bluuebunny/crossref_metadata_2025 huggingface dataset:
The TRRAP transcription cofactor represses interferon-stimulated genes in colorectal cancer cells
Authors: Detilleux Dylane , Raynaud Peggy , Pradet-Balade Bérengère , Helmlinger Dominique
Published: April 2021

Transcription is essential for cells to respond to signaling cues and involves factors with multiple distinct activities. One such factor, TRRAP, functions as part of two large complexes, SAGA and TIP60, which have crucial roles during transcription activation. Structurally, TRRAP belongs to the PIKK family but is the only member classified as a pseudokinase. Recent studies established that a dedicated HSP90 co-chaperone, the TTT complex, is essential for PIKK stabilization and activity. Here, using endogenous auxin- inducible degron alleles, we show that the TTT subunit TELO2 promotes TRRAP assembly into SAGA and TIP60 in human colorectal cancer cells (CRC). Transcriptomic analysis revealed that TELO2 contributes to TRRAP regulatory roles in CRC cells, most notably of MYC target genes. Surprisingly, TELO2 and TRRAP depletion also induced the expression of type I interferon genes. Using a combination of nascent RNA, antibody-targeted chromatin profiling (CUT&RUN) and kinetic analyses, we show that TRRAP directly represses the expression of IRF9, which is a master regulator of interferon stimulated genes. We have therefore uncovered a new, unexpected transcriptional repressor role for TRRAP, which may contribute to its tumorigenic activity.
Pairing to the microRNA 3′ region occurs through two alternative binding modes, with affinity shaped by nucleotide identity as well as pairing position
Authors: McGeary Sean E. , Bisaria Namita , Bartel David P.
Published: April 2021

MicroRNAs (miRNAs), in association with Argonaute (AGO) proteins, direct repression by pairing to sites within mRNAs. Compared to pairing preferences of the miRNA seed region (nucleotides 2–8), preferences of the miRNA 3′ region are poorly understood, due to the sparsity of measured affinities for the many pairing possibilities. We used RNA bind-n-seq with purified AGO2–miRNA complexes to measure relative affinities of >1,000 3′-pairing architectures for each miRNA. In some cases, optimal 3′ pairing increased affinity by >500-fold. Some miRNAs had two high-affinity 3′-pairing modes—one of which included additional nucleotides bridging seed and 3′ pairing to enable high-affinity pairing to miRNA nucleotide 11. The affinity of binding and the position of optimal pairing both tracked with the occurrence of G or oligo(G/C) nucleotides within the miRNA. These and other results advance understanding of miRNA targeting, providing insight into how optimal 3′ pairing is determined for each miRNA.

HIGHLIGHTS

RNA bind-n-seq reveals relative affinities of >1,000 3′-pairing architectures

Two distinct 3′-binding modes can enhance affinity, by >500-fold in some instances

G and oligo(G/C) residues help define the miRNA 3′ segment most critical for pairing

Seed mismatch identity can influence the contribution of compensatory 3′ pairing
α3β4* Nicotinic acetylcholine receptors strongly modulate the excitability of VIP neurons in the mouse inferior colliculus
Authors: Rivera-Perez Luis M. , Kwapiszewski Julia T. , Roberts Michael T.
Published: April 2021

The inferior colliculus (IC), the midbrain hub of the central auditory system, receives extensive cholinergic input from the pontomesencephalic tegmentum. Activation of nicotinic acetylcholine receptors (nAChRs) in the IC can alter acoustic processing and enhance auditory task performance. However, how nAChRs affect the excitability of specific classes of IC neurons remains unknown. Recently, we identified vasoactive intestinal peptide (VIP) neurons as a distinct class of glutamatergic principal neurons in the IC. Here, in experiments using male and female mice, we show that cholinergic terminals are routinely located adjacent to the somas and dendrites of VIP neurons. Using whole-cell electrophysiology in brain slices, we found that acetylcholine drives surprisingly strong and long-lasting excitation and inward currents in VIP neurons. This excitation was unaffected by the muscarinic receptor antagonist atropine. Application of nAChR antagonists revealed that acetylcholine excites VIP neurons mainly via activation of α3β4* nAChRs, a nAChR subtype that is rare in the brain. Furthermore, we show that cholinergic excitation is intrinsic to VIP neurons and does not require activation of presynaptic inputs. Lastly, we found that low frequency trains of acetylcholine puffs elicited temporal summation in VIP neurons, suggesting that in vivo-like patterns of cholinergic input can reshape activity for prolonged periods. These results reveal the first cellular mechanisms of nAChR regulation in the IC, identify a functional role for α3β4* nAChRs in the auditory system, and suggest that cholinergic input can potently influence auditory processing by increasing excitability in VIP neurons and their postsynaptic targets.

