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Targeted protein degradation is a drug modality represented by compounds that recruit a target to an E3 ubiquitin ligase to promote target ubiquitination and proteasomal degradation. Historically, the field distinguishes monovalent degraders from bifunctional degraders (PROTACs) that connect target and ligase via separate binding ligands joined via a linker1–4. Here, we elucidate the mechanism of action of a PROTAC-like degrader of the transcriptional coactivator BRD4, composed of a BRD4 ligand linked to a ligand for the E3 ligase CRL4DCAF15. Using orthogonal CRISPR/Cas9 screens we identify the degrader activity is independent of DCAF15, and relies on a different CRL4 substrate receptor, DCAF16. We demonstrate an intrinsic affinity between BRD4 and DCAF16, which is dependent on the tandem bromodomains of BRD4 and further increased by the degrader without physically engaging DCAF16 in isolation. Structural characterization of the resulting ternary complex reveals both BRD4 bromodomains are bivalently engaged in cis by the degrader and are bound to DCAF16 through several interfacial BRD4-DCAF16 and degrader-DCAF16 contacts. Our findings demonstrate that intramolecularly bridging domains can confer glue-type stabilization of intrinsic target-E3 interactions, and we propose this as a general strategy to modulate the surface topology of target proteins to nucleate co-opting of E3 ligases or other cellular effector proteins for effective proximity-based pharmacology.
Glutathione (GSH) is the main determinant of intracellular redox potential and participates in multiple cellular signaling pathways. Achieving a detailed understanding of intracellular GSH trafficking and regulation depends on the development of tools to map GSH compartmentalization and intra-organelle fluctuations. Herein, we present a new GSH sensing platform, TRaQ-G, for live-cell imaging. This small-molecule/protein hybrid sensor possesses a unique reactivity turn-on mechanism that ensures that the small molecule is only sensitive to GSH in the desired location. Furthermore, TRaQ-G can be fused to a fluorescent protein of choice to give a ratiometric response. Using TRaQ-G-mGold, we demonstrated that the nuclear and cytosolic GSH pools are independently regulated during cell proliferation. We also used this sensor, in combination with roGFP, to quantify redox potential and GSH concentration simultaneously in the endoplasmic reticulum. Finally, by exchanging the fluorescent protein, we created a near-infrared, targetable and quantitative GSH sensor.
Tracking influenza a virus infection in the lung from hematological data with machine learning
(2022)
The tracking of pathogen burden and host responses with minimal-invasive methods during respiratory infections is central for monitoring disease development and guiding treatment decisions. Utilizing a standardized murine model of respiratory Influenza A virus (IAV) infection, we developed and tested different supervised machine learning models to predict viral burden and immune response markers, i.e. cytokines and leukocytes in the lung, from hematological data. We performed independently in vivo infection experiments to acquire extensive data for training and testing purposes of the models. We show here that lung viral load, neutrophil counts, cytokines like IFN-γ and IL-6, and other lung infection markers can be predicted from hematological data. Furthermore, feature analysis of the models shows that blood granulocytes and platelets play a crucial role in prediction and are highly involved in the immune response against IAV. The proposed in silico tools pave the path towards improved tracking and monitoring of influenza infections and possibly other respiratory infections based on minimal-invasively obtained hematological parameters.
We have investigated the systematic differences introduced when performing a Bayesian-inference analysis of the equation of state of neutron stars employing either variable- or constant-likelihood functions. The former have the advantage that it retains the full information on the distributions of the measurements, making an exhaustive usage of the data. The latter, on the other hand, have the advantage of a much simpler implementation and reduced computational costs. In both approaches, the EOSs have identical priors and have been built using the sound-speed parameterization method so as to satisfy the constraints from X-ray and gravitationalwaves observations, as well as those from Chiral Effective Theory and perturbative QCD. In all cases, the two approaches lead to very similar results and the 90%-confidence levels are essentially overlapping. Some differences do appear, but in regions where the probability density is extremely small and are mostly due to the sharp cutoff set on the binary tidal deformability Λ˜ ≤ 720 employed in the constant-likelihood analysis. Our analysis has also produced two additional results. First, a clear inverse correlation between the normalized central number density of a maximally massive star, nc,TOV/ns, and the radius of a maximally massive star, RTOV. Second, and most importantly, it has confirmed the relation between the chirp mass Mchirp and the binary tidal deformability Λ˜. The importance of this result is that it relates a quantity that is measured very accurately, Mchirp, with a quantity that contains important information on the micro-physics, Λ˜. Hence, once Mchirp is measured in future detections, our relation has the potential of setting tight constraints on Λ˜.
