The properties of (1,3)-β-glucans (i.e., callose) remain largely unknown despite their importance in plant development and defence. Here we use mixtures of (1,3)-β-glucan and cellulose, in ionic liquid solution and hydrogels, as proxies to understand the physico-mechanical properties of callose. We show that after callose addition the stiffness of cellulose hydrogels is reduced at a greater extent than predicted from the ideal mixing rule (i.e., the weighted average of the individual components' properties). In contrast, yield behaviour after the elastic limit is more ductile in cellulose-callose hydrogels compared with sudden failure in 100% cellulose hydrogels. The viscoelastic behaviour and the diffusion of the ions in mixed ionic liquid solutions strongly indicate interactions between the polymers. Fourier-transform infrared analysis suggests that these interactions impact cellulose organisation in hydrogels and cell walls. We conclude that polymer interactions alter the properties of callose-cellulose mixtures beyond what it is expected by ideal mixing.

Previous studies have reported that microglia depletion leads to impairment of synapse formation and these cells rapidly repopulate from CNS progenitors. However, the impact of microglia depletion and repopulation in the long-term state of the CNS environment has not been characterized. Here, we report that acute and synchronous microglia depletion and subsequent repopulation induces gray matter microgliosis, neuronal death in the somatosensory cortex and ataxia-like behavior. We find a type 1 interferon inflammatory signature in degenerating somatosensory cortex from microglia-depleted mice. Transcriptomic and mass cytometry analysis of repopulated microglia demonstrates an interferon regulatory factor 7-driven activation state. Minocycline and anti-IFNAR1 antibody treatment attenuate the CNS type 1 interferon-driven inflammation, restore microglia homeostasis and reduce ataxic behavior. Neither microglia depletion nor repopulation impact neuropathology or T-cell responses during experimental autoimmune encephalomyelitis. Together, we found that acute microglia ablation induces a type 1 interferon activation state of gray matter microglia associated with acute neurodegeneration.

Osteoarthritis is one of the leading causes of pain and disability in the aged population due to articular cartilage damage. This warrants investigation of signaling mechanisms that could protect cartilage from degeneration and degradation. Here we show in a murine model of experimental osteoarthritis that YAP activation by transgenic overexpression or by deletion of its upstream inhibitory kinases Mst1/2 preserves articular cartilage integrity, whereas deletion of YAP in chondrocytes promotes cartilage disruption. Our work shows that YAP is both necessary and sufficient for the maintenance of cartilage homeostasis in osteoarthritis. Mechanistically, inflammatory cytokines, such as TNFα or IL-1β, trigger YAP/TAZ degradation through TAK1-mediated phosphorylation. Furthermore, YAP directly interacts with TAK1 and attenuates NF-κB signaling by inhibiting substrate accessibility of TAK1. Our study establishes a reciprocal antagonism between Hippo-YAP/TAZ and NF-κB signaling in regulating the induction of matrix-degrading enzyme expression and cartilage degradation during osteoarthritis pathogenesis.

Lung adenocarcinomas (LUADs) with mutations in the K-ras oncogene display dismal prognosis. Proinflammatory and immunomodulatory events that drive development of K-ras mutant LUAD are poorly understood. Here, we develop a lung epithelial specific K-ras mutant/Stat3 conditional knockout (LR/Stat3Δ/Δ) mouse model. Epithelial Stat3 deletion results in intriguing sex-associated discrepancies; K-ras mutant tumors are decreased in female LR/Stat3Δ/Δ mice whereas tumor burdens are increased in males. RNA-sequencing and tumor microenvironment (TME) analysis demonstrate increased anti-tumor immune responses following Stat3 deletion in females and, conversely, elevated pro-tumor immune pathways in males. While IL-6 blockade in male LR/Stat3Δ/Δ mice reduces lung tumorigenesis, inhibition of estrogen receptor signaling in female mice augments K-ras mutant oncogenesis and reprograms lung TME toward a pro-tumor phenotype. Our data underscore a critical sex-specific role for epithelial Stat3 signaling in K-ras mutant LUAD, thus paving the way for developing personalized (e.g. sex-based) immunotherapeutic strategies for this fatal disease.

RNA localization is a crucial process for cellular function and can be quantitatively studied by single molecule FISH (smFISH). Here, we present an integrated analysis framework to analyze sub-cellular RNA localization. Using simulated images, we design and validate a set of features describing different RNA localization patterns including polarized distribution, accumulation in cell extensions or foci, at the cell membrane or nuclear envelope. These features are largely invariant to RNA levels, work in multiple cell lines, and can measure localization strength in perturbation experiments. Most importantly, they allow classification by supervised and unsupervised learning at unprecedented accuracy. We successfully validate our approach on representative experimental data. This analysis reveals a surprisingly high degree of localization heterogeneity at the single cell level, indicating a dynamic and plastic nature of RNA localization.