Key points summary

The inferior colliculus (IC), the midbrain hub of the central auditory system, receives extensive cholinergic input and expresses a variety of nicotinic acetylcholine receptor (nAChR) subunits.

In vivo activation of nAChRs alters the input-output functions of IC neurons and influences performance in auditory tasks. However, how nAChR activation affects the excitability of specific IC neuron classes remains unknown.

Here we show in mice that cholinergic terminals are located adjacent to the somas and dendrites of VIP neurons, a class of IC principal neurons.

We find that acetylcholine elicits surprisingly strong, long-lasting excitation of VIP neurons and this is mediated mainly through activation of α3β4* nAChRs, a subtype that is rare in the brain.

Our data identify a role for α3β4* nAChRs in the central auditory pathway and reveal a mechanism by which cholinergic input can influence auditory processing in the IC and the postsynaptic targets of VIP neurons.
From Structure to Sequence: Identification of polyclonal antibody families using cryoEM
Authors: Antanasijevic Aleksandar , Bowman Charles A. , Kirchdoerfer Robert N. , Cottrell Christopher A. , Ozorowski Gabriel , Upadhyay Amit A. , Cirelli Kimberly M. , Carnathan Diane G. , Enemuo Chiamaka A. , Sewall Leigh M. , Nogal Bartek , Zhao Fangzhu , Groschel Bettina , Schief William R. , Sok Devin , Silvestri Guido , Crotty Shane , Bosinger Steven E. , Ward Andrew B.
Published: April 2021

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next generation sequencing of immune repertoires we were able to specifically identify clonal family members, synthesize the monoclonal antibodies and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.

Graphical Abstract

One Sentence Summary

CryoEM and next generation sequencing were used to identify monoclonal antibodies elicited by HIV Env vaccine candidates.
Single-cell immune profiling with TCR clonotype barcoding identifies biomarker signatures that predict response to immune checkpoint blockade
Authors: Lee Dean , Fulton Ross , Manne Monika , Schweizer Liang , Raue Andreas
Published: April 2021

The identification of predictive biomarkers for patient treatment response is urgently needed to increase the probability of success of existing and novel experimental therapies. Single-cell profiling has provided novel biological insights into drug responses in the tumor microenvironment, but its potential for biomarker discovery has not been fully explored for therapeutic purposes. We describe a novel approach to discover predictive response biomarkers from single-cell data from a small patient cohort using the T cell receptor sequence intrinsic to each T cell to match clonotypes between pre- and post-treatment tumor samples. As a result, we have identified a predictive gene expression signature for immune checkpoint blockade and validated its predictive performance using data from three larger clinical studies. Our results demonstrated that applying clonotyping with single-cell genomic profiling is a promising novel approach for biomarker identification that does not require data collected from large patient cohorts. This could increase success rates, reduce clinical trial size, and significantly impact future clinical developments of immunomodulatory therapeutics.
Occult polyclonality of preclinical pancreatic cancer models drives in vitro evolution
Authors: Monberg Maria E. , Geiger Heather , Lee Jaewon J. , Sharma Roshan , Semaan Alexander , Bernard Vincent , Swartzlander Daniel B. , Stephens Bret M. , Chen Ken , Katz Matthew HG , Robine Nicolas , Guerrero Paola A. , Maitra Anirban
Published: April 2021

Intratumoral heterogeneity (ITH) is a hallmark of cancer. The advent of single-cell technologies has helped uncover ITH in a high-throughput manner in different cancers across varied contexts. Here we apply single-cell sequencing technologies to reveal striking ITH in assumptively oligoclonal pancreatic ductal adenocarcinoma (PDAC) cell lines. Our findings reveal a high degree of both genomic and transcriptomic heterogeneity in established and globally utilized PDAC cell lines, custodial variation induced by growing apparently identical PDAC cell lines in different laboratories, and profound transcriptomic shifts in transitioning from 2D to 3D spheroid growth models. Our findings also call into question the validity of widely available immortalized, non-transformed pancreatic lines as contemporaneous “control” lines in experiments. Further, while patient-derived organoid (PDOs) are known to reflect the cognatein vivobiology of the parental tumor, we identify transcriptomic shifts duringex vivopassage that might hamper their predictive abilities over time. The impact of these findings on rigor and reproducibility of experimental data generated using established preclinical PDAC models between and across laboratories is uncertain, but a matter of concern.
Non-clinical evidence supports anti-inflammatories as more effective medication than antihistamines against tarantula local effects envenomation
Authors: Alves Bruno Ricardo , Sutti Rafael , da Silva Pedro Ismael , Bertani Rogerio , Santos-Junior Jair Guilherme , Rocha e Silva Thomaz Augusto Alves , Linardi Alessandra
Published: April 2021