Viewpoint effects on object recognition interact with object-scene consistency effects. While recognition of objects seen from “accidental” viewpoints (e.g., a cup from below) is typically impeded compared to processing of objects seen from canonical viewpoints (e.g., the string-side of a guitar), this effect is reduced by meaningful scene context information. In the present study we investigated if these findings established by using photographic images, generalise to 3D models of objects. Using 3D models further allowed us to probe a broad range of viewpoints and empirically establish accidental and canonical viewpoints. In Experiment 1, we presented 3D models of objects from six different viewpoints (0°, 60°, 120°, 180° 240°, 300°) in colour (1a) and grayscaled (1b) in a sequential matching task. Viewpoint had a significant effect on accuracy and response times. Based on the performance in Experiments 1a and 1b, we determined canonical (0°-rotation) and non-canonical (120°-rotation) viewpoints for the stimuli. In Experiment 2, participants again performed a sequential matching task, however now the objects were paired with scene backgrounds which could be either consistent (e.g., a cup in the kitchen) or inconsistent (e.g., a guitar in the bathroom) to the object. Viewpoint interacted significantly with scene consistency in that object recognition was less affected by viewpoint when consistent scene information was provided, compared to inconsistent information. Our results show that viewpoint-dependence and scene context effects generalize to depth rotated 3D objects. This supports the important role object-scene processing plays for object constancy.
In a dynamic environment, the already limited information that human working memory can maintain needs to be constantly updated to optimally guide behaviour. Indeed, previous studies showed that working memory representations are continuously being transformed during delay periods leading up to a response. This goes hand-in-hand with the removal of task-irrelevant items. However, does such removal also include veridical, original stimuli, as they were prior to transformation? Here we aimed to assess the neural representation of task-relevant transformed representations, compared to the no-longer-relevant veridical representations they originated from. We applied multivariate pattern analysis to electroencephalographic data during maintenance of orientation gratings with and without mental rotation. During maintenance, we perturbed the representational network by means of a visual impulse stimulus, and were thus able to successfully decode veridical as well as imaginary, transformed orientation gratings from impulse-driven activity. On the one hand, the impulse response reflected only task-relevant (cued), but not task-irrelevant (uncued) items, suggesting that the latter were quickly discarded from working memory. By contrast, even though the original cued orientation gratings were also no longer task-relevant after mental rotation, these items continued to be represented next to the rotated ones, in different representational formats. This seemingly inefficient use of scarce working memory capacity was associated with reduced probe response times and may thus serve to increase precision and flexibility in guiding behaviour in dynamic environments.
Background: Blood donation saves lives. Provided they are in good health, male volunteers can donate as often as six times per year from the age of 18 into their late sixties. The burden of blood donation is very unevenly distributed, with a small minority of altruistic individuals providing this critical resource. While the consequences of persistent iron depletion in blood donors have been studied in the context of cancer and coronary heart disease, potential effects of the erythropoietic stress from repetitive large-volume phlebotomy remain unexplored. We sought to investigate if and how repeated blood donations affect the clonal composition of the hematopoietic stem and progenitor cell (HSPC) compartment.
Methods: 105 healthy, male individuals with an extensive blood donation history (median of 120 donations per donor; median age of 66 yrs.) were screened for the presence of clonal hematopoiesis (CH) using a sequencing panel covering 141 genes commonly mutated in human myeloid neoplasms. The control cohort consisted of 103 healthy, male donors with a median of 5 donations per donor and a median age of 63. Donors positive for CH were subsequently studied longitudinally. The pathogenicity of detected variants was compared using established scoring systems. Finally, to assess the functional consequences of blood-donation induced CH, selected CH mutations were introduced by CRISPR-mediated editing into HSPCs from human cord blood (CB) or bone marrow (BM). The effect of these mutations was tested under different stress stimuli using functional ex vivo long-term culture initiating cells (LTC-IC) assays.