Diverse γ-aminobutyric acid (GABA)-ergic interneurons provide different modes of inhibition to support circuit operation in the neocortex. However, the cellular and molecular mechanisms underlying the systematic generation of assorted neocortical interneurons remain largely unclear. Here we show that NKX2.1-expressing radial glial progenitors (RGPs) in the mouse embryonic ventral telencephalon divide progressively to generate distinct groups of interneurons, which occupy the neocortex in a time-dependent, early inside-out and late outside-in, manner. Notably, the late-born chandelier cells, one of the morphologically and physiologically highly distinguishable GABAergic interneurons, arise reliably from continuously dividing RGPs that produce non-chandelier cells initially. Selective removal of Partition defective 3, an evolutionarily conserved cell polarity protein, impairs RGP asymmetric cell division, resulting in premature depletion of RGPs towards the late embryonic stages and a consequent loss of chandelier cells. These results suggest that consecutive asymmetric divisions of multipotent RGPs generate diverse neocortical interneurons in a progressive manner.

Perceptual constancy requires neural representations that are selective for object identity, but also tolerant across identity-preserving transformations. How such representations arise in the brain and support perception remains unclear. Here, we study tolerant representation of sound identity in the auditory system by recording neural activity in auditory cortex of ferrets during perceptual constancy. Ferrets generalize vowel identity across variations in fundamental frequency, sound level and location, while neurons represent sound identity robustly across acoustic variations. Stimulus features are encoded with distinct time-courses in all conditions, however encoding of sound identity is delayed when animals fail to generalize and during passive listening. Neurons also encode information about task-irrelevant sound features, as well as animals' choices and accuracy, while population decoding out-performs animals' behavior. Our results show that during perceptual constancy, sound identity is represented robustly in auditory cortex across widely varying conditions, and behavioral generalization requires conserved timing of identity information.

CrAssphages are an extensive and ubiquitous family of tailed bacteriophages, predicted to infect bacteria of the order Bacteroidales. Despite being found in ~50% of individuals and representing up to 90% of human gut viromes, members of this viral family have never been isolated in culture and remain understudied. Here, we report the isolation of a CrAssphage (ΦCrAss001) from human faecal material. This bacteriophage infects the human gut symbiont Bacteroides intestinalis, confirming previous in silico predictions of the likely host. DNA sequencing demonstrates that the bacteriophage genome is circular, 102 kb in size, and has unusual structural traits. In addition, electron microscopy confirms that ΦcrAss001 has a podovirus-like morphology. Despite the absence of obvious lysogeny genes, ΦcrAss001 replicates in a way that does not disrupt proliferation of the host bacterium, and is able to maintain itself in continuous host culture during several weeks.

All-inorganic perovskites have a special place in halide perovskite family because of their potential for better stability. However, the representative cesium lead iodide (CsPbI3) is metastable and spontaneously converts to the non-perovskite structure at room temperature. Here, we demonstrate that what appears to be all-inorganic CsPbI3 stabilized in its perovskite form using the purported intermediate known as hydrogen lead iodide (HPbI3) is, in fact, the hybrid perovskite cesium dimethylammonium lead iodide (Cs1-xDMAxPbI3, x = 0.2 to 0.5). Thus, many of the reported all-inorganic perovskites are actually still hybrid organic-inorganic perovskites, as strongly evidenced by a wide battery of experimental techniques presented here. Solar cells based on the representative composition Cs0.7DMA0.3PbI3 can achieve an average power conversion efficiency of 9.27 ± 1.28% (max 12.62%). These results provide an alternative angle to look at previous results pertaining all-inorganic CsPbI3 while the DMA cation is now revealed as an alternative A site cation.

BRAF inhibitors target the BRAF-V600E/K mutated kinase, the driver mutation found in 50% of cutaneous melanoma. They give unprecedented anti-tumor responses but acquisition of resistance ultimately limits their clinical benefit. The master regulators driving the expression of resistance-genes remain poorly understood. Here, we demonstrate that the Aryl hydrocarbon Receptor (AhR) transcription factor is constitutively activated in a subset of melanoma cells, promoting the dedifferentiation of melanoma cells and the expression of BRAFi-resistance genes. Typically, under BRAFi pressure, death of BRAFi-sensitive cells leads to an enrichment of a small subpopulation of AhR-activated and BRAFi-persister cells, responsible for relapse. Also, differentiated and BRAFi-sensitive cells can be redirected towards an AhR-dependent resistant program using AhR agonists. We thus identify Resveratrol, a clinically compatible AhR-antagonist that abrogates deleterious AhR sustained-activation. Combined with BRAFi, Resveratrol reduces the number of BRAFi-resistant cells and delays tumor growth. We thus propose AhR-impairment as a strategy to overcome melanoma resistance.