Background

Tarantulas are the most common invertebrates pets, especially in North America and Europe. The most commercialized genera are from Southern Asia and tropical Americas, represented byVitalius, found in Southeastern Brazil, andBrachypelma, common in Mexican desert. Bites by these spiders in humans occurs during manipulation and generally result in clinical manifestations such local pain, erythema and oedema, with the possibility of secondary local infection. Hence, the cases are usually treated with prescription free drugs such as antihistamines and anti-inflammatories.

Methods

In this work, we investigated the post treatment with commercial nonsteroidal and steroidal anti-inflammatories and anti-histamines administered by oral and intraperitoneal routes on rat paw oedema induced by venoms ofV. dubiusandB. smithi. Hydroplethysmometer standard oedema measurement and Evans blue extravasation were performed. Dose standardization experiments showed that theV. dubiusis more potent thanB. smithi, and doses were established at 30 μg/paw and 60 μg/paw respectively.

Results

The oral post-administration of ketoprofen (non-selective cyclooxygenase inhibitor) and prednisolone (steroidal anti-inflammatory) markedly reduced a paw oedema evoked by onlyV. dubiusvenom, but loratadine (H1-antihistamine) had negligible effect on rat paw oedema induced by both venoms. Intraperitoneal administration, ketoprofen (20 mg Kg−1) and loratadine (5 mg Kg−1) reduced the rat paw oedema induced byV. dubiusandB. smithiwhile methylprednisolone (10 mg Kg−1) only inhibited the oedema induced byV. dubius.

Conclusion

These results suggest that the pos-treatment with nonsteroidal and steroidal anti-inflammatory drugs are more potent than antihistamines in attenuating the local effect induced byV. dubiusandB. smithivenoms.
Selective prefrontal-amygdala circuit interactions underlie social and nonsocial valuation in rhesus macaques
Authors: Pujara Maia S. , Ciesinski Nicole K. , Reyelts Joseph F. , Rhodes Sarah E.V. , Murray Elisabeth A.
Published: April 2021

Lesion studies in macaques suggest dissociable functions of the orbitofrontal cortex (OFC) and medial frontal cortex (MFC), with OFC being essential for goal-directed decision making and MFC supporting social cognition. Bilateral amygdala damage results in impairments in both of these domains. There are extensive reciprocal connections between these prefrontal areas and the amygdala; however, it is not known whether the dissociable roles of OFC and MFC depend on functional interactions with the amygdala. To test this possibility, we compared the performance of male rhesus macaques (Macaca mulatta) with crossed surgical disconnection of the amygdala and either MFC (MFC x AMY,n=4) or OFC (OFC x AMY,n=4) to a group of unoperated controls (CON,n=5). All monkeys were assessed for their performance on two tasks to measure: (1) food-retrieval latencies while viewing videos of social and nonsocial stimuli in a test of social interest, and (2) object choices based on current food value using reinforcer devaluation in a test of goal-directed decision making. Compared to the CON group, the MFC x AMY group, but not the OFC x AMY group, showed significantly reduced food-retrieval latencies while viewing videos of conspecifics, indicating reduced social valuation and/or interest. By contrast, on the devaluation task, group OFC x AMY, but not group MFC x AMY, displayed deficits on object choices following changes in food value. These data indicate that the MFC and OFC must functionally interact with the amygdala to support normative social and nonsocial valuation, respectively.

Significance Statement

Ascribing value to conspecifics (social) vs. objects (nonsocial) may be supported by distinct but overlapping brain networks. Here we test whether two nonoverlapping regions of the prefrontal cortex, the medial frontal cortex and the orbitofrontal cortex, must causally interact with the amygdala to sustain social valuation and goal-directed decision making, respectively. We found that these prefrontal-amygdala circuits are functionally dissociable, lending support for the idea that medial frontal and orbital frontal cortex make independent contributions to cognitive appraisals of the environment. These data provide a neural framework for distinct value assignment processes and may enhance our understanding of the cognitive deficits observed following brain injury or in the development of mental health disorders.
Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay
Authors: Yu Zhong-Qiu , Liu Xiao-Man , Zhao Dan , Xu Dan-Dan , Du Li-Lin
Published: April 2021

Protein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeastSchizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins, transmembrane proteins, and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.
The second wave of SARS-CoV-2 infections and COVID-19 deaths in Germany – driven by values, social status and migration background? A county-scale explainable machine learning approach
Authors: Doblhammer Gabriele , Reinke Constantin , Kreft Daniel
Published: April 2021

There is a general consensus that SARS-CoV-2 infections and COVID-19 deaths have hit lower social groups the hardest, however, for Germany individual level information on socioeconomic characteristics of infections and deaths does not exist. The aim of this study was to identify the key features explaining SARS-CoV-2 infections and COVID-19 deaths during the upswing of the second wave in Germany.