Results: Compared to the control cohort, frequent donors were significantly more likely to have mutations in genes encoding for epigenetic modifiers (44.7 vs. 22.3 %), most specifically in the two genes most commonly mutated in CH, DNMT3A and TET2 (35.2 vs. 20.3 %). However, no difference in the variant allele frequency (VAF) of detected mutations was found between the groups. Longitudinal analysis revealed that the majority of the mutations remained at a stable VAF over an observation period of approximately one year. Three DNMT3A variants from the frequent donor cohort were introduced into healthy HSPCs and functionally analyzed: All expanded in response to EPO, but none responded to LPS or IFNγ stimulation. This contrasted with the leukemogenic DNMT3A R882H mutation, which did not expand in the presence of EPO but instead responded strongly to inflammatory stimuli.
Conclusions: Frequent whole blood donation is associated with a higher prevalence of CH driven by mutations in genes encoding for epigenetic modifiers, with DNMT3A and TET2 being the most common. This increased CH prevalence is not associated with a higher pathogenicity of the associated variants and is likely a result of the selection of clones with improved responsiveness to EPO under the condition of bleeding stress. Our data show that even highly frequent blood donations over many years is not increasing the risk for malignant clones further underscoring the safety of repetitive blood donations. To our knowledge, this is the first CH study analyzing a cohort of individuals known for their superior health and survival, able to donate blood until advanced age. Thus, our analysis possibly identified mutations associated with beneficial outcomes, rather than a disease condition, such as mutations in DNMT3A that mediated the improved expansion of HSPCs in EPO enriched environments. Our data support the notion of ongoing Darwinian evolution in humans at the somatic stem cell level and present EPO as one of the environmental factors to which HSPCs with specific mutations may respond with superior fitness.
Selective attention implements preferential routing of attended stimuli, likely through increasing the influence of the respective synaptic inputs on higher-area neurons. As the inputs of competing stimuli converge onto postsynaptic neurons, presynaptic circuits might offer the best target for attentional top-down influences. If those influences enabled presynaptic circuits to selectively entrain postsynaptic neurons, this might lead to selective routing. Indeed, when two visual stimuli induce two gamma rhythms in V1, only the gamma induced by the attended stimulus entrains gamma in V4. Here, we modeled this selective entrainment with a Dynamic Causal Model for Cross-Spectral Densities and found that it can be explained by attentional modulation of intrinsic V1 connections. Specifically, local inhibition was decreased in the granular input layer and increased in the supragranular output layer of the V1 circuit that processed the attended stimulus. Thus, presynaptic attentional influences and ensuing entrainment were sufficient to mediate selective routing.
RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large messenger ribonucleoprotein particle (mRNP) assemblies, these proteins often function in. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components in S. cerevisiae. For functional analysis, we chose Npl3, for which we determined crosslinks in its two RNA recognition motifs (RRM) and in the flexible linker region connecting the two. Using NMR and structural analyses, we show that both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of further mRNP components into nuclear mRNPs, establishing a function of Npl3 in nuclear mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of the nuclear mRNP component Npl3, an approach that can be applied to many RBPs in any RNA metabolic process.
Vaccines are central to controlling the coronavirus disease 2019 (COVID-19) pandemic but the durability of protection is limited for currently approved COVID-19 vaccines. Further, the emergence of variants of concern (VoCs) that evade immune recognition has reduced vaccine effectiveness, compounding the problem. Here, we show that a single dose of a murine cytomegalovirus (MCMV)-based vaccine, which expresses the spike (S) protein of the virus circulating early in the pandemic (MCMVS), protects highly susceptible K18-hACE2 mice from clinical symptoms and death upon challenge with a lethal dose of D614G SARS-CoV-2. Moreover, MCMVS vaccination controlled two immune-evading VoCs, the Beta (B.1.135) and the Omicron (BA.1) variants in BALB/c mice, and S-specific immunity was maintained for at least 5 months after immunization, where neutralizing titers against all tested VoCs were higher at 5-months than at 1-month post-vaccination. Thus, cytomegalovirus (CMV)-based vector vaccines might allow for long-term protection against COVID-19.