Controlled switching of the spin state of transition metal ions, particularly of FeII and FeIII, is a prerequisite to achieve selectivity, efficiency, and catalysis in a number of metalloenzymes. Here we report on an iron(III) porphyrin with a photochromic axial ligand which, upon irradiation with two different wavelengths reversibly switches its spin state between low-spin (S = 1/2) and high-spin (S = 5/2) in solution (DMSO-acetone, 2:598). The switching efficiency is 76% at room temperature. The system is neither oxygen nor water sensitive, and no fatigue was observed after more than 1000 switching cycles. Concomitant with the spin-flip is a change in redox potential by ~60 mV. Besides serving as a simple model for the first step of the cytochrome P450 catalytic cycle, the spin switch can be used to switch the spin-lattice relaxation time T1 of the water protons by a factor of 15.

Many cellular programs of neural development are under combinatorial regulation by different chemoattractive or chemorepulsive factors. Here, we describe a microfluidic platform that utilizes well-controlled three-dimensional (3D) diffusion to generate molecular gradients of varied steepness in a large array of hydrogel cylinders, allowing high-throughput 3D chemotactic assays for mechanistic dissection of steepness-dependent neuronal chemotaxis. Using this platform, we examine neuronal sensitivity to the steepness of gradient composed of netrin-1, nerve growth factor, or semaphorin3A (Sema3A) proteins, and reveal dramatic diversity and complexity in the associated chemotactic regulation of neuronal development. Particularly for Sema3A, we find that serine/threonine kinase-11 and glycogen synthase kinase-3 signaling pathways are differentially involved in steepness-dependent chemotactic regulation of coordinated neurite repellence and neuronal migration. These results provide insights to the critical role of gradient steepness in neuronal chemotaxis, and also prove the technique as an expandable platform for studying other chemoresponsive cellular systems.

Obesity is a major threat to global health and metabolically associated with glycerol homeostasis. Here we demonstrate that in human adipocytes, the decreased pH observed during lipolysis (fat burning) correlates with increased glycerol release and stimulation of aquaglyceroporin AQP10. The crystal structure of human AQP10 determined at 2.3 Å resolution unveils the molecular basis for pH modulation-an exceptionally wide selectivity (ar/R) filter and a unique cytoplasmic gate. Structural and functional (in vitro and in vivo) analyses disclose a glycerol-specific pH-dependence and pinpoint pore-lining His80 as the pH-sensor. Molecular dynamics simulations indicate how gate opening is achieved. These findings unravel a unique type of aquaporin regulation important for controlling body fat mass. Thus, targeting the cytoplasmic gate to induce constitutive glycerol secretion may offer an attractive option for treating obesity and related complications.

Current methods for generating liquid-liquid interfaces with either controlled composition or coverage often rely on adsorption equilibria which limits the freedom to design such multiphase materials, in particular when different components are used. Moreover, when interfaces become densely populated, slowing down of adsorption may impose additional constraints. Up to now, it is not possible to control surface coverage and composition of droplet interfaces at will. Here, we report a generic and versatile method to create designer liquid-liquid interfaces, using transient double emulsions. We demonstrate how the surface coverage in Pickering emulsions can be controlled at will, even for dense particulate layers going up to multilayers. Moreover, composite droplet interfaces with compositional control can be generated, even with particles which would have intrinsically different or even opposite adsorption characteristics. Given its simplicity, this method offers a general approach for control of composition of liquid-liquid interfaces in a variety of multiphase systems.

The type VI secretion system (T6SS) is a multi-protein complex that injects bacterial effector proteins into target cells. It is composed of a cell membrane complex anchored to a contractile bacteriophage tail-like apparatus consisting of a sharpened tube that is ejected by the contraction of a sheath against a baseplate. We present structural and biochemical studies on TssA subunits from two different T6SSs that reveal radically different quaternary structures in comparison to the dodecameric E. coli TssA that arise from differences in their C-terminal sequences. Despite this, the different TssAs retain equivalent interactions with other components of the complex and position their highly conserved N-terminal ImpA_N domain at the same radius from the centre of the sheath as a result of their distinct domain architectures, which includes additional spacer domains and highly mobile interdomain linkers. Together, these variations allow these distinct TssAs to perform a similar function in the complex.

