title: Near Infrared-II Fluorescent protein for In-vivo Imaging

abstract: In vivo fluorescent imaging in the second near-infrared window (NIR-II) provides an excellent approach for understanding the biological processes in substantially scattered tissue environments with reasonable temporal-spatial resolution. In spite of an enormous amount of organic and inorganic NIR-II fluorophores developed, there is no NIR-II fluorescent protein reported. Here, we present the first NIR-II fluorescent protein, IRFP1032 which exhibits strong exciton absorption and emission in the NIR-II region, with exciton extinction coefficient about 4.1 x106 M-1cm-1 at the excitation maximum 1008 nm, emission maximum of 1032 nm, and emission quantum yield about 0.84%. The IRFP1032 is found to be the brightest NIR-II fluorophore ever reported (brightness of 3.4 x 104 M-1cm-1 in PBS) which is thousands-fold brighter than IR26 in DCM. Taking the advantage of the excellent photo-properties of the NIR-II fluorescent proteins, a collection of high-quality in vivo imaging research was realized, for instance, real time observation of blood flow dynamics, dual-channel imaging of the lymphatic/blood vessel network and the trajectories of single bacterial cell travelling in blood vessels. Moreover, a mammalian expression vector was constructed for the IRFP1032, and the corresponding NIR-II fluorescence was able to be recorded unambiguously. The promising NIR-II in vivo imaging properties of IRPF1032 demonstrated here would open a new scene in fluorescent protein-based imaging.


title: Improved mammalian retromer cryo-EM structures reveal a new assembly interface

abstract: Retromer (VPS26/VPS35/VPS29 subunits) assembles with multiple sorting nexin (SNX) proteins on membranes to mediate endosomal recycling of transmembrane protein cargoes. Retromer has been implicated in other cellular events, including mitochondrial homeostasis, nutrient sensing, autophagy, and fission events. Mechanisms for mammalian retromer assembly remain undefined, and retromer engages multiple sorting nexin proteins to sort cargoes to different destinations. Published structures demonstrate mammalian retromer forms oligomers in vitro, but several structures were poorly resolved. We report here improved retromer oligomer structures using single particle cryo-electron microscopy (cryo-EM) by combining data collected from tilted specimens with multiple improvements in data processing, including using a three-dimensional (3D) starting model for improved automated particle picking in RELION. A retromer mutant (3KE retromer) that breaks VPS35-mediated interfaces was used to determine a structure of a new assembly interface formed by the VPS26A and VPS35 N-termini. The interface reveals how an N-terminal VPS26A arrestin saddle can link retromer chains by engaging a neighboring VPS35 N-terminus, on the opposite side from the well-characterized C-VPS26/N-VPS35 interaction observed within heterotrimers. The new interaction interface exhibits substantial buried surface area ([~]7,000 [A]2) and further suggests metazoan retromer may serve as an adaptable scaffold.

category: biochemistry


title: Biologically-derived neoproteoglycans for profiling protein-glycosaminoglycan interactions

abstract:
Glycosaminoglycans (GAGs) are a class of highly negatively charged membrane associated and extracellular matrix polysaccharides involved in the regulation of myriad biological functions, including cell adhesion, migration, signaling and differentiation, among others. GAGs are typically attached to core proteins, termed proteoglycans (PGs), and can engage >500 binding proteins, making them prominent relays for sensing external stimuli and transducing cellular responses. However, their unique substructural protein-recognition domains that confer their binding specificity remain elusive. While the emergence of glycan arrays has rapidly enabled the profiling of ligand specificities of a range of glycan-binding proteins, their adaptation for the analysis of GAG-binding proteins has been considerably more challenging. Current GAG-microarrays primarily employ synthetically defined oligosaccharides, which capture only a fraction of the structural diversity of native GAG polysaccharides. Augmenting existing array platforms to include GAG structures purified from tissues or produced in cells with engineered glycan biosynthetic pathways may significantly advance the understanding of structure-activity relationships in GAG-protein interactions. Here, we demonstrate an efficient and tunable strategy to mimic cellular proteoglycan architectures by conjugating biologically-derived GAG chains to a protein scaffold, defined as neoproteoglycans (neoPGs). The use of a reactive fluorogenic linker enabled real-time monitoring of the conjugation reaction efficiency and tuning of the neoPG valency. Immobilization of the reagents on a 96-well array platform allowed for efficient probing of ligand binding and enzyme substrate specificity, including growth factors and the human sulfatase 1. The neoPGs can also be used directly as soluble probes to evaluate GAG-dependent growth factor signaling in cells.

Graphical Abstract

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title: Discovery of small molecule inhibitors of Plasmodium falciparum apicoplast DNA polymerase

abstract: Malaria is caused by infection with protozoan parasites of the Plasmodium genus, which is part of the phylum Apicomplexa. Most organisms in this phylum contain a relic plastid called the apicoplast. The apicoplast genome is replicated by a single DNA polymerase (apPOL), which is an attractive target for anti-malarial drugs. We screened small-molecule libraries (206,504 compounds) using a fluorescence-based high-throughput DNA polymerase assay. Dose/response analysis and counter-screening identified 186 specific apPOL inhibitors. Toxicity screening against human HepaRG human cells removed 84 compounds and the remaining were subjected to parasite killing assays using chloroquine resistant P. falciparum parasites. Nine compounds were potent inhibitors of parasite growth and may serve as lead compounds in efforts to discover novel malaria drugs.


title: Alternative 3' UTRs contributes to post-transcriptional gene expression regulation under high salt stress

abstract:
High salt stress continually challenges growth and survival of many plants, but the underlying molecular basis is not fully explored. Alternative polyadenylation (APA) produces mRNAs with different 3'UTRs (alternative 3'UTRs) to regulate gene expression at post-transcriptional level. However, the roles of such process in response to salt stress remain elusive. Here, we reported that alternative 3'UTRs responded to high salt stress in the halophyte-Spartina alterniflora, which tolerant to hash salt environment. High salt stress induced the global APA and increased the prevalence of APA events. Strikingly, high salt stress significantly led to 3' UTR lengthening of 207 transcripts through increasing the usage of distal poly(A) sites. Transcripts with alternative 3' UTRs were mainly enriched in salt stress related ion transporters. Alternative 3' UTRs of SaHKT1 increased RNA stability and protein synthesis in vivo. Regulatory AU-rich elements were identified in the alternative 3' UTRs and alternative 3' UTRs increased protein level of SaHKT1 in an AU-rich element dependent manner. Finally, 3' UTR lengthening might result from variations in poly(A) signals and poly(A) factors. Overall, these results suggest that APA is a potential novel high salt stress responsive mechanism by modulating mRNA 3' UTR length. These results also reveal complex regulator roles of alternative 3' UTRs coupling alternative polyadenylation and regulatory elements at post-transcriptional level in plants.

One sentence summaryAlternative 3' UTRs acts as a potential novel mechanism in gene expression regulation of high salt tolerant genes

category: plant biology


title: Information Decay and Enzymatic Information Recovery for DNA Data Storage

abstract: Synthetic DNA has been proposed as a storage medium for digital information due to its high theoretical storage density and anticipated long storage horizons. However, under all ambient storage conditions, DNA undergoes a slow chemical decay process resulting in nicked (broken) DNA strands, and the information stored in these strands is no longer readable. In this work we design an enzymatic repair procedure, which is applicable to the DNA pool prior to readout and can partially reverse the damage. Through a chemical understanding of the decay process, an overhang at the 3 end of the damaged site is identified as obstructive to repair via the base excision-repair (BER) mechanism. The obstruction can be removed via the enzyme apurinic/apyrimidinic endonuclease I (APE1), thereby enabling repair of hydrolytically damaged DNA via Bst polymerase and Taq ligase. Simulations of damage and repair reveal the benefit of the enzymatic repair step for DNA data storage, especially when data is stored in DNA at high storage densities (= low physical redundancy) and for long time durations.

category: synthetic biology


title: A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

abstract: The TGF{beta} superfamily of secreted growth factors comprises more than 30 members including TGF{beta}s, BMPs and Activins. While all TGF{beta} superfamily members signal through heteromeric receptor complexes to regulate a plethora of developmental and homeostatic processes, each ligand possesses a unique affinity towards a subset of BMP and TGF{beta} type I and type II receptors. Whereas the Activin and TGF{beta} class display a higher affinity towards type II receptors, BMPs and GDFs preferentially bind to type I receptors. Sofar, the lack of specific antibodies and chemical biology tools hampered simultaneous testing of ligand binding towards all BMP and TGF{beta} receptors. Here we present a N-terminally Halo- and SNAP-tagged TGF{beta}/BMP receptor library to visualize the receptor complexes in dual color. In combination with novel fluorescently labeled TGF{beta} superfamily ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent growth factor binding for Activin A, TGF{beta}1 and BMP9 in a cellular context. We confirm ligand-receptor interface specificity by identifying BMPR2- or ALK2-mutants that switch from a low-affinity Activin A- or BMP9-receptor to a high-affinity receptor, respectively.

category: cell biology


title: An amphipathic helix in Brl1 is required for membrane fusion during nuclear pore complex biogenesis in S. cerevisiae

abstract: The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-ET analyses reveal multi-layered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.