The free energy of TAP-solutions for the SK-model of mean field spin glasses can be expressed as a nonlinear functional of local terms: we exploit this feature in order to contrive abstract REM-like models which we then solve by a classical large deviations treatment. This allows to identify the origin of the physically unsettling quadratic (in the inverse of temperature) correction to the Parisi free energy for the SK-model, and formalizes the true cavity dynamics which acts on TAP-space, i.e. on the space of TAP-solutions. From a non-spin glass point of view, this work is the first in a series of refinements which addresses the stability of hierarchical structures in models of evolving populations.
The Kitaev material α-RuCl3 is among the most prominent candidates to host a quantum spin-liquid state endowed with fractionalized excitations. Recent experimental and theoretical investigations have separately revealed the importance of both the magnetoelastic coupling and the magnetic anisotropy, in dependence of the applied magnetic field direction. In this combined theoretical and experimental research, we investigate the anisotropic magnetic and magnetoelastic properties for magnetic fields applied along the main crystallographic axes as well as for fields canted out of the honeycomb plane. We found that the magnetostriction anisotropy is unusually large compared to the anisotropy of the magnetization, which is related to the strong magnetoelastic Γ′˜-type coupling in our \textit{ab-initio} derived model. We observed large, non-symmetric magnetic anisotropy for magnetic fields canted out of the honeycomb ab-plane in opposite directions, namely towards the +c∗ or −c∗ axes, respectively. The observed directional anisotropy is explained by considering the relative orientation of the magnetic field with respect to the co-aligned RuCl6 octahedra. Magnetostriction measurements in canted fields support this non-symmetric magnetic anisotropy, however these experiments are affected by magnetic torque effects. Comparison of theoretical predictions with experimental findings allow us to recognize the significant contribution of torque effects in experimental setups where α-RuCl3 is placed in canted magnetic fields.
The phase diagram of the (1+1)-dimensional Gross-Neveu model is reanalyzed for (non-)zero chemical potential and (non-)zero temperature within the mean-field approximation. By investigating the momentum dependence of the bosonic two-point function, the well-known second-order phase transition from the Z2 symmetric phase to the so-called inhomogeneous phase is detected. In the latter phase the chiral condensate is periodically varying in space and translational invariance is broken. This work is a proof of concept study that confirms that it is possible to correctly localize second-order phase transition lines between phases without condensation and phases of spatially inhomogeneous condensation via a stability analysis of the homogeneous phase. To complement other works relying on this technique, the stability analysis is explained in detail and its limitations and successes are discussed in context of the Gross-Neveu model. Additionally, we present explicit results for the bosonic wave-function renormalization in the mean-field approximation, which is extracted analytically from the bosonic two-point function. We find regions -- a so-called moat regime -- where the wave function renormalization is negative accompanying the inhomogeneous phase as expected.
The phase diagram of the (1+1)-dimensional Gross-Neveu model is reanalyzed for (non-)zero chemical potential and (non-)zero temperature within the mean-field approximation. By investigating the momentum dependence of the bosonic two-point function, the well-known second-order phase transition from the Z2 symmetric phase to the so-called inhomogeneous phase is detected. In the latter phase the chiral condensate is periodically varying in space and translational invariance is broken. This work is a proof of concept study that confirms that it is possible to correctly localize second-order phase transition lines between phases without condensation and phases of spatially inhomogeneous condensation via a stability analysis of the homogeneous phase. To complement other works relying on this technique, the stability analysis is explained in detail and its limitations and successes are discussed in context of the Gross-Neveu model. Additionally, we present explicit results for the bosonic wave-function renormalization in the mean-field approximation, which is extracted analytically from the bosonic two-point function. We find regions -- a so-called moat regime -- where the wave function renormalization is negative accompanying the inhomogeneous phase as expected.
The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing, and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately-100-kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA- and RNA-binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerise, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.
Background: Driven by globalization, urbanization and climate change, the distribution range of invasive vector species has expanded to previously colder ecoregions. To reduce health-threatening impacts on humans, insect vectors are extensively studied. Population genomics can reveal the genomic basis of adaptation and help to identify emerging trends of vector expansion.