We considered information on COVID-19 diagnoses and deaths from 1. October to 15. December 2020 on the county-level, differentiating five two-week time periods. We used 155 indicators to characterize counties in nine geographic, social, demographic, and health domains. For each period, we calculated directly age-standardized COVID-19 incidence and death rates on the county level. We trained gradient boosting models to predict the incidence and death rates with the 155 characteristics of the counties for each period. To explore the importance and the direction of the correlation of the regional indicators we used the SHAP procedure. We categorized the top 20 associations identified by the Shapley values into twelve categories depicting the correlation between the feature and the outcome.

We found that counties with low SES were important drivers in the second wave, as were those with high international migration and a high proportion of foreigners and a large nursing home population. During the period of intense exponential increase in infections, the proportion of the population that voted for the Alternative for Germany (AfD) party in the last federal election was among the top characteristics correlated with high incidence and death rates.

We concluded that risky working conditions with reduced opportunities for social distancing and a high chronic disease burden put populations in low-SES counties at higher risk of SARS-CoV-2 infections and COVID-19 deaths. In addition, noncompliance with Corona measures and spill-over effects from neighbouring counties increased the spread of the virus. To further substantiate this finding, we urgently need more data at the individual level.
Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast
Authors: Kalita R. , Flanagan W. , Lightley J. , Kumar S. , Alexandrov Y. , Garcia E. , Hintze M. , Barkoulas M , Dunsby C. , French P.M.W.
Published: April 2021

We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire 4 images from which the phase gradients and quantitative phase image can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be interleaved with single-shot imaging polarimetry.
A Paradigm Free Regularization Approach to Recover Brain Activation from Functional MRI Data
Authors: Costantini Isa , Deriche Rachid , Deslauriers-Gauthier Samuel
Published: April 2021

Context

Functional MRI is a non-invasive imaging technique that provides an indirect view into brain activity, via the blood-oxygen-level-dependent (BOLD) response. In particular, resting-state fMRI poses challenges to the recovery of brain activity without prior knowledge on the experimental paradigm, as it is the case for task-fMRI. Conventional methods to infer brain activity from the fMRI signals, for example the general linear model (GLM), require the knowledge of the experimental paradigm to define regressors and estimate the contribution of each voxel’s time course to the task. To overcome this limitation, approaches to deconvolve the BOLD response and recover the underlying neural activation without a priori information on the task have been proposed. State-of-the-art techniques, and in particular the Total Activation (TA), formulates the deconvolution as an optimization problem with decoupled spatial and temporal regularization terms. This increases the number of hyperparameters to be set and requires an optimization strategy that alternates between the constraints.

Approach

In this work, we propose a paradigm-free regularization algorithm named Paradigm-Free fMRI (PF-fMRI) that is applied on the 4-D fMRI image, acting simultaneously in the 3-D space and 1-D time dimensions. Based on the idea that large image variations should be preserved as they occur during brain activation, whereas small variations considered as noise should be removed, the PF-fMRI applies an anisotropic regularization, thus recovering the location and the duration of brain activation.

Results

Using the experimental paradigm as ground truth, the PF-fMRI is validated on synthetic and real task-fMRI data from 51 subjets, and its performance is compared to the TA. Results show higher correlations of the recovered time-courses with the ground truth compared to the TA and lower computational times. In addition, we show that the PF-fMRI recovers activity that agrees with the GLM, without requiring or using any knowledge of the experimental paradigm.
The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult gut
Authors: Ramadan Rana , van Neerven Sanne M. , Wouters Valerie M. , Martins Garcia Tania , Muncan Vanesa , Franklin Olivia D. , Battle Michele , Carlson Karen Sue , Leach Joshua , Sansom Owen J. , Vermeulen Louis , Medema Jan Paul , Huels David J
Published: April 2021