title: The multi-level regulation of clownfish metamorphosis by thyroid hormones

abstract: Most marine organisms have a biphasic life cycle during which a pelagic larva is transformed into a radically different juvenile. In vertebrates the role of thyroid hormones (TH) in triggering this transition is well known, but how the morphological and physiological changes are integrated in a coherent way with the ecological transition remains poorly explored. To gain insight into this question, we performed an integrative analysis of metamorphosis of a marine teleost, the clownfish Amphiprion ocellaris. We reveal how TH coordinate a change in color vision as well as a major metabolic shift in energy production, hence highlighting its central integrative role in regulating this transformation. By manipulating the activity of LXR, a major regulator of metabolism, we also reveal a tight link between metabolic changes and metamorphosis progression. Strikingly, we observed that these regulations are at play in the wild revealing how hormones coordinate energy needs with available resources during life cycle.

category: developmental biology


title: Optical control of CRISPR-Cas editing with cyclically caged guide RNAs

abstract: The CRISPR/Cas system has been proved as one of the most powerful tools for precise gene editing. However, the approaches for precise control over the genome editing and regulatory events are still desirable. Here, we reported a spatiotemporal and efficient CRISPR/Cas9 and Cpf1-mediated editing with photo-sensitive circular gRNAs. This approach relies on only two or three pre-installed photolabile substituents followed by a simple covalent cyclization, which provides a robust synthesize approach in comparison to heavily modified gRNAs. In established cells stably expressing Cas9, the circular gRNA in coordination with light irradiation could direct a precise cleavage of GFP and VEGFA within a pre-defined cutting region. We have also achieved light-mediated MSTN gene editing in embryos, whereas a new bow-knot-type gRNA showed no background editing in the absence of light irradiation. Together, our work provides a significantly improved method to precisely manipulate where and when genes are edited.


title: The ion permeability of DNA nanotube channels

abstract: Techniques from structural DNA nanotechnology make it possible to assemble complex 3-dimensional nanostructures with virtually arbitrary control over their sizes, shapes and features at length scales of 3-100 nm, providing a flexible means for constructing nanoscale devices and machines. Here, we assemble micron-length DNA nanotubes and assess their performance as pipes for controlled ion transport. DNA nanotubes grow via assembly of DNA tiles from a seed pore, a 12-helix DNA origami cylinder functionalized with cholesterol, to form a DNA nanotube channel. The central channel of a nanotube can be obstructed via Watson-Crick hybridization of a channel cap, a second DNA origami structure, to the end of a nanotube channel or a nanotube seed pore. Single-channel electrophysiological characterization shows that both nanotube seed pores and nanotube channels display ohmic ion conductance consistent with their central channels diameters. Binding of the channel cap reduces the conductances of both DNA nanotube channels and seed pores, demonstrating control of ion-transport through these micron-length channels. Because these channels could be assembled into branched architectures or routed between specific molecular terminals, these results suggest a route to self-assembling nanofluidic devices and circuits in which transport can be controlled using dynamic biomolecular interactions.

category: biophysics


title: Type III interferons induce pyroptosis in gut epithelial cells and delay tissue restitution upon acute intestinal injury.

abstract: Tissue damage and repair are hallmarks of the inflammatory process. Despite a wealth of information focused on the mechanisms that govern tissue damage, mechanistic insight on how inflammatory immune mediators affect the restitution phase is lacking. Here, we investigated how interferons influence tissue restitution after damage of the intestinal mucosa driven by inflammatory or physical injury. We found that type III, but not type I, interferons serve a central role in the restitution process. Type III interferons induce the upregulation of ZBP1, caspase activation, and cleavage of gasdermin C, and drive epithelial cell death by pyroptosis, thus delaying tissue restitution. We also found that this pathway is transcriptionally regulated in IBD patients. Our findings highlight a new molecular signaling cascade initiated by the immune system that affects the outcome of the immune response by delaying tissue repair and that may have important implications for human inflammatory disorders.

category: immunology


title: Specimen alignment with limited point-based homology: 3D morphometrics of disparate bivalve shells (Mollusca: Bivalvia)

abstract: 1.Comparative morphology fundamentally relies on the orientation and alignment of specimens. In the era of geometric morphometrics, point-based homologies are commonly deployed to register specimens and their landmarks in a shared coordinate system. However, the number of point-based homologies commonly diminishes with increasing phylogenetic breadth. These situations invite alternative, often conflicting, approaches to alignment. The bivalve shell (Mollusca: Bivalvia) exemplifies a homologous structure with few universally homologous points--only one can be identified across the Class, the shell  beak. Here, we develop an axis-based framework, grounded in the homology of shell features, to orient shells for landmark-based, comparative morphology. As the choice of homologous points for alignment can affect shape differences among specimens, so can the choice of orientation axes. Analysis of forty-five possible alignment schemes finds general conformity among the shape differences of  typical equilateral shells, but the shape differences among atypical shells can change considerably, particularly those with distinctive modes of growth. Each alignment implies a hypothesis about the ecological, developmental, or evolutionary basis of morphological differences, but we recognize one alignment in particular as a continuation of the historical approaches to morphometrics of shell form: orientation via the hinge line. Beyond bivalves, this axis-based approach to aligning specimens facilitates the comparison of continuous differences in shape among many other phylogenetically broad and morphologically disparate samples.

category: evolutionary biology


title: Tumor purity adjusted beta values improve biological interpretability of high-dimensional DNA methylation data

abstract: A common issue affecting DNA methylation analysis in tumor tissue is the presence of a substantial amount of non-tumor methylation signal derived from the surrounding microenvironment. Although approaches for quantifying and correcting for the infiltration component have been proposed previously, we believe these have not fully addressed the issue in a comprehensive and universally applicable way. We present a multi-population framework for adjusting DNA methylation beta values on the Illumina 450/850K platform using generic purity estimates to account for non-tumor signal. Our approach also provides an indirect estimate of the aggregate methylation state of the surrounding normal tissue. Using whole exome sequencing derived purity estimates and Illumina 450K methylation array data generated by The Cancer Genome Atlas project (TCGA), we provide a demonstration of this framework in breast cancer illustrating the effect of beta correction on the aggregate methylation beta value distribution, clustering accuracy, and global methylation profiles.

category: genomics


title: A dual-target herbicidal inhibitor of lysine biosynthesis

abstract: Herbicides with novel modes of action are urgently needed to safeguard global agricultural industries against the damaging effects of herbicide-resistant weeds. We recently developed the first herbicidal inhibitors of lysine biosynthesis, which provided proof-of-concept for a promising novel herbicide target (Soares da Costa et al., 2021). In this study, we expanded upon our understanding of the mode of action of herbicidal lysine biosynthesis inhibitors. We previously postulated that these inhibitors may act as proherbicides (Soares da Costa et al., 2021). Here we show this is not the case. We report an additional mode of action of these inhibitors, through their inhibition of a second lysine biosynthesis enzyme, and investigate the molecular determinants of inhibition. Furthermore, we extend our herbicidal activity analyses to include a weed species of global significance.


title: Activated I-BAR IRSp53 clustering controls the formation of VASP-actin-based membrane protrusions

abstract: Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodia initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro and in silico to unravel the mechanism of filopodia initiation driven by the membrane curvature sensor IRSp53. We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodia initiation.


title: Stimulus-dependent depth constancy during head tilt

abstract:
Stereopsis is traditionally measured with noise-based stereo tests while the observer views the test in primary gaze. We investigated the effect of stimulus sparseness and axial variations of interocular disparity induced via head rotations.

First, we measured stereoacuity using a 4-Alternative-Forced-Choice (4-AFC) task with three uncrossed and one crossed disparity bandpass-filtered circles on a passive-3-D-monitor. Ten binocularly-normal adults fixated a central cross and clicked on the circle withcrossed disparity for forty trials/condition. Observers adopted head tilts of 0{degrees} or {+/-}20{degrees} pitch, roll, or yaw, enforced with an innertial measurement unit and fixation enforced with an eye tracker. Next, we measured stereoacuity in 8 adults while either the head (H), monitor (M), or both (B) were tilted 0{degrees}, {+/-}22.5{degrees}, or {+/-}45{degrees} roll in random order (eighty trials/condition) using a 4-AFC task and random-dot stimuli. Head tilts did not signifcantly alter stereoacuity using narrow-band stimuli(p>0.05), despite that IPDs and the axis of disparity were differentially affected by the tilts. However, for random dot stimuli, stereoacuity decreased with increasing orientation difference between the head and monitor (H and M: p<0.05; B: p>0.05].

Head tilt decreases IPD and rotates the axis of interocular disparity, however, these manipulations affect stereoacuity when measured with noise stimuli but not with sparse stimuli. The results are consistent with a vestibular input to stereoscopic disparity processing that can be detected by sparse stimuli but is masked by dense stimuli. The results have implications for natural vision and for clinical screening in patients with abnormal head posture.

Significance statementDepth perception is a critical feature of human vision and it is thought that the ability to perceive stereoscopic depth is bound to an essentially eye-fixed, horizontal disparity of each image that rapidly deteriorates away from that limited horizontal axis. In a set of head tilt experiments, we varied the orientations of stereoscopic images and demonstrate that stereoacuity remains constant when deploying sparse narrow-band stimuli, and only worsens when using fine-detailed noise stimuli that mask off-axis disparities. These results shine new light upon the debate of neuroplasticity of stereo vision. Moreover, the results are consequential for diagnosis and treatment in people with atypical head- and eye alignment, such as for patients with torticollis or strabismus.

category: neuroscience


title: Lipobiotin-capture magnetic bead assay for isolation, enrichment and detection of Mycobacterium tuberculosis from saliva

abstract:
BackgroundPulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and the poor sensitivity. We present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test.

MethodsLipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and estimate the amount of mycobacterial DNA in different sample types.

ResultsWe found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to 8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts.

ConclusionsImplementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.

category: microbiology


title: Listeria monocytogenes gene essentiality under laboratory conditions and during macrophage infection

abstract: The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth in- and outside the host. We here have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach classified 394 open reading frames as essential for growth under standard laboratory conditions and identified 42 further genes, which become additionally essential during intracellular growth in macrophages. Most essential genes encode components of the translation machinery, act in chromosome-related processes, cell division and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions were also essential, opening novel options for the development of anti- listerial drugs. Among the genes specifically required during intracellular growth were known virulence factors, genes compensating intracellular auxotrophies and several cell division genes. Our experiments also highlight the importance of PASTA kinase signalling, glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.


title: Profiling Genetic Diversity Reveals the Molecular Basis for Balancing Function with Misfolding in Alpha-1 Antitrypsin

abstract: Genetic variation of alpha-1 antitrypsin (AAT) is responsible for alpha-1-antitrypsin deficiency (AATD) leading to gain-of-toxic aggregation in the liver and loss-of-function on neutrophil elastase (NE) inhibitory activity in the lung contributing to chronic obstructive pulmonary disease (COPD) during aging. To probe the molecular basis for how biology designs the protein fold to achieve balance between sequence, function and structure contributing to AATD in the population, we measured the intracellular monomer and polymer, secreted monomer and polymer and NE inhibitory activity of 75 alpha-1-antitrypsin (AAT) variants. To address the complex folding dynamics affecting the form and function of the protein fold that is differentially impacted by variants in the population, we applied a Gaussian process regression (GPR) based machine learning approach termed variation spatial profiling (VSP). By using a sparse collection of extant variants to link genotype to phenotype, VSP maps spatial covariance (SCV) relationships that quantitate the functional value of every residue in the wild-type (WT) AAT sequence with defined uncertainty in the context of its protein fold design. The SCV-based uncertainty allows us to pinpoint critical short- and long-range residue interactions involving 3 regions-the N-terminal (N1), middle (M2) and carboxyl-terminal (C3) of AAT polypeptide sequence that differentially contribute to the balance between function and misfolding of AAT, thus providing an unanticipated platform for precision therapeutic development for liver and lung disease. By understanding mechanistically the complex fold design of the metastable WT AAT fold, we posit that GPR-based SCV provides a foundation for understanding the evolutionary design of the fold from the ensemble of structures found in the population driving biology for precision management of AATD in the individual.

category: molecular biology


title: Hepatic Deletion of X-box Binding Protein 1 in Farnesoid X Receptor Null Mice Leads to Enhanced Liver Injury

abstract:
Background & AimsFarnesoid X receptor (FXR) regulates bile acid metabolism and FXR null (Fxr-/-) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) pathway is a protective pathway of the unfolded protein response (UPR) that is activated in response to ER stress. In this study we sought to determine the role of the UPR in Fxr-/- mice.

Approach & ResultsWe examined hepatic UPR gene and protein expression in 10- and 24-week-old wild type (WT) and Fxr-/- mice. Hepatic XBP1 and other UPR pathways were activated in 24-week-old Fxr-/- mice, but not WT mice. To further determine the role of the liver UPR activation in Fxr-/- mice, we generated mice with FXR and liver-specific XBP1 double knockout (DKO, Fxr-/-Xbp1LKO) and Fxr-/- Xbp1fl/fl single knockout (SKO) mice and characterized their phenotypes at different ages. DKO mice demonstrated enhanced liver injury, apoptosis and fibrosis compared with SKO mice. RNA-seq revealed increased gene expression in apoptosis, inflammation and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein (CHOP) pathway was activated in DKO mice. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors.

ConclusionsThe hepatic XBP1 pathway is activated in older Fxr-/- mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.


title: RNA G-quadruplex structure contributes to cold adaptation in plants

abstract: Nucleotide composition is suggested to infer gene functionality and ecological adaptation of species to distinct environments. However, the underlying biological function of nucleotide composition dictating environmental adaptations is largely unknown. Here, we systematically analyzed the nucleotide compositions of transcriptomes across 1000 plants (1KP) and their corresponding habitats. Intriguingly, we found that plants growing in cold climates have guanine (G)-enriched transcriptomes, which are prone to forming RNA G-quadruplex structures. Both immunofluorescence detection and in vivo structure profiling revealed that RNA G-quadruplex formation in plants was globally enhanced in response to cold. Cold-responsive RNA G-quadruplexes strongly enhanced mRNA stability, rather than affecting translation. Disruption of individual RNA G-quadruplex promoted mRNA decay in the cold, leading to impaired plant cold response. Therefore, we propose that plants adopted RNA G-quadruplex structure as a molecular marker to facilitate their adaptation to the cold during evolution.


title: Lung injury epigenetically primes mesenchyme for amplified activation upon re-injury

abstract: The lungs have a remarkable capacity to repair. However, repetitive injury can lead to progressive fibrosis and end-stage organ failure. Whether tissue-resident mesenchymal cell populations retain epigenetic memory of prior injuries that contribute to this pathological process is unknown. Here we used a genetic lineage labeling approach to mark the lung mesenchyme prior to injury, then performed multi-modal analyses on isolated lung mesenchyme during the initiation, progression and resolution of the fibrotic response. Our results demonstrate the remarkable epigenetic and transcriptional plasticity of the lung mesenchyme during fibrogenic activation and de-activation. Despite this plasticity, we also find that the lung mesenchyme retains specific epigenetic traits (memory) of prior activation, resulting in amplified induction of a fibrogenic program upon re-injury. We identify Runx1 as a critical driver of both fibrogenic activation and epigenetic memory. Comparison of fresh isolated and cultured lung mesenchyme demonstrates that Runx1 is spontaneously activated in standard culture conditions, previously masking these roles of Runx1. Genetic and pharmacological targeting of Runx1 dampens fibrogenic mesenchymal cell activation in cell and tissue models, confirming its functional importance. Finally, publicly available scRNAseq data reveal selective expression of Runx1 in the fibrogenic cell subpopulations that emerge in mouse and human fibrotic lung tissue. Collectively, our findings implicate Runx1 in both the initiation and memory of fibrogenic mesenchymal cell activation that together prime amplified mesenchymal cell responses upon repeated injury.


title: ReLo: a simple colocalization assay to identify and characterize physical protein-protein interactions

abstract: Characterizing protein-protein interactions (PPIs) is fundamental for understanding biochemical processes. Many methods have been established to identify and study direct PPIs; however, screening and investigating PPIs involving large and poorly soluble proteins remain challenges. As a result, we developed ReLo, a simple cell culture-based method to detect and investigate interactions in a cellular context. ReLo allows both the identification of protein domains that mediate the formation of complexes and screening of interfering point mutations. ReLo is sensitive to drugs that mediate or interfere with a specific interaction. Furthermore, protein conformation- and protein arginine methylation-dependent interactions can be studied. Importantly, ReLo can be used for specifically detecting direct but not indirect PPIs and can be applied for describing the binding topology of subunits within multiprotein complexes. Because of these attributes, ReLo is a simple, quick and versatile tool for identifying and studying binary PPIs, as well as for characterizing multisubunit complexes.


title: Confinement of unliganded EGFR by tetraspanin nanodomains gates EGFR ligand binding and signaling

abstract: The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology. EGFR is activated by ligand binding, triggering receptor dimerization, activation of kinase activity, and intracellular signaling. EGFR is transiently confined within various plasma membrane nanodomains, yet how this may contribute to regulation of EGFR ligand binding is poorly understood. To resolve how EGFR nanoscale compartmentalization gates ligand binding, we developed single-particle tracking methods to track the mobility of ligand-bound and total EGFR, in combination with modeling of EGFR ligand binding. In comparison to unliganded EGFR, ligand-bound EGFR was more confined and distinctly regulated by clathrin and tetraspanin nanodomains. Ligand binding to unliganded EGFR occurred preferentially in tetraspanin nanodomains, and disruption of tetraspanin nanodomains impaired EGFR ligand binding and altered the conformation of the receptors ectodomain. We thus reveal a novel mechanism by which EGFR confinement within tetraspanin nanodomains regulates receptor signaling at the level of ligand binding.


title: Connecting the Dots: PHF13 and cohesin promote polymer-polymer phase separation of chromatin into chromosomes

abstract: How interphase chromatin compacts into mitotic chromosomes has eluded researchers for over a century. Here we show that PHF13, a tightly regulated H3K4me epigenetic reader, and cohesin, a mediator of chromatin architecture, cooperate to drive polymer-polymer-phase-separation (PPPS) and higher order compaction of chromatin into chromosomes. PHF13 interacts with cohesin, shows similar dynamics during mitosis, and their co-depletion dramatically impairs mitotic condensation. Mechanistically, we demonstrate that PHF13 stabilizes cohesin chromatin interactions and that itself oligomerizes, resulting in a polymer with increased chromatin avidity and the ability to bridge neighboring and distant nucleosomes. Consistently, molecular dynamic simulations modelling the ability of PHF13 and cohesin to drive chromatin phase separations recapitulated our in vivo observations and are in line with 2-step condensation models.


title: Conserved Pib2 regions have distinct roles in TORC1 regulation at the vacuole

abstract: TORC1 is a critical controller of cell growth in eukaryotes. In yeast, the presence of nutrients is signaled to TORC1 by several upstream regulatory sensors that together coordinate TORC1 activity. TORC1 localizes to both vacuolar and endosomal membranes, where differential signaling occurs. This localization is mimicked by Pib2, a key upstream TORC1 regulator that is essential for TORC1 reactivation after nutrient starvation or pharmacological inhibition. Pib2 has both positive and negative effects on TORC1 activity, but the mechanisms remain poorly understood. Here, we pinpoint the Pib2 inhibitory function on TORC1 to residues within short, conserved N-terminal regions. We also show that Pib2 C-terminal regions, helical region E and tail, are essential for TORC1 reactivation. Further, the Pib2 FYVE domain is essential for vacuolar localization, however, it is surprisingly unnecessary for recovery from rapamycin exposure. Using chimeric Pib2 targeting constructs, we show that endosomal localization is not necessary for TORC1 reactivation and cell growth after rapamycin treatment. Thus, a comprehensive molecular dissection of Pib2 demonstrates that each of its conserved regions differentially contribute to Pib2 regulation of TORC1 activity.


title: Temporal and spatial dynamics mapping reveals follicle development regulated by different stromal cell populations

abstract: Follicle development is a complex dynamic process. The follicles are encapsulated in the stroma, and once the follicle develops, the follicle moves from the cortex to the medulla and finally to the cortex to ovulate in the process of continuous growth. Many of these processes cannot be explained with the follicle alone. Through single-cell and spatial transcriptome sequencing at key time points of follicular development in mice after birth, we found that ovarian stromal cells are not only one of the main cell groups that make up the ovary but that their cell population and spatial location are also closely related to follicular development. Through analysis of cell communication, it was found that ovarian stromal cells were the main transmitters of intercellular communication, and many of the signals they sent were received by granulosa cells and oocytes to participate in follicle development. Ovarian stromal cells are not a homogeneous cell population. We combined single cell types with their spatial location information to divide ovarian stromal cells into four types, namely, structural stromal cells, perifollicular stromal cells, stromal progenitor cells, and steroidogenic stromal cells, each of which plays a different function in follicle development. Indepth studies of the different spatial locations and different types of stromal cells will expand our understanding of follicle development dynamics, leading to new targets and novel approaches for the treatment of ovarian-related diseases.


title: Efficient quantification of lipid packing defect sensing by amphipathic peptides; comparing Martini 2 & 3 with CHARMM36

abstract:
In biological systems, proteins can be attracted to curved or stretched regions of lipid bilayers by sensing hydrophobic defects in the lipid packing on the membrane surface. Here, we present an efficient end-state free energy calculation method to quantify such sensing in molecular dynamics simulations. We illustrate that lipid packing defect sensing can be defined as the difference in mechanical work required to stretch a membrane with and without a peptide bound to the surface. We also demonstrate that a peptides ability to concurrently induce excess leaflet area (tension) and elastic softening - a property we call the  characteristic area of sensing (CHAOS) - and lipid packing sensing behavior are in fact two sides of the same coin. In essence, defect sensing displays a peptides propensity to generate tension. The here-proposed mechanical pathway is equally accurate yet, computationally, about 40 times less costly than the commonly used alchemical pathway (thermodynamic integration), allowing for more feasible free energy calculations in atomistic simulations. This enabled us to directly compare the Martini 2 and 3 coarse-grained and the CHARMM36 atomistic force-fields in terms of relative binding free energies for six representative peptides including the curvature sensor ALPS and two antiviral amphipathic helices (AH). We observed that Martini 3 qualitatively reproduces experimental trends, whilst producing substantially lower (relative) binding free energies and shallower membrane insertion depths compared to atomistic simulations. In contrast, Martini 2 tends to overestimate (relative) binding free energies. Finally, we offer a glimpse into how our end-state based free energy method can enable the inverse design of optimal lipid packing defect sensing peptides when used in conjunction with our recently developed Evolutionary Molecular Dynamics (Evo-MD) method. We argue that these optimized defect sensors - aside from their biomedical and biophysical relevance - can provide valuable targets for the development of lipid force-fields.

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title: Loss of the adhesion protein Kindlin-1 stimulates tumor clearance via modulation of Tregs

abstract: The adhesion protein Kindlin-1 is over-expressed in breast cancer where it has been shown to be associated with metastasis-free survival, however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in a mouse model of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells leads to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs and impairment of their immune-suppressive activities in Kindlin-1 depleted tumors. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of mammary tumorigenesis. Analysis of cytokines secreted from the Met-1 cells identified a significant increase in IL-6 secretion when Kindlin-1 was depleted. Conditioned media from Kindlin-1 depleted cells lead to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6 and depletion of CD25+ Tregs resulted in a reduction of Met-1 tumor growth in mice. Overall, these data identify a novel function for Kindlin-1 in the regulation of anti-tumor immunity through cytokine regulation of Treg number and function.

category: cancer biology


title: The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes

abstract:
O_LINitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2-fixing clade retained nodulation and diversified as stable nodulators while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades.
C_LIO_LIWe characterize two nodule anatomy types across 128 species in 56 of the 150 genera of the legume subfamily Caesalpinioideae: 1) fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads and 2) symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes.
C_LIO_LIUsing a robust phylogenomic tree based on 997 genes from 146 caesalpinioid genera, we show that losses of nodulation are more prevalent in lineages with FTs.
C_LIO_LIWe propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through greater compartmentalisation of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical clade of legumes.
C_LI


title: The evolution of ageing in cooperative breeders

abstract: Cooperatively breeding animals live longer than their solitary counterparts. The traditional explanation for this is that cooperative breeding evolves more readily in long-lived species. Here, we reverse this argument and show that long lifespans are an evolutionary consequence of cooperative breeding. Natural selection favours a delayed onset of senescence in cooperative breeders, relative to solitary breeders, because cooperative breeders have a delayed age of first reproduction due to reproductive queueing. Especially long lifespans evolve in cooperative breeders with age-dependent reproductive queueing. Finally, we show that lower genetic relatedness among group members leads to the evolution of longer lifespans. This is because selection against higher mortality is weaker when mortality reduces competition between relatives. Our results link the evolutionary theory of ageing with kin selection theory, demonstrating that the evolution of ageing in cooperative breeders is driven by the timing of reproduction and kin structure within breeding territories.


title: High salt intake activates the hypothalamic-pituitary-adrenal axis, amplifies the stress response, and alters tissue glucocorticoid exposure in mice

abstract:
High salt intake is common and contributes to poor cardiovascular health. Sustained cortisol excess also induces an adverse cardiovascular profile. Urinary cortisol excretion positively correlates with urinary sodium excretion. We hypothesised that this was due to hypothalamic-pituitary-adrenal axis activation by high salt intake.

In male C57BL6/J mice, 2 weeks of high salt intake increased Crh and Pomc mRNA abundance in the hypothalamus and anterior pituitary, respectively and caused a sustained rise in plasma corticosterone. Plasma copeptin and anterior pituitary V1b receptor mRNA expression was elevated, which may contribute to basal HPA axis activation. Additionally, high salt intake amplified glucocorticoid response to restraint stress, indicative of enhanced HPA axis sensitivity. In the periphery, high salt intake reduced the binding capacity of corticosteroid-binding globulin, enhancing glucocorticoid bioavailability. Within several tissues, the expression of glucocorticoid-regenerating enzyme, 11{beta}-hydroxysteroid dehydrogenase type 1, was increased and the glucocorticoid receptor downregulated. Overall, high salt intake increased glucocorticoid exposure in the hippocampus, anterior pituitary and liver.

Chronic high salt intake amplifies basal and stress-induced glucocorticoid levels and resets glucocorticoid biology centrally, peripherally and within cells. This shows direct connectivity between salt homeostasis and HPA axis function. The cumulative effect is likely maladaptive and may contribute to the long-term health consequences of a high salt diet.

category: physiology


title: Pinch2 is a novel regulator of myelination in the Central Nervous System

abstract:
The extensive morphological changes of oligodendrocytes during axon ensheathment and myelination involve assembly of the Ilk-Parvin-Pinch (IPP) heterotrimeric complex of proteins to relay essential mechanical and biochemical signals between integrins and the actin cytoskeleton. Binding of Pinch 1 and 2 isoforms to Ilk is mutually exclusive and allows the formation of distinct IPP complexes with specific signaling properties. Using tissue-specific conditional gene ablation in mice, we reveal an essential role for Pinch2 during central nervous system myelination. Unlike Pinch1-gene ablation, loss of Pinch2 in oligodendrocytes results in hypermyelination and in the formation of pathological myelin outfoldings in white matter regions. These structural changes concurred with inhibition of Rho GTPases RhoA and Cdc42 activities and phenocopied aspects of myelin pathology observed in corresponding mouse mutants. We propose a dual role for Pinch2 in preventing excess of myelin wraps through RhoA-dependent control of membrane growth and in fostering myelin stability via Cdc42-dependent organization of cytoskeletal septins. Together, these findings indicate that IPP-containing Pinch2 is a novel critical cell-autonomous molecular hub ensuring synchronous control of key signaling networks during developmental myelination.