Results: By applying whole genome analyses and genotype-environment associations to populations of the main dengue vector Ae. aegypti, sampled along an altitudinal temperature gradient in Nepal (200- 1300m), we identify adaptive traits and describe the species’ genomic footprint of climate adaptation to colder ecoregions. We found two clusters of differentiation with significantly different allele frequencies in genes associated to climate adaptation between the highland population (1300m) and all other lowland populations (≤ 800 m). We revealed non-synonymous mutations in 13 of the candidate genes associated to either altitude, precipitation or cold tolerance and identified an isolation-by-environment differentiation pattern.
Conclusion: Other than the expected gradual differentiation along the altitudinal gradient, our results reveal a distinct genomic differentiation of the highland population. This finding either indicates a differential invasion history to Nepal or local high-altitude adaptation explaining the population’s phenotypic cold tolerance. In any case, this highland population can be assumed to carry pre-adapted alleles relevant for the species’ invasion into colder ecoregions worldwide that way expanding their climate niche.
Objects that are congruent with a scene are recognised more efficiently than objects that are incongruent. Further, semantic integration of incongruent objects elicits a stronger N300/N400 EEG component. Yet, the time course and mechanisms of how contextual information supports access to semantic object information is unclear. We used computational modelling and EEG to test how context influences semantic object processing. Using representational similarity analysis, we established that EEG patterns dissociated between objects in congruent or incongruent scenes from around 300 ms. By modelling semantic processing of objects using independently normed properties, we confirm that the onset of semantic processing of both congruent and incongruent objects is similar (∼150 ms). Critically, after ∼275 ms, we discover a difference in the duration of semantic integration, lasting longer for incongruent compared to congruent objects. These results constrain our understanding of how contextual information supports access to semantic object information.
Respiratory complex I in mitochondria and bacteria catalyzes the transfer of electrons from NADH to quinone (Q). The free energy available from the reaction is used to pump protons and to establish a membrane proton electrochemical gradient, which drives ATP synthesis. Even though several high-resolution structures of complex I have been resolved, how Q reduction is linked with proton pumping, remains unknown. Here, microsecond long molecular dynamics (MD) simulations were performed on Yarrowia lipolytica complex I structures where Q molecules have been resolved in the ~30 Å long Q tunnel. MD simulations of several different redox/protonation states of Q reveal the coupling between the Q dynamics and the restructuring of conserved loops and ion pairs. Oxidized quinone stabilizes towards the N2 FeS cluster, a binding mode not previously described in Yarrowia lipolytica complex I structures. On the other hand, reduced (and protonated) species tend to diffuse towards the Q binding sites closer to the tunnel entrance. Mechanistic and physiological relevance of these results are discussed.
Neuroscience studies in non-human primates (NHP) often follow the rule of thumb that results observed in one animal must be replicated in at least one other. However, we lack a statistical justification for this rule of thumb, or an analysis of whether including three or more animals is better than including two. Yet, a formal statistical framework for experiments with few subjects would be crucial for experimental design, ethical justification, and data analysis. Also, including three or four animals in a study creates the possibility that the results observed in one animal will differ from those observed in the others: we need a statistically justified rule to resolve such situations. Here, I present a statistical framework to address these issues. This framework assumes that conducting an experiment will produce a similar result for a large proportion of the population (termed ‘representative’), but will produce spurious results for a substantial proportion of animals (termed ‘outliers’); the fractions of ‘representative’ and ‘outliers’ animals being defined by a prior distribution. I propose a procedure in which experimenters collect results from M animals and accept results that are observed in at least N of them (‘N-out-of-M’ procedure). I show how to compute the risks α (of reaching an incorrect conclusion) and β (of failing to reach a conclusion) for any prior distribution, and as a function of N and M. Strikingly, I find that the N-out-of-M model leads to a similar conclusion across a wide range of prior distributions: recordings from two animals lowers the risk α and therefore ensures reliable result, but leaves a large risk β; and recordings from three animals and accepting results observed in two of them strikes an efficient balance between acceptable risks α and β. This framework gives a formal justification for the rule of thumb of using at least two animals in NHP studies, suggests that recording from three animals when possible markedly improves statistical power, provides a statistical solution for situations where results are not consistent between all animals, and may apply to other types of studies involving few animals.
Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 Å resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including ∼70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated transmembrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.