The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. In recent years, the signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. Here, we set out to systematically compare the effect of two major ECM classes, fibrillar collagen type I and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all differentiated and undifferentiated cell types present in both cultures, albeit at different ratios. Specific to the collagen culture was a subset of cells with a fetal-like gene expression program. In contrast, laminin, but not collagen IV, increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture, resembles the gut development in vivo. Here, the ECM is dramatically remodelled by mesenchymal cells, which is accompanied by a specific and local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.
Behavior-dependent spatial maps enable efficient theta phase coding
Authors: Parra-Barrero Eloy , Diba Kamran , Cheng Sen
Published: April 2021

Navigation through space involves learning and representing relationships between past, current and future locations. In mammals, this might rely on the hippocampal theta phase code, where in each cycle of the theta oscillation, spatial representations start behind the animal’s location and then sweep forward. However, the exact relationship between phase and represented and true positions remains unclear. Developing a quantitative framework for the theta phase code, we formalize two previous notions: in spatial sweeps, different phases of theta encode positions at fixed distances behind or ahead of the animal, whereas in temporal sweeps, they encode positions reached at fixed time intervals into the past or future. These two schemes predict very different position representations during theta depending on the animal’s running speed. Paradoxically, in some studies sweep length has been shown to increase proportionally with running speed, consistent with temporal sweeps, whereas in other studies individual place field parameters such as field size and phase precession slope were shown to remain constant with speed, consistent with spatial sweeps. Here, we introduce a third option: behavior-dependent sweeps, according to which sweep length and place field properties vary across the environment depending on the running speed characteristic of each location. Analyzing single-cell and population variables in parallel in recordings from rat CA1 place cells and comparing them to model simulations, we show that behavior-dependent sweeps uniquely account for all relevant variables. This coding scheme combines features and advantages of both spatial and temporal sweeps, revealing an efficient hippocampal code.

Significance

To learn the structure of the world and the consequences of our actions, information about the past must be carried through to the present and linked to what is currently happening. To plan, desired future states and the predicted outcomes of actions must be represented. In mammals, including humans, hippocampal neurons are thought to encode such representations of past, present and future states at different phases of the theta oscillation. However, the precise hippocampal phase code remains unknown. We show that two previous ideas are incompatible with each other and with rat experimental data. So, we propose a new coding scheme that synthesizes features from both ideas and accounts for all relevant observations.
Enabling the execution of large scale workflows for molecular dynamics simulations
Authors: Andrio Pau , Hospital Adam , Ramon-Cortes Cristian , Conejero Javier , Lezzi Daniele , Ejarque Jorge , Gelpi Josep LL. , Badia Rosa M.
Published: April 2021

The usage of workflows has led to progress in many fields of science, where the need to process large amounts of data is coupled with difficulty in accessing and efficiently using High Performance Computing platforms. On the one hand, scientists are focused on their problem and concerned with how to process their data. On top of that, the applications typically have different parts and use different tools for each part, thus complicating the distribution and the reproducibility of the simulations. On the other hand, computer scientists concentrate on how to develop frameworks for the deployment of workflows on HPC or HTC resources; often providing separate solutions for the computational aspects and the data analytic ones.

In this paper we present an approach to support biomolecular researchers in the development of complex workflows that i) allow them to compose pipelines of individual simulations built from different tools and interconnected by data dependencies, ii) run them seamlessly on different computational platforms, and iii) scale them up to the large number of cores provided by modern supercomputing infrastructures. Our approach is based on the orchestration of computational building blocks for Molecular Dynamics simulations through an efficient workflow management system that has already been adopted in many scientific fields to run applications on multitudes of computing backends.

Results demonstrate the validity of the proposed solution through the execution of massively parallel runs in a supercomputer facility.
Investigating the two regimes of fibrin clot lysis: an experimental and computational approach
Authors: Raynaud Franck , Chopard Bastien , Rousseau Alexandre , Boudjeltia Karim Zouaoui , Monteyne Daniel , Perez-Morga David
Published: April 2021

It has been observedin vitrothat complete clot lysis is generally preceded by a period of latency during which the degradation seems to be inefficient. However, this latency was merely notified but not yet quantitatively discussed. In our experiments we observed that the lysis ubiquitously occurred in two distinct regimes, a slow and a fast lysis regime. We quantified extensively the duration of these regimes for a wide spectrum of experimental conditions and found that on average the slow regime lasts longer than the fast one, meaning that during most of the process the lysis is ineffective. We proposed a computational model in which the two regimes result from a spatially constrained kinetic of clot lysis: first the biochemical reactions take place at the outer core of the fibrin fibers composing the clot, then in the bulk resulting in the observed fast lysis regime. This simple hypothesis appeared to be sufficient to reproduce with a great accuracy the lysis profiles obtained experimentally. Our results shed light on new insights regarding the dynamical aspects of the lysis of fibrin rich clots in a context where the timing is so critical for patient treatment and outcome.

