bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intrinsic Network Activity Reflects the Ongoing Experience of Chronic Pain
Authors: Jahn Pauline , Deak Bettina , Mayr Astrid , Stankewitz Anne , Keeser Daniel , Griffanti Ludovica , Witkovsky Viktor , Irving Stephanie , Schulz Enrico 
Published: July 2021

AbstractAnalyses of intrinsic network activity have been instrumental in revealing cortical processes that are altered in chronic pain patients. However, such studies have not accounted for variable time courses of network activity and subjective pain experience. In a novel approach, we aimed to elucidate how intrinsic functional networks evolve in regard to the fluctuating intensity of the experience of chronic pain.In a longitudinal study with 156 fMRI sessions, 20 chronic back pain patients and 20 chronic migraine patients were asked to continuously rate the intensity of their endogenous pain. Using group independent component analysis and dual-regression, we extracted the time courses of 100 independent components separately for chronic back pain and chronic migraine. We investigated the relationship between the fluctuation of intrinsic network activity with the time course of subjective pain ratings.For chronic back pain, we found increased cortical network activity for the salience network and a local pontine network, as well as decreased network activity in the anterior and posterior default mode network for higher pain intensities. Higher pain intensities in chronic migraine were accompanied with lower activity in a prefrontal cortical network.By taking the perspective of the individual, we focused on the processes that matter for each patient, which are phases of relatively low pain and more straining phases of relatively high pain. The present design of ongoing assessment of the endogenous pain can be a powerful and promising tool to assess the signature of a patient’s endogenous pain encoding over weeks and months.
The influence of adaptation to life at high-altitude on condition dependent sexual shape and size dimorphism in Drosophila melanogaster
Authors: Pesevski Maria , Dworkin Ian 
Published: July 2021

AbstractSexual dimorphism is common despite factors such as inter-sex genetic correlations and sex-specific patterns of selection that might limit its evolution. Sexual dimorphism can be phenotypically plastic and condition dependent, that themselves may be targets of selection. However, it remains unclear how sexual dimorphism, its plasticity and condition dependence evolves, in particular during rapid adaptation to a new environment. Furthermore, the interplay between SSD and other forms of dimorphism, such as shape dimorphism co-evolves. Using Sub-Saharan populations of Drosophila melanogaster that vary for size and shape as a result of adaptation to high altitude environments, we examined sex specific patterns of developmental plasticity. We raised strains of Drosophila from low (Zambia) and high (Ethiopia) altitude populations varying for food quality or rearing temperature. We observed expected differences in wing size and shape due to population, sex and plasticity. While larval mass showed substantial evolved changes for sex specific condition dependence, effects on wing size and shape were modest. We examined shape-size allometric effects between groups. Allometric effects were generally similar across sexes, but differed substantially due to population of origin and plasticity. We discuss findings within the context of the evolution of plasticity for SSD, condition dependence and allometric relationships.
Conversion of mammalian cell culture media waste to microbial fermentation feed efficiently supports production of recombinant protein by Escherichia coli
Authors: Lynch Ciara D , O’Connell David J 
Published: July 2021

AbstractDeriving new value from waste streams through secondary processes is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be recycled and used as a feed in secondary microbial fermentation to produce new recombinant protein products. Our results show that CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich microbiological media (LB). The titre of recombinant protein produced following induction in a 4-hour expression screen was approximately equivalent in the CDSM fed cultures to that of baseline, and this was maintained in a 16-hr preparative fermentation. To understand the protein production achieved in CDSM fed culture we performed a quantitative analysis of proteome changes in the E. coli using mass spectrometry. This analysis revealed significant upregulation of protein synthesis machinery enzymes and significant downregulation of carbohydrate metabolism enzymes. We conclude that spent cell culture media, which represents 100s of millions of litres of waste generated by the bioprocessing industry annually, may be valorized as a feed resource for the production of recombinant proteins in secondary microbial fermentations.
Bayesian inference of clonal expansions in a dated phylogeny
Authors: Helekal David , Ledda Alice , Volz Erik , Wyllie David , Didelot Xavier 
Published: July 2021

ABSTRACTMicrobial population genetics models often assume that all lineages are constrained by the same population size dynamics over time. However, many neutral and selective events can invalidate this assumption, and can contribute to the clonal expansion of a specific lineage relative to the rest of the population. Such differential phylodynamic properties between lineages result in asymmetries and imbalances in phylogenetic trees that are sometimes described informally but which are difficult to analyse formally. To this end, we developed a model of how clonal expansions occur and affect the branching patterns of a phylogeny. We show how the parameters of this model can be inferred from a given dated phylogeny using Bayesian statistics, which allows us to assess the probability that one or more clonal expansion events occurred. For each putative clonal expansion event we estimate their date of emergence and subsequent phylodynamic trajectories, including their long-term evolutionary potential which is important to determine how much effort should be placed on specific control measures. We demonstrate the applicability of our methodology on simulated and real datasets.
The versatile regulation of K2P channels by polyanionic lipids of the phosphoinositide (PIP2) and fatty acid metabolism (LC-CoA)
Authors: Riel Elena B. , Jürs Björn C. , Cordeiro Sönke , Musinszki Marianne , Schewe Marcus , Baukrowitz Thomas 
Published: July 2021

AbstractWork of the past three decades provided tremendous insight into the regulation of K+ channels - in particular Kir channels - by polyanionic lipids of the phosphoinositide (e.g. PIP2) and fatty acid metabolism (e.g. oleoyl-CoA). However, comparatively little is known regarding the phosphoinositide regulation in the K2P channel family and the effects of long-chain fatty acid CoA esters (LC-CoA, e.g. oleoyl-CoA) are so far unexplored. By screening most mammalian K2P channels (12 in total), we report strong effects of polyanionic lipids (activation and inhibition) for all tested K2P channels. In most cases the effects of PIP2 and oleoyl-CoA were similar causing either activation or inhibition depending on the respective subgroup. Activation was observed for members of the TREK, TALK and THIK subfamily with the strongest activation by PIP2 seen for TRAAK (~110-fold) and by oleoyl-CoA for TALK-2 (~90-fold). In contrast, inhibition was observed for members of the TASK and TRESK subfamilies up to ~85 %. In TASK-2 channels our results indicated an activatory as well as an inhibitory PIP2 site with different affinities. Finally, we provided evidence that PIP2 inhibition in TASK-1 and TASK-3 channels is mediated by closure of the recently identified lower X-gate as critical mutations within the gate (i.e. L244A, R245A) prevent PIP2 induced inhibition. Our results disclosed K2P channels as a family of ion channels highly sensitive to polyanionic lipids (PIP2 and LC-CoA), extended our knowledge on the mechanisms of lipid regulation and implicate the metabolisms of these lipids as possible effector pathways to regulate K2P channel activity.
The method of choice to knock-in large inserts via CRISPR
Authors: Marks David , Bachmann Lisa , Villarejo Lucia Gallego , Geidies Alexander , Heinen Natalie , Anstatt Jannis , Müller Thorsten 
Published: July 2021

AbstractCRISPR/Cas9 gene editing is a revolutionary method used to study gene function by transcript silencing, knock-out, or activation. The knock-in of DNA fragments to endogