strand to the leader TRS. The mechanism is essential and one single mutation at the RNA binding site of the N-NTD impairs the viral replication. Here, we describe a counterintuitive mechanism of action of N-NTD based on molecular dynamics simulation and kinetic modelling of the experimental melting activity of N-NTD. This data impacts directly in the understanding of the way N protein acts in the cell and will guide future experiments.
Insulin-like growth factor 1 signaling in the placenta requires endothelial nitric oxide synthase to support trophoblast function and normal fetal growth

Currently, there is no effective treatment for placenta dysfunction in utero. In a ligated mouse model of fetal growth restriction (FGR), nanoparticle-mediated human insulin-like 1 growth factor (hIGF1) gene delivery (NP-Plac1-hIGF1) increased hIGF1 expression and maintained fetal growth. However, whether it can restore fetal growth remains to be determined. Using the endothelial nitric oxide synthase knockout (eNOS−/−) mouse model, a genetic model of FGR, we found that despite inducing expression of hIGF1 in the placentas treated with NP-Plac1-hIGF1 (P=0.0425), FGR did not resolve. This was associated with no change to the number of fetal capillaries in the placenta labyrinth; an outcome increased with NP-Plac1-hIGF1 treatment in the ligated mouse model, despite increased expression of angiopoietin 1 (P=0.05) and suggested IGF1 signaling in the placenta requires eNOS to modulate placenta angiogenesis. To further assess this hypothesis, we used the BeWo Choriocarcinoma cell line and human placenta explant cultures that were treated with NP-Plac1-hIGF1, oxidative stress was induced with hydrogen peroxide (H2O2) and NOS activity inhibited using the inhibitor L-NMMA. In both BeWo and explants, the protective effect of NP-Plac1-hIGF1 treatment against H2O2 induced cell death/lactate dehydrogenase release was prevented by eNOS inhibition (P=0.003 and P<0.0001, respectively). This was associated with an increase in mRNA expression of oxidative stress markers HIF1α (P<0.0001) and ADAM10 (P=0.0002) in the NP-Plac1-hIGF1+H2O2+L-NMMA treated BeWo cells. These findings show for the first time the requirement of eNOS/NOS in IGF1 signaling in placenta cells that may have implications for placental angiogenesis and fetal growth.
Intracellular Staphylococcus aureus perturbs the host cell Ca2+-homeostasis to promote cell death

The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases ranging from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction and spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide shRNA screen and identified the calcium signaling pathway to be involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+-increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by decrease in endoplasmic reticulum Ca2+-concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+-increase. As a consequence, we observed that the cytoplasmic Ca2+-rise led to increase in mitochondrial Ca2+-concentration, the activation of calpains and caspases and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+-homeostasis and induces cytoplasmic Ca2+-overload, which results in both apoptotic and necrotic cell death in parallel or succession. Importance Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered as hide-out from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+-overload, which results in host cell death. Thus, this study firstly showed how an intracellular bacterium perturbs the host cell Ca2+-homeostasis.
Novel cryo-electron tomography structure of Arp2/3 complex in cells reveals mechanisms of branch formation

The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9. 0Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Our structure indicates a central role for the ArpC3 subunit in stabilizing the active conformation and suggests that in the branch junction relocation of the ArpC5 N-terminus and the C-terminal tail of Arp3 is important to fix Arp2 and Arp3 in an actin dimer-like conformation. Notably, our model of the branch junction in cells significantly differs from the previous in vitro branch junction model.
Active perception during angiogenesis: filopodia speed up Notch selection of tip cells in silico and in vivo

How do cells make efficient collective decisions during tissue morphogenesis? Humans and other organisms utilize feedback between movement and sensing known as ‘sensorimotor coordination’ or ‘active perception’ to inform behaviour, but active perception has not before been investigated at a cellular level within organs. Here we provide the first proof of concept in silico/in vivo study demonstrating that filopodia (actin-rich, dynamic, finger like cell-membrane protrusions) play an unexpected role in speeding up collective endothelial decisions during the time-constrained process of ‘tip cell’ selection during blood vessel formation (angiogenesis). We first validate simulation predictions in vivo with live imaging of zebrafish intersegmental vessel growth. Further simulation studies then indicate the effect is due to the coupled positive feedback between movement and sensing on filopodia conferring a bistable switch-like property to Notch lateral inhibition, ensuring tip selection is a rapid and robust process. We then employ measures from computational neuroscience to assess whether filopodia function as a primitive (‘basal’) form of active perception and find evidence in support. By viewing cell behaviour in tissues through the ‘basal cognitive lens’ we acquire a fresh perspective on not only the well-studied tip cell selection process, revealing a hidden, yet vital, time-keeping role for filopodia, but on how to interpret and understand cell behaviour in general, opening up a myriad of new and exciting research directions.
BAX is necessary for neuronal death following exposure to isoflurane during the neonatal period

Exposure to volatile anesthetics during the neonatal period results in acute neuronal death in rodent and non-human primate models, potentially leading to lasting cognitive deficits. We used Bax-/- mice to show that neuronal death following neonatal exposure to isoflurane is mediated by the apoptotic pathway, and that GABAergic interneurons are selectively vulnerable. Neonatal Bax-/- mice also showed attenuated microglial activation after exposure to isoflurane, indicating that neuroinflammatory response is secondary to neuronal apoptosis. Isoflurane-induced neuronal apoptosis in neonates appeared to have little effect on seizure threshold or cognitive function later in life. Collectively, these findings define the acute injury mechanism of volatile anesthetics during the neonatal period.