Significance

While the interplay between the main components of the fibrinolytic system is well understood, some dynamical aspects of the fibrinolysis remain unclear. Notably, we observe thatin vitrofibrin rich clots undergo a slow and inefficient phase of degradation when subject to endogenous fibrinolysis. In fact, it turns out that a large part of the lysis process operates in this slow regime. To explain this observation, we proposed a computational model in which the properties of the binding of the proteins change during the lysis. First plasminogen and tissue plasminogen activator bind at the surface of the fibers, resulting in a slow lysis, then in the bulk of the fibers thus speeding up the degradation of the clot..
Computational and biochemical analysis of type IV Pilus dynamics and stability
Authors: Karami Yasaman , López-Castilla Aracelys , Ori Andrea , Thomassin Jenny-Lee , Bardiaux Benjamin , Malliavin Therese , Izadi-Pruneyre Nadia , Francetic Olivera , Nilges Michael
Published: April 2021

Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend, retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including EnterohemorrhagicEscherichia coli(EHEC). The structure of the EHEC T4P has been determined by integrating Nuclear Magnetic Resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability and function, we performed a total of 108 μs all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH and ionic strength was complemented by targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments revealed a conserved set of residues defining a novel calcium-binding site at the interface between three pilin subunits. Calcium binding enhanced T4P stabilityex vivoandin vitro, supporting the role of this binding site as a potential pocket for drug design.
Arf6 is necessary for high level Wingless signalling duringDrosophilawing development
Authors: Marcetteau Julien , Matusek Tamàs , Luton Frédéric , Thérond Pascal P.
Published: April 2021

Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa.In vitrostudies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findingsin vivo, we analyse the consequence of the absence of Arf6 activity onDrosophilawing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling.
Opening of a Cryptic Pocket in β-lactamase Increases Penicillinase Activity
Authors: Knoverek Catherine R , Mallimadugula Upasana L , Singh Sukrit , Rennella Enrico , Frederick Thomas E , Yuwen Tairan , Raavicharla Shreya , Kay Lewis E , Bowman Gregory R
Published: April 2021

Understanding the functional role of protein excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in β-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across β-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM β-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR CEST experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket’s functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.
Facilitation between ecosystem engineers, salt marsh grass and mussels, produces pattern formation on salt marsh shorelines
Authors: Lipcius Romuald N. , Matthews David G. , Shaw Leah , Shi Junping , Zaytseva Sofya
Published: April 2021

Interspecific facilitation between ecosystem engineers, such as salt marsh grass and mussel aggregations, is a key process that structures communities and enhances biodiversity. Scale-dependent pattern formation via self-organization is ubiquitous in terrestrial, aquatic and marine ecosystems. Despite their prevalence and ecological importance, these two phenomena have rarely been linked. We provide empirical evidence that the facilitative interaction in salt marshes between smooth cordgrassSpartina alternifloraand the ribbed musselGeukensia demissaproduces distinct spatial patterns along marsh shorelines. These findings advance our understanding of linkages between facilitation and pattern formation in nature, and are particularly relevant to conservation and restoration of salt marshes threatened by climate change and sea-level rise.
Soil composition and plant genotype determine benzoxazinoid-mediated plant-soil feedbacks in cereals
Authors: Cadot Selma , Gfeller Valentin , Hu Lingfei , Singh Nikhil , Sánchez-Vallet Andrea , Glauser Gaétan , Croll Daniel , Erb Matthias , van der Heijden Marcel G. A. , Schlaeppi Klaus
Published: April 2021

Plant-soil feedbacks refer to effects on plants that are mediated by soil modifications caused by the previous plant generation. Maize conditions the surrounding soil by secretion of root exudates including benzoxazinoids (BXs), a class of bioactive secondary metabolites. Previous work found that a BX- conditioned soil microbiota enhances insect resistance while reducing biomass in the next generation of maize plants. Whether these BX-mediated and microbially driven feedbacks are conserved across different soils and response species is unknown. We found the BX-feedbacks on maize growth and insect resistance conserved between two arable soils, but absent in a more fertile grassland soil, suggesting a soil-type dependence of BX feedbacks. We demonstrated that wheat also responded to BX-feedbacks. While the negative growth response to BX-conditioning was conserved in both cereals, insect resistance showed opposite patterns, with an increase in maize and a decrease in wheat. Wheat pathogen resistance was not affected. Finally and consistent with maize, we found the BX-feedbacks to be cultivar specific. Taken together, BX- feedbacks affected cereal growth and resistance in a soil and genotype dependent manner. Cultivar-specificity of BX-feedbacks is a key finding, as it hides the potential to optimize crops that avoid negative plant-soil feedbacks in rotations.
Custom long non-coding RNA capture enhances detection sensitivity in different human sample types
Authors: Morlion Annelien , Everaert Celine , Nuytens Justine , Hulstaert Eva , Vandesompele Jo , Mestdagh Pieter
Published: April 2021

Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.
Anomalous SAXS at P12 beamline EMBL Hamburg: instrumentation and applications
Authors: Gruzinov Andrey Yu. , Schroer Martin A. , Manalastas-Cantos Karen , Kikhney Alexey G. , Hajizadeh Nelly R. , Schulz Florian , Franke Daniel , Svergun Dmitri I. , Blanchet Clement E.
Published: April 2021

Small-angle X-ray scattering (SAXS) is an established method for studying nanostructured systems and in particular biological macromolecules in solution. To obtain element-specific information about the sample, anomalous SAXS (ASAXS) exploits changes of the scattering properties of selected atoms when the energy of the incident X-rays is close to the binding energy of their electrons. While ASAXS is widely applied to condensed matter and inorganic systems, its use for biological macromolecules is challenging because of the weak anomalous effect. Biological objects are often only available in small quantities and are prone to radiation damage, which makes biological ASAXS measurements very challenging. The BioSAXS beamline P12 operated by the European Molecular Biology Laboratory (EMBL) at the PETRA III storage ring (DESY, Hamburg) is dedicated to studies of weakly scattering objects. Here, recent developments at P12 allowing for ASAXS measurements are presented. The beamline control, data acquisition and data reduction pipeline of the beamline were adapted to conduct ASAXS experiments. Modelling tools were developed to compute ASAXS patterns from atomic models, which can be used to analyze the data and to help designing appropriate data collection strategies. These developments are illustrated with ASAXS experiments on different model systems performed at the P12 beamline.
Control of strain in subgrains of protein crystals by the introduction of grown-in dislocations
Authors: Koizumi Haruhiko , Uda Satoshi , Suzuki Ryo , Tachibana Masaru , Kojima Kenichi , Tsukamoto Katsuo , Yoshizaki Izumi , Fukuyama Seijiro , Suzuki Yoshihisa
Published: April 2021

It is important to reveal the exact cause of poor diffractivity in protein crystals in order to determine the accurate structure of protein molecules. It is shown that there is a large amount of local strain in subgrains of glucose isomerase crystals even though the overall crystal quality is rather high, as shown by clear equal-thickness fringes in X-ray topography. Thus, a large stress is exerted on the subgrains of protein crystals, which could significantly lower the resistance of the crystals to radiation damage. It is also demonstrated that this local strain can be reduced through the introduction of dislocations in the crystal. This suggests that the introduction of dislocations in protein crystals can be effective in enhancing the crystal quality of subgrains of protein crystals. By exploiting this effect, the radiation damage in subgrains could be decreased, leading to the collection of X-ray diffraction data sets with high diffractivity.
Structural and functional studies of SAV1707 from Staphylococcus aureus elucidate its distinct metal-dependent activity and a crucial residue for catalysis
Authors: Kim Dong-Gyun , Lee Kyu-Yeon , Lee Sang Jae , Cheon Seung-Ho , Choi Yuri , Lee Hyung Ho , Ahn Hee-Chul , Lee Bong-Jin
Published: April 2021

The metallo-β-lactamase fold is the most abundant metal-binding domain found in two major kingdoms: bacteria and archaea. Despite the rapid growth in genomic information, most of these enzymes, which may play critical roles in cellular metabolism, remain uncharacterized in terms of structure and function. In this study, X-ray crystal structures of SAV1707, a hypothetical metalloenzyme from Staphylococcus aureus, and its complex with cAMP are reported at high resolutions of 2.05 and 1.55 Å, respectively, with a detailed atomic description. Through a functional study, it was verified that SAV1707 has Ni2+-dependent phosphodiesterase activity and Mn2+-dependent endonuclease activity, revealing a different metal selectivity depending on the reaction. In addition, the crystal structure of cAMP-bound SAV1707 shows a unique snapshot of cAMP that reveals the binding mode of the intermediate, and a key residue Phe511 that forms π–π interactions with cAMP was verified as contributing to substrate recognition by functional studies of its mutant. Overall, these findings characterized the relationship between the structure and function of SAV1707 and may provide further understanding of metalloenzymes possessing the metallo-β-lactamase fold.
Serum levels of tissue factor pathway inhibitor: Potential association with Raynaud’s phenomenon and telangiectasia in patients with systemic sclerosis
Authors: Norimatsu Yuta , Miyagawa Takuya , Fukui Yuki , Omatsu Jun , Toyama Satoshi , Awaji Kentaro , Ikawa Tetsuya , Watanabe Yusuke , Yoshizaki Ayumi , Sato Shinichi , Asano Yoshihide
Published: April 2021