Summary statementPinch proteins are core components of a ternary protein complex comprising Ilk and Parvin (IPP). This work identifies Pinch2 as key regulator of the formation and maturation of CNS myelin.


title: Neural representations of vicarious rewards are linked to interoception and prosocial behaviour

abstract:
From  likes on social media, to seeing other people eat in a restaurant, every day we constantly observe other people receiving rewards. Theoretical accounts posit that vicarious processing of these rewards might be linked to peoples sensitivity to internal body states (interoception) and facilitates a tendency to act prosocially to obtain positive outcomes for others. However, the neural processes underlying the links between vicarious reward processing, interoception and prosocial behaviour are poorly understood. Previous research has linked vicarious reward processing to the anterior cingulate gyrus (ACCg) and the anterior insula (AI). Can we predict someones tendency to be prosocial, or their propensity to be aware of interoceptive signals, from variability in how the ACCg and AI process other peoples rewards? Here, participants monitored rewards being delivered to themselves or an anonymous stranger during functional magnetic resonance imaging (fMRI). In a separate session, they performed a task measuring their willingness to be prosocial and exert effort to obtain rewards for others, and a task measuring their propensity to be aware and use interoceptive signals. Using multivariate similarity analysis, we show that peoples willingness to be prosocial is predicted by greater similarity between self and other representations in the ACCg. Moreover, greater dissimilarity in self-other representations in the AI is linked to a greater interoceptive propensity. These findings highlight that vicarious reward representations are linked to bodily signals in AI, and foster prosocial tendencies through the ACCg, with such information represented when simply seeing stimuli that indicate other peoples rewards.

Significance StatementEvery day we see other people receiving rewards. Theories suggest that how we think about these stimuli is linked to our tendency to help others (prosociality), and how strongly we rely on bodily (interoceptive) signals to guide behaviour. Can we predict someones degree of prosociality, or propensity to be aware of interoceptive signals, from how similarly their brain processes other peoples rewards to their own? Here, we show that responses in the anterior cingulate cortex and anterior insula when simply being told that an anonymous other person or oneself is receiving rewards, can predict how prosocial someone is and their level of interoceptive propensity, respectively. Simply viewing others getting rewarded fosters prosociality and varies with bodily signals.


title: Lyophilization induces alterations in cryptococcal exopolysaccharide resulting in reduced antibody binding

abstract: The structural, antigenic, and immunological characterization of microbial polysaccharides requires purification that often involves detergent precipitation and lyophilization. Here we examine physicochemical changes induced by lyophilization on exopolysaccharide (EPS) of the pathogenic fungus Cryptococcus neoformans. Solution 1H NMR reveals significant anomeric signal attenuation following lyophilization of native EPS while 1H ssNMR shows few changes, suggesting diminished molecular motion and consequent broadening of 1H NMR polysaccharide resonances. 13C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense rosette-like aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide binds the native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.


title: Post-weaning parvalbumin expression in left orbitofrontal cortex shapes sociability in female mice

abstract: The adolescent social experience is essential for the maturation of the prefrontal cortex (PFC) in mammalian species. However, it is still unclear which cortical circuits mature with such experience and how it shapes adult social behaviors in a sex-specific manner. Here, we found that the post-weaning social isolation during adolescence (PWSI) caused hypersociability in female mice only and impaired both the parvalbumin (PV) expression and social sniffing-induced activity in the left orbitofrontal cortex (OFCL). Similarly, the CRISPR/Cas9-mediated knock-down of PV in the OFCL enhanced sociability and reduced the social sniffing-induced activity in female mice via decreased excitability of PV+ neurons and reduced synaptic inhibition in the OFCL. Moreover, optogenetic activation of OFCL excitatory neurons enhanced sociability in female mice. Our data demonstrate that the adolescent social experience is critical for the maturation of PV+ inhibitory circuits in the OFCL; this maturation shapes female social behavior via enhancing social representation in the OFCL.

category: animal behavior and cognition


title: Mouse Genomic Associations with Ex Vivo Sensitivity to Simulated Space Radiation

abstract: Exposure to ionizing radiation is considered by NASA to be a major health hazard for deep space exploration missions. Ionizing radiation sensitivity is modulated by both genomic and environmental factors. Understanding their contributions is crucial for designing experiments in model organisms, evaluating the risk of deep space (i.e. high-linear energy transfer, or LET, particle) radiation exposure in astronauts, and also selecting therapeutic irradiation regimes for cancer patients. We identified single nucleotide polymorphisms in 15 strains of mice, including 10 collaborative cross model strains and 5 founder strains, associated with spontaneous and ionizing radiation-induced ex vivo DNA damage quantified based on immunofluorescent 53BP1+ nuclear foci. Statistical analysis suggested an association with pathways primarily related to cellular signaling, metabolism, tumorigenesis and nervous system damage. We observed different genomic associations in early (4 and 8 hour) responses to different LET radiation, while later (24 hour) DNA damage responses showed a stronger overlap across all LETs. Furthermore, a subset of pathways was associated with spontaneous DNA damage, suggesting 53BP1+ foci as a potential biomarker for DNA integrity in mouse models. Based on our results, we suggest several mouse strains as new models to further study the impact of ionizing radiation and validate the identified genetic loci. We also highlight the importance of future human ex vivo studies to refine the association of genes and pathways with the DNA damage response to ionizing radiation and identify targets for space travel countermeasures.


title: Nanopore quality score resolution can be reduced with little effect on downstream analysis

abstract: We investigate the effect of quality score information loss on downstream analysis from nanopore sequencing FASTQ files. We polished denovo assemblies for a mock microbial community and a human genome, and we called variants on a human genome. We repeated these experiments using various pipelines, under various coverage level scenarios, and various quality score quantizers. In all cases we found that the quantization of quality scores cause little difference on (or even improves) the results obtained with the original (non-quantized) data. This suggests that the precision that is currently used for nanopore quality scores is unnecessarily high, and motivates the use of lossy compression algorithms for this kind of data. Moreover, we show that even a non-specialized compressor, like gzip, yields large storage space savings after quantization of quality scores.

category: bioinformatics


title: Tyrosine phosphorylation tunes chemical and thermal sensitivity of TRPV2 ion channel

abstract: Transient receptor potential vanilloid 2 (TRPV2) is a multimodal ion channel widely regulating central and peripheral functions. Its important involvement in immune responses has been suggested such as in the macrophages phagocytosis process. However, the endogenous signaling cascades controlling the gating of TRPV2 remain to be understood. Here, we report that enhancing tyrosine phosphorylation remarkably alters the chemical and thermal sensitivities of TRPV2 endogenously expressed in rat bone marrow-derived macrophages. We identify that the protein tyrosine kinase JAK1 mediates TRPV2 phosphorylation at the molecular sites Tyr(335), Tyr(471), and Tyr(525). JAK1 phosphorylation is required for maintaining TRPV2 activity and the phagocytic ability of macrophages. We further show that TRPV2 phosphorylation is dynamically balanced by protein tyrosine phosphatase non-receptor type 1 (PTPN1). PTPN1 inhibition increases TRPV2 phosphorylation, further reducing the activation temperature threshold to [~]40 {degrees}C. Our data thus unveil an intrinsic mechanism where the phosphorylation/dephosphorylation dynamic balance sets the basal chemical and thermal sensitivity of TRPV2. Targeting this pathway will aid therapeutic interventions in physiopathological contexts.


title: Molecular mechanisms of long-term light adaptation of an extremophilic alga Cyanidioschyzon merolae

abstract: Oxygenic phototrophs have evolved a remarkable plethora of strategies to react to changes in light intensity and spectral range, which allows them to thrive in a wide range of environmental conditions. Varying light quality and quantity influences the balance between solar energy capture and utilisation in photosynthesis, affecting concomitantly the downstream processes of central carbon and nitrogen metabolism as well as cellular growth and division. Here, we performed a comprehensive analysis of the mechanisms of long-term photoacclimation of an extremophilic red alga Cyanidioschyzon merolae that grows in sulphuric hot springs at high temperatures and low pH. By using spectroscopic, confocal fluorescence microscopy, photosynthetic performance measurements and global transcriptome analyses, we identified several molecular mechanisms underlying the long-term adaptation of this acido-thermophilic red alga to varying light intensity and spectral quality. These include: (1) remodelling of the functional antenna size of both photosystems; (2) rearrangement of the PSB/PSII/PSI microdomains within thylakoids; (3) modulation of the photosynthetic performance parameters, especially at the level of non-photochemical quenching, and (4) transcriptional regulation of photosynthesis and its regulatory components as well as downstream metabolic pathways related to ROS detoxification, cell/organelle division, and central carbon and nitrogen metabolism. Such an intricate network of interplay between light-driven reactions and downstream metabolic pathways provides the necessary basis for maintaining the highest photosynthetic performance under light-limiting conditions.


title: Parallel CRISPR-Cas guided mutagenesis of chromosome and virulence plasmid in Shigella flexneri by cytosine base editing

abstract:
Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrhoeal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by sgRNA to introduce targeted CT transitions, resulting in internal STOP codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter assay, we were able to generate loss-of-function mutants in both E. coli and Shigella with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion is in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Further, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella genes and found the mutant phenotype to be similar to that of the reported gene deletion mutants, with no apparent polar effects. This method can be used in a 96-well plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days.