Intrinsically disordered proteins (IDPs) usually fold during binding to target proteins. In contrast to interactions between folded proteins, this additional folding step makes the binding process more complex. Understanding the mechanism of coupled binding and folding of IDPs requires analysis of binding pathways that involve formation of the transient complex (TC). However, experimental characterization of TC is challenging because it only appears for a very brief period during binding. Here, we use single-molecule fluorescence spectroscopy to investigate the mechanism of diffusion-limited association of an IDP. A large enhancement of the association rate is observed due to the stabilization of TC by non-native electrostatic interactions. Moreover, photon-by-photon analysis reveals that the lifetime of TC for IDP binding is at least two orders of magnitude longer than that for binding of two folded proteins. This result suggests the long lifetime of TC is generally required for folding of IDPs during binding processes.

TH17 cells originating from regulatory T (Treg) cells upon loss of the Treg-specific transcription factor Foxp3 accumulate in sites of inflammation and aggravate autoimmune diseases. Whether an active mechanism drives the generation of these pathogenic 'ex-Foxp3 TH17' cells, remains unclear. Here we show that pro-inflammatory cytokines enhance the expression of transcription regulator Id2, which mediates cellular plasticity of Treg into ex-Foxp3 TH17 cells. Expression of Id2 in in vitro differentiated iTreg cells reduces the expression of Foxp3 by sequestration of the transcription activator E2A, leading to the induction of TH17-related cytokines. Treg-specific ectopic expression of Id2 in mice significantly reduces the Treg compartment and causes immune dysregulation. Cellular fate-mapping experiments reveal enhanced Treg plasticity compared to wild-type, resulting in exacerbated experimental autoimmune encephalomyelitis pathogenesis or enhanced anti-tumor immunity. Our findings suggest that controlling Id2 expression may provide a novel approach for effective Treg cell immunotherapies for both autoimmunity and cancer.

There have been very few reports on protein domains that specifically recognize sulfur. Here we present the crystal structure of the sulfur-binding domain (SBD) from the DNA phosphorothioation (PT)-dependent restriction endonuclease ScoMcrA. SBD contains a hydrophobic surface cavity that is formed by the aromatic ring of Y164, the pyrolidine ring of P165, and the non-polar side chains of four other residues that serve as lid, base, and wall of the cavity. The SBD and PT-DNA undergo conformational changes upon binding. The S187RGRR191 loop inserts into the DNA major groove to make contacts with the bases of the GPSGCC core sequence. Mutating key residues of SBD impairs PT-DNA association. More than 1000 sequenced microbial species from fourteen phyla contain SBD homologs. We show that three of these homologs bind PT-DNA in vitro and restrict PT-DNA gene transfer in vivo. These results show that SBD-like PT-DNA readers exist widely in prokaryotes.

At the initial stage of carcinogenesis single mutated cells appear within an epithelium. Mammalian in vitro experiments show that potentially cancerous cells undergo live apical extrusion from normal monolayers. However, the mechanism underlying this process in vivo remains poorly understood. Mosaic expression of the oncogene vSrc in a simple epithelium of the early zebrafish embryo results in extrusion of transformed cells. Here we find that during extrusion components of the cytokinetic ring are recruited to adherens junctions of transformed cells, forming a misoriented pseudo-cytokinetic ring. As the ring constricts, it separates the basal from the apical part of the cell releasing both from the epithelium. This process requires cell cycle progression and occurs immediately after vSrc-transformed cell enters mitosis. To achieve extrusion, vSrc coordinates cell cycle progression, junctional integrity, cell survival and apicobasal polarity. Without vSrc, modulating these cellular processes reconstitutes vSrc-like extrusion, confirming their sufficiency for this process.

Marking functionally distinct neuronal ensembles with high spatiotemporal resolution is a key challenge in systems neuroscience. We recently introduced CaMPARI, an engineered fluorescent protein whose green-to-red photoconversion depends on simultaneous light exposure and elevated calcium, which enabled marking active neuronal populations with single-cell and subsecond resolution. However, CaMPARI (CaMPARI1) has several drawbacks, including background photoconversion in low calcium, slow kinetics and reduced fluorescence after chemical fixation. In this work, we develop CaMPARI2, an improved sensor with brighter green and red fluorescence, faster calcium unbinding kinetics and decreased photoconversion in low calcium conditions. We demonstrate the improved performance of CaMPARI2 in mammalian neurons and in vivo in larval zebrafish brain and mouse visual cortex. Additionally, we herein develop an immunohistochemical detection method for specific labeling of the photoconverted red form of CaMPARI. The anti-CaMPARI-red antibody provides strong labeling that is selective for photoconverted CaMPARI in activated neurons in rodent brain tissue.