Vasculopathy is a critical step of systemic sclerosis (SSc) development, bridging between autoimmune inflammation and tissue fibrosis. Impaired coagulation system is a part of SSc vasculopathy, but the role of tissue factor pathway inhibitor (TFPI), a critical regulator of the extrinsic coagulation pathway, remained unknown. Therefore, we evaluated the clinical correlation of serum TFPI levels in SSc patients. Serum TFPI levels were comparable between SSc and control participants, but SSc patients with Raynaud’s phenomenon and telangiectasia had significantly lower serum TFPI levels than those without. Importantly, there was a significant positive correlation between serum TFPI levels and protein S activity. These results support the critical role of impaired coagulation system in SSc.
The best of both worlds: Apomixis and sexuality co‐occur in species of Microlicia, Melastomataceae
Authors: Viana Matheus Lacerda , Oliveira Paulo Eugênio , Romero Rosana , Caetano Ana Paula Souza
Published: April 2021

Apomixis, the asexual formation of embryos and seeds, occurs in approximately 18% of angiosperm families. Melastomataceae exhibits a remarkable number of apomictic species, distributed among different tribes. This mode of reproduction has been elucidated in Miconieae, but remains unclarified for other groups, such as Microlicieae. Although apomixis has been previously described for Microlicieae species, the cytological basis for this phenomenon is entirely unknown in this group. Thus, populations of Microlicia fasciculata and M. polystemma were used in order to (a) investigate the presence of autonomous apomixis; (b) verify if this mode of reproduction leads to polyembryony; and (c) investigate whether apomixis may occur in parallel with the sexual process. We tested these species for autonomous fruit set and polyembryony, and pollen viability, and analyzed pollen tube growth. Anatomical techniques were used to elucidate the micro‐ and megasporogenesis and gametogenesis. The species showed autonomous fruit and seed formation and exhibited polyembryony. Apospory and adventitious embryony were the developmental mechanisms of apomixis in M. fasciculata and M. polystemma, respectively. Both species exhibited low pollen viability. However, some viable pollen, reduced embryo sac formation, natural pollination and pollen tube growth enable sexual reproduction and characterize these species as facultative apomicts. The independence of pollinators for fruit set, uniparental reproduction and the possibility of sexual reproduction, confer reproductive assurance and flexibility, bringing together advantages of sexual and asexual reproduction. In this sense, apomixis may have played an important role in the evolution and diversification of Microlicia, a widely distributed genus in the Brazilian Cerrado.
Microbiology of a NaCl stalactite ‘salticle’ in Triassic halite
Authors: Thompson Thomas P. , Kelly Stephen A. , Skvortsov Timofey , Plunkett Gill , Ruffell Alastair , Hallsworth John E. , Hopps Jason , Gilmore Brendan F.
Published: April 2021

Large regions of Earth's surface are underlain by salt deposits that evaporated from ancient oceans and are populated by extreme halophilic microbes. Some of these halophiles may have been preserved over geological timescales within hypersaline fluid inclusions, but ingresses of water and/or anthropogenic activities can lead to the formation of alternative habitats, including NaCl stalactites or other speleothems. While the microbiology of ancient evaporites has been well studied, the ecology of these recently formed structures is less‐well understood. Here, the microbiology of a NaCl stalactite (‘salticle’) in a Triassic halite mine is characterized. The specific aims were to determine the presence of fluid inclusions, determine the microbial structure of the salticle compared with a nearby brine‐pool and surficial soil, and characterize the ecophysiological capabilities of this unique ecosystem. The salticle contained fluid inclusions, and their microbiome was composed of Euryarchaetota, Proteobacteria, and Actinobacteria, with Haloarchaea in greater abundance than brine‐pool or soil microbiomes. The salticle metagenome exhibited a greater abundance of genes involved in osmoregulation, anaerobic respiration, UV resistance, oxidative stress, and stress‐protein synthesis relative to the soil microbiome. We discuss the potential astrobiological implications of salticles as enclosed salt‐saturated habitats that are protected from ionizing radiation and have a stable water activity.