ImportanceLoss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches system biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen - Shigella. This pathogen causes high morbidity and mortality in children and antibiotic resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method can be programmed to generated multiple mutants in a single, massively parallel, reaction. By the virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.


title: FAM172A controls the nuclear import and alternative splicing function of AGO2

abstract: The poorly characterized protein FAM172A is mutated in some individuals affected by a disorder of neural crest development called CHARGE syndrome. We also know that FAM172A can interact with the main CHARGE syndrome-associated protein CHD7 and the small RNA-binding protein AGO2 at the chromatin-spliceosome interface. Focusing on this intriguing FAM172A-AGO2 interaction, we now report that FAM172A is one of the long sought-after regulator of AGO2 nuclear import. This FAM172A function relies on its nuclear localization signal, being enhanced by CK2-mediated phosphorylation and abrogated by a CHARGE syndrome-associated missense mutation. Accordingly, Fam172a and Ago2 genetically interact in mice, and neural crest-specific depletion of Ago2 is sufficient to phenocopy CHARGE syndrome without impacting post-transcriptional gene silencing. Rapamycin-mediated rescue suggests that observed morphological anomalies are instead due to alternative splicing defects. This work thus demonstrates that non-canonical nuclear functions of AGO2 and associated regulatory mechanisms may be clinically relevant.


title: Deep Learning for MYC binding site recognition

abstract:
MotivationThe definition of the genome distribution of the Myc transcription factor is extremely important since it may help predict its transcriptional activity particularly in the context of cancer. Myc is among the most powerful oncogenes involved in the occurrence and development of more than 80% of different types of pediatric and adult cancers. Myc regulates thousands of genes which can be in part different, depending on the type of tissues and tumours. Myc distribution along the genome has been determined experimentally through chromatin immunoprecipitation This approach, although powerful, is very time consuming and cannot be routinely applied to tumours of individual patients. Thus, it becomes of paramount importance to develop in-silico tools that can effectively and rapidly predict its distribution on a given cell genome. New advanced computational tools (DeeperBind) can then be successfully employed to determine the function of Myc in a specific tumour, and may help to devise new directions and approaches to experiments first and personalized and more effective therapeutic treatments for a single patient later on.

ResultsThe use of DeeperBind with DeepRAM on Colab platform can effectively predict the binding sites for the MYC factor with an accuracy above 0.96 AUC, when trained with multiple cell lines. The analysis of the filters in DeeperBind trained models shows, besides the consensus sequence CACGTG classically associated to the MYC factor, also the other consensus sequences G/C box or TGGGA, respectively bound by the SP1 and MIZ-1 transcription factors, which are known to mediate the MYC repressive response. Overall, our findings suggest a stronger sinergy between the machine learning tools as DeeperBind and biological experiments, which may reduce the time consuming experiments by providing a direction to guide them.


title: Visual Perception of 3D Space and Shape in Time - Part I: 2D Space Perception by 2D Linear Translation

abstract:
Visual perception plays a critical role in navigating space and extracting useful semantic information crucial to survival. To identify distant landmarks, we constantly shift gaze vectors through saccades, while still maintaining the visual perception of stable allocentric space. How can we sustain stable allocentric space so effortlessly? To solve this question, we have developed a new concept of NHT (Neural Holography Tomography). This model states that retinotopy is invisible (not available to consciousness) and must be converted to a time code by traveling alpha brainwaves to perceive objects consciously. According to this framework, if identical alpha phases are continually assigned to a landmark, we perceive its exact and consistent allocentric location.

To test this hypothesis, we designed reaction time (RT) experiments to observe evidence of the predicted space-to-time conversion. Various visual stimuli were generated at a wide range of eccentricities either on a large TV (up to 40{degrees}) or by LED strips on a hemispherical dome (up to 60{degrees}). Participants were instructed to report the observed patterns promptly under either covert (no eye movement) or overt (with eye movement) conditions. As predicted, stimuli presented at the center of fixation always produced the fastest RTs. The additional RT delay was precisely proportional to the eccentricity of the peripheral stimulus presentation. Furthermore, both covert and overt attention protocols created the same RT delays, and trajectories of saccadic eye motions were in parallel to the overt RT vs. eccentricity. These findings strongly support our NHT model, in which the observed RT-eccentricity dependence is indicative of the spatiotemporal conversion required for maintaining a stable allocentric frame of reference. That is, we perceive space by time.


title: A method to estimate the effective point spread function of static single molecule localization microscopy images

abstract:
Single molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM dataset is an essential aspect of the image processing and analysis pipeline. Here we present a simple method that uses the pair autocorrelation function evaluated both in space and time to measure the time-interval dependent effective point spread function of SMLM images of static samples. Using this approach, we demonstrate that experimentally obtained images typically have effective point spread functions that are broader than expected from the localization precision alone, due to additional uncertainty arising from factors such as drift and drift correction algorithms. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labelled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins.

STATEMENT OF SIGNIFICANCESingle molecule localization microscopy (SMLM) is a class of imaging methods that resolve fluorescently labeled structures beyond the optical resolution limit of visible light. SMLM detects stochastically blinking labels over time, and localizes each blink with precision of order 10 nm. The effective resolution depends on factors such as signal-to-noise ratio, localization algorithm, and several post-processing steps such as stage drift correction. We present a method to evaluate this effective resolution by taking advantage of temporal correlations of fluorophore blinking to separate the distribution of pairs of localizations from the same molecule from those from different molecules. The method is robust on useful timescales for a range of SMLM probes.


title: Theoretical Quantification of the Polyvalent Binding of Nanoparticles Coated with Peptide-MHC to TCR-Nanoclusters

abstract: Nanoparticles (NPs) coated with pMHCs can reprogram a specific type of CD4+ T cells into diseasesuppressing T regulatory type 1 cells by binding to their TCRs expressed as TCR-nanoclusters (TCRnc). NP size and number of pMHCs coated on them (called valence) can be adjusted to increase their efficacy. Here we explore how this polyvalent interaction is manifested and examine if it can facilitate T cell activation. This is done by developing a multiscale biophysical model that takes into account the complexity of this interaction. Using the model, we quantify pMHC insertion probabilities, dwell time of NP binding, TCRnc carrying capacity, the distribution of covered and bound TCRs by NPs, and cooperativity in the binding of pMHCs within the contact area. Model fitting and parameter sweeping further reveal that moderate jumps between IFN{gamma} dose-response curves at low valences can occur, suggesting that the geometry of NP binding can prime T cells for activation.


title: Rapid in vivo multiplexed editing (RIME) of the adult mouse liver

abstract:
Background & AimsAssessing mammalian gene function in vivo has traditionally relied on manipulation of the mouse genome in embryonic stem cells or peri-zygotic embryos. These approaches are time consuming and require extensive breeding when simultaneous mutations in multiple genes is desired. The aim of this study is to introduce a Rapid In vivo Multiplexed Editing (RIME), and to provide a proof-of-concept of this system.

Approach & ResultsRIME, a system wherein CRISPR/Cas9 technology, paired with adeno-associated viruses (AAVs), permits the inactivation of one or more genes in the adult mouse liver. The method is quick, requiring as little as 1 month from conceptualization to knockout (KO), and highly efficient, enabling editing in >95% of target cells. To highlight its utility, we used this system to inactivate, alone or in combination, genes with functions spanning metabolism, mitosis, mitochondrial maintenance, and cell proliferation.

ConclusionRIME enables the rapid, efficient, and inexpensive analysis of multiple genes in the mouse liver in vivo.


title: Four tumor micro-environmental niches explain a continuum of inter-patient variation in the macroscopic cellular composition of breast tumors.

abstract:
The tumor microenvironment is a complex, self-organising tissue whose architecture determines prognostic and response to therapy. There is significant variation in the cellular and spatial architecture of the tumor-microenvironment within and across patients. Are there rules that constrain the architecture of the tumor-microenvironment?

To find out, we develop a quantitative framework of tumor architecture inspired by ideas from satellite imaging, which we apply on deep single-cell profiling and multiplex imaging data in breast tumors. Data analysis shows that inter-patient variation in the macroscopic cellular composition of tumors is structured as a continuum explained by four tumor niches: cancer, inflammatory, tertiary lymphoid structure and fibrotic/necrotic. These niches have their origin in tumor micro-architecture and are shared across patients and tumor subtypes. Niche prevalence depends strongly on the patient, which constraints and explains inter-patient variation.

The present framework will facilitate interpreting inter-patient variation in terms of a tractable number of micro-environmental niches which serve as organizational entities at the meso-scale to bridge the micro- and macro-scales of tumor architecture.

category: systems biology


title: The onset of hearing triggers nuclear translocation in inner hair cells

abstract:
Nuclear position is precisely orchestrated during cell division, migration, and maturation of cells and tissues. Here we report a previously unrecognized, programmed movement of the nucleus in rat cochlear inner hair cells. This cell-type specific nuclear movement among hair cells of the inner ear offers a new glimpse into the cellular mechanisms involved in the functional maturation of inner hair cells. In early post-natal days, the nucleus sits at the very basal pole of the hair cell, far from the apically located mechano-transducing stereocilia, but close to where synapses with primary afferent and efferent neurons are forming. By maturity, the nucleus moves to occupy a new position half-way along the length of the cell. Remarkably, nuclear translocation happens quickly over the course of 1-2 post-natal days, coinciding with the onset of hearing. The movement of the nucleus likely signals the closing of a critical period correlated with the forming and refining of synapses at the onset of hearing.

Significance StatementThe misplacement of a cells nucleus has been implicated in hearing, vision, and muscular pathologies. In this study, we report a remarkably abrupt change in nuclear position in cochlear inner hair cells that coincides with the onset of hearing in rats, when hair cells switch from producing sensory-independent action potentials to sensory-dependent graded potentials. This work suggests that post-mitotic nuclear migration may be critical to the final phase of post-natal development in mammalian hearing.


title: The 13A4 monoclonal antibody to the mouse PROM1 protein recognizes a structural epitope

abstract:
PurposeWe endeavored to map the epitope of the rat monoclonal antibody mAB 13A4 to the mouse PROM1 (CD133, AC133) protein. mAB 13A4 is the main reagent used to detect the mouse PROM1 protein. PROM1 is required for the maintenance of primary cilia and mutations in the Prom1 gene are associated with retinal degeneration.

MethodsEpitope tagged clones of splice variants and tiled deletion mutants were used to map the mAB 13A4 epitope and test the predicted tertiary structure of PROM1. The proteins were expressed in Neuro 2a cells and analyzed by Western blot with antibodies to PROM1 and the epitope tag.

ResultsDeletions in the second and third extracellular domains of the PROM1 protein disrupted the mAB 13A4 epitope. Furthermore, the affinity of mAB 13A4 to the major PROM1 isoform in photoreceptor cells is significantly reduced due to the inclusion of a photoreceptor-specific alternative exon in the third extracellular domain. Interestingly, a deletion in the photoreceptor specific isoform of six amino acids adjacent to the alternative exon restored the affinity of mAB 13A4 to PROM1.

ConclusionmAB 13A4 recognizes a structural epitope that is stabilized by two of the extracellular domains of PROM1. The results of our mutagenesis are consistent with the computationally predicted helical bundle structure of PROM1 and point to the utility of mAB 13A4 for evaluating the effect of mutations on the PROM1 structure. We show that the PROM1 isoform composition needs to be considered when interpreting tissue and developmental expression data produced by mAB 13A4.


title: NKG2A and HLA-E define a novel alternative immune checkpoint axis in bladder cancer

abstract: PD-1/PD-L1-blockade immunotherapies have limited efficacy in the treatment of muscle-invasive bladder cancer (MIBC) and metastatic urothelial carcinoma. Here, we show that KLRC1 (NKG2A) expression associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in CD8Ahigh bladder tumors. The loss of antigen presentation is a common mechanism for tumor escape in bladder cancer. NKG2A+ CD8 T cells are able to circumvent HLA-ABC loss through TCR-independent cytotoxicity, which is partly mediated by DNAM-1. In bladder tumors, NKG2A is acquired on a subset of PD-1+ CD8 T cells, alongside stronger tissue-residency memory features, TCR-independent cytotoxicity and evidence of recent proliferation. HLA-E is low but variably expressed on bladder tumors. When expressed, NKG2A+ CD8 T cell anti-tumor responses to HLA-ABC-deficient tumors are inhibited and partly restored upon NKG2A blockade. Overall, our study identifies an alternative path for CD8 T cell exhaustion, that is mediated by NKG2A upregulation and TCR-independent cytotoxicity.


title: Gut microbiota-derived tryptamine impairs insulin sensitivity

abstract:
Gut-microbiota plays a pivotal role in development of type 2 diabetes (T2D), yet the molecular mechanism remains elusive. Here, we show that tryptamine, a microbial metabolite of tryptophan, impairs glucose tolerance and insulin sensitivity. Tryptamine presents a higher level in monkeys with spontaneous diabetes and human with T2D and positively correlated with the glucose tolerance. In parallel, tryptamine level was suppressed by dietary fibers intervention in T2D subjects and negatively correlated with improvement of glucose tolerance. The inhibitory effect of tryptamine on insulin signaling as shown was dependent on a trace amine-associated receptor 1 (TAAR1)-extracellular signal-regulated kinase (ERK) signaling axis. Monoassociation of T2D-associated tryptamine-producing bacteria Ruminococcus gnavus impairs insulin sensitivity in pseudo germ-free mice. Our findings indicate gut microbiota-derived tryptamine contributes to the development of insulin resistance in T2D and may serve as a new target for intervention.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Phenethylamine-producing gut bacteria induces diarrhea-predominant irritable bowel syndrome by increasing serotonin biosynthesis

abstract:
Despite the strong association between gut microbial dysbiosis, serotonin (5-HT) dysregulation and diarrhea-predominant irritable bowel syndrome (IBS-D), the mechanism by which changes in the gut microbiota contribute to the pathogenesis of IBS-D, particularly the role of dysregulated 5-HT production, remains unclear. The present study identified Ruminococcus gnavus in the human gut microbiota as a key risk factor of IBS-D. R. gnavus was significantly enriched in IBS-D patients and exhibited positive correlation with serum 5- HT level and severity of diarrhea symptoms. We showed that R. gnavus induced diarrhea-like symptoms in mice by promoting microbial shunting of essential aromatic amino acids to aromatic trace amines including phenethylamine and tryptamine, thereby stimulating the biosynthesis of peripheral 5-HT, a potent stimulator for gastrointestinal transit. This study identify gut-microbial metabolism of dietary amino acids as a cause of IBS-D and lays a foundation for developing novel therapeutic target for the treatment of IBS-D.

Graphical abstract

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title: Moving Yeasts: Resolving the Mystery

abstract: We have recently discovered a novel dimorphic yeast Aureobasidium tremulum sp.nov. The species name tremulum indicates its trembling movement. With keen microscopic observations and various experimentation, we concluded that the movement is of two types, trembling movement, and actual displacement. The movement is because of two reasons; 1. Due to the dynamic movement of intracellular lipid granules, and; 2. Due to the movement of an intracellular bacterial population which is present as an endosymbiont. The mechanism of displacement has been hypothesized. The presence of both lipid granules and bacteria has been confirmed using microscopic analysis (Bright field, Phase contrast, confocal, Cryo-SEM, and HR-TEM). The bacterial presence was confirmed using whole-genome metagenomic analysis. Endosymbiotic bacteria have also been isolated and identified by 16s rRNA gene sequencing. Interestingly, some of them possibly are novel species. Also, the whole genome of A.tremulum has been announced. We also report on, restriction of the mode of bacterial mycophagy in presence of an antibiotic.


title: Medial prefrontal cortex Notch1 signalling mediates methamphetamine-induced psychosis via Hes1-dependent suppression of GABAB1 receptor expression.

abstract: Methamphetamine (METH), a widely abused stimulant drug, induces psychosis in approximately half of abusers; this effect is becoming a major concern for society. Although the Notch1 signalling pathway has been shown to play a part in the pathogenesis of some psychiatric disorders, its role in METH-induced psychosis (MIP) is still unknown. Here, we showed that Notch1 signalling was downregulated in the medial prefrontal cortex (mPFC) in a MIP mouse model. Direct genetic and pharmacological manipulations of Notch1 signalling bidirectionally altered MIP-related behaviours and governed neuronal activity in the mPFC. Moreover, Notch1 signalling negatively regulated GABAB1 receptor expression in the mPFC of MIP mice through Hes1, a transcriptional repressor in Notch1 signalling. Further, we show that Hes1 can directly bind to the GABAB1 receptor promoter. Notably, pharmacological regulation of the GABAB receptor in the mPFC reversed the changes in MIP-related behaviours caused by the dysfunction of Notch1 signalling. Together, our findings uncover a previously unrecognised Notch1-Hes1-GABAB1 receptor-dependent mechanism involved in regulating mPFC neuronal activity and behavioural phenotypes in MIP. Our work provides mechanistic insight into the aetiology and pathophysiology of MIP.


title: Seed Germination, Post-fire Plant Growth and Conservation of the Rare Endemic and Endangered Chamaecrista glandulosa var. mirabilis (Fabaceae)

abstract: Conservation has been challenged by biodiversity loss drivers. Also, fire disturbance can temporarily change ecosystems. Fire effects in soil nutrients and pH, plant abundance, reproduction, seed weight, seed germination, and plant growth were assessed. Four seed starting systems were used for germination and growth under laboratory conditions. We found significant differences in the means of iron, manganese, nickel, soil pH, and plant height, and a significant positive linear relationship between seed weight and plant height. Results indicate that fires increase soil pH and cause changes in micronutrients that can increase plant growth. Large size plants are produced from high weight seeds. Finally, ex situ conservation and species reintroduction were feasible conservation strategies that should be integrated with in situ conservation.

category: ecology


title: Optimized Workflow for Enrichment and Identification of Biotinylated Peptides using Tamavidin 2-REV for BioID and Cell Surface Proteomics

abstract: Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the low abundance of protein biotinylation in cells required large amounts of cellular proteins to detect enough biotinylated peptides. Here we optimized the workflow for efficient enrichment and identification of biotinylated peptides. The most efficient recovery was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and the optimal temperature and salt concentration for elution. Using the optimized workflow, over 2-fold more biotinylated peptides were identified with higher purity from RAW264.7 macrophages expressing TurboID-fused STING. In addition, sequential digestion with Glu-C and trypsin led to the identification of biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon EGF stimulation. This workflow would be useful not only for BioID and cell surface proteomics but also for various other applications based on protein biotinylation.

category: biochemistry


title: Towards resolution of the intron retention paradox in breast cancer

abstract:
After many years of neglect in the field of alternative splicing, the importance of intron retention (IR) in cancer has come into focus following landmark discoveries of aberrant IR patterns in cancer. Many solid and liquid tumours are associated with drastic increases in IR and such patterns have been pursued as both biomarkers and therapeutic targets. Paradoxically, breast cancer (BrCa) is the only tumour type in which IR is reduced compared to adjacent normal breast tissue.

In this study, we have conducted a pan-cancer analysis of IR with emphasis on BrCa and its subtypes. We explored mechanisms that could cause aberrant and pathological IR and clarified why normal breast tissue has unusually high IR.

Strikingly, we found that reduced IR in BrCa can be largely attributed to normal breast tissue having the highest occurrence of IR events compared to other healthy tissues. Our analyses suggest that low numbers of IR events in breast tumours are associated with poor prognosis, particularly in the luminal B subtype. Interestingly, we found that IR frequencies negatively correlate with cell proliferation in BrCa cells, i.e. rapidly dividing tumour cells have the lowest number of IR events. Aberrant RNA binding protein (RBP) expression and changes in tissue composition are among the causes of low IR in BrCa.

Our results suggest that IR should be considered for therapeutic manipulation in BrCa patients with aberrantly low IR levels and that further work is needed to understand the cause and impact of high IR in other tumour types.


title: DNA methylation dynamics during stress-response in woodland strawberry (Fragaria vesca)

abstract:
O_LIEnvironmental stresses can result in a wide range of physiological and molecular responses in plants. These responses can also impact epigenetic information in genomes especially at the level of DNA methylation. DNA methylation is the hallmark heritable epigenetic modification and plays a key role in silencing transposable elements (TEs). Although DNA methylation is an essential epigenetic mechanism, fundamental aspects of its contribution to stress responses and adaptation remain obscure.
C_LIO_LIWe investigated epigenome dynamics of wild strawberry (Fragaria vesca) in response to variable environmental conditions at DNA methylation level. F. vesca methylome responded with great plasticity to ecologically relevant abiotic and hormonal stresses. Thermal stress resulted in substantial genome-wide loss of DNA methylation. Notably, all tested stress conditions resulted in marked hot spots of differential DNA methylation near centromeric or pericentromeric regions, particularly in non-symmetrical DNA methylation context. Additionally, we identified differentially methylated regions (DMRs) within promoter regions of transcription factor (TF) superfamilies involved in plant stress-response and assessed the effects of these changes on gene expression.
C_LIO_LIThese findings improve our understanding on stress-response at the epigenome level by highlighting the correlation between DNA methylation, TEs and gene expression regulation in plants subjected to a broad range of environmental stresses.
C_LI

category: plant biology


title: Differential effects of group III metabotropic glutamate receptors on spontaneous inhibitory synaptic currents in spine-innervating double bouquet and parvalbumin-expressing dendrite-targeting GABAergic interneurons in human neocortex

abstract: Diverse neocortical GABAergic neurons specialise in synaptic targeting and their effects are modulated by presynaptic metabotropic glutamate receptors (mGluRs) suppressing neurotransmitter release in rodents, but their effects in human neocortex are unknown. We tested whether activation of group III mGluRs by L-AP4 changes GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in two distinct dendritic spine-innervating GABAergic interneurons recorded in vitro in human neocortex. Calbindin-positive double bouquet cells (DBC) had columnar "horsetail" axons descending through layers II-V innervating dendritic spines (48%) and shafts, but not somata of pyramidal and non-pyramidal neurons. Parvalbumin-expressing dendrite-targeting cell (PV-DTC) axons extended in all directions innervating dendritic spines (22%), shafts (65%) and somata (13%). As measured, 20% of GABAergic neuropil synapses innervate spines, hence DBCs, but not PV-DTCs, preferentially select spine targets. Group III mGluR activation paradoxically increased the frequency of sIPSCs in DBCs (to median 137% of baseline), but suppressed it in PV-DTCs (median 92%), leaving the amplitude unchanged. The facilitation of sIPSCs in DBCs may result from their unique GABAergic input being disinhibited via network effect. We conclude that dendritic spines receive specialised, diverse GABAergic inputs, and group III mGluRs differentially regulate GABAergic synaptic transmission to distinct GABAergic cell types in human cortex.


title: Design of the mammalian cone photoreceptor to Off bipolar cell synapse

abstract: Graded synapses in sensory systems reliably transmit small signals in the presence of continuous quantal noise. To understand how signaling is optimized during graded transmission, we counted the number of vesicles released by a mammalian cone terminal and compared it to the simultaneous responses in each Off bipolar cell type. Off bipolar cells contacting the terminal base comprised two groups depending on how they sampled transmitter release. In both groups, responses initially grew non-linearly with the number of released vesicles implicating a role for cooperativity during sparse release. One group sampled release from most of a cones [~]20 ribbons and can exploit averaging to improve signal reliability. The other, less-sensitive group made 1-3 contacts at the terminal center and responded to pooled transmitter, a consequence of membrane depolarization, using an insensitive kainate receptor. Off bipolar cells use different strategies to minimize transmission noise and encode cone output over different ranges.


title: A-type FHFs mediate resurgent currents through TTX-resistant voltage-gated sodium channels

abstract: Resurgent currents (INaR) produced by voltage-gated sodium channels are required for many neurons to maintain high-frequency firing, and contribute to neuronal hyperexcitability and disease pathophysiology. Here we show, for the first time, that INaR can be reconstituted in a heterologous system by co-expression of sodium channel -subunits and A-type fibroblast growth factor homologous factors (FHFs). Specifically, A-type FHFs induces INaR from Nav1.8, Nav1.9 tetrodotoxin-resistant neuronal channels and, to a lesser extent, neuronal Nav1.7 and cardiac Nav1.5 channels. Moreover, we identified the N-terminus of FHF as the critical molecule responsible for A-type FHFs-mediated INaR. Among the FHFs, FHF4A is the most important isoform for mediating Nav1.8 and Nav1.9 INaR. In nociceptive sensory neurons, FHF4A knockdown significantly reduces INaR amplitude and the percentage of neurons that generate INaR, substantially suppressing excitability. Thus, our work reveals a novel molecular mechanism underlying TTX-resistant INaR generation and provides important potential targets for pain treatment.


title: Circulating mast cell progenitors migrate to atopic dermatitis-like skin then adapt locally

abstract: BackgroundMast cells (MCs) are tissue-resident immune cells that are classified into two subsets in mice: connective tissue-type MCs (CTMCs) and mucosal type MCs (MMCs). Although both CTMCs and MMCs can be induced from bone marrow (BM)-derived hematopoietic stem cells (HSCs) in vitro, recent research on MC ontogeny has revealed that MMCs are maintained with a supply of BM-derived HSCs, while CTMCs are maintained locally by self-proliferation in steady state in vivo. However, how CTMCs, such as skin MCs, are maintained in an inflammatory state such as atopic dermatitis (AD) remains to be fully elucidated. Methods: MC903-induced AD model was used to identify BM-derived MCs in the skin. The infiltration and proliferation of MCs were evaluated by flow cytometry using CD45.1 BM-chimera mice and parabiosis. BM-derived MCs in AD-like skin were compared to resident MCs (rMCs) in gene expressions by RNA sequence analysis. The fate of BM-derived MCs in AD-like skin was investigated for expressions of CTMC markers and responses to compound 48/80. Results: In AD-like skin, significant increase of both rMCs and BM-derived MCs was observed. BM-derived MCs were derived from circulating MC progenitors (MCps) and were distinguished from rMCs by integrin{beta}7 expression, which was gradually downregulated in the skin. RNA sequence analysis showed that integrin{beta}7+ MCs in the skin shared characteristics of both MMC and CTMC. Integrin{beta}7+ MCs proliferated in situ and acquired the CTMC phenotypes in AD-like skin. Conclusions: Skin MCs are maintained in AD-like skin by both local proliferation of rMCs and infiltration/proliferation of BM-derived MCs, which differentiate toward CTMC in the skin.


title: Genomic and Transcriptomic Profiling of Phoenix Colonies

abstract: Pseudomonas aeruginosa is a Gram-negative bacterium responsible for numerous human infections. Previously, novel antibiotic tolerant variants known as phoenix colonies as well as variants similar to viable but non-culturable (VBNC) colonies were identified in response to high concentrations of aminoglycosides. In this study, the mechanisms behind phoenix colony and VBNC-like colony emergence were further explored using both whole genome sequencing and RNA sequencing. Phoenix colonies were found to have a single nucleotide polymorphism (SNP) in the PA4673 gene, which is predicted to encode a GTP-binding protein. No SNPs were identified within VBNC-like colonies compared to the founder population. RNA sequencing did not detect change in expression of PA4673 but revealed multiple differentially expressed genes that may play a role in phoenix colony emergence. One of these differentially expressed genes, PA3626, encodes for a tRNA pseudouridine synthase which when knocked out led to a complete lack of phoenix colonies. Although not immediately clear whether the identified genes in this study may have interactions which have not yet been recognized, they may contribute to the understanding of how phoenix colonies are able to emerge and survive in the presence of antibiotic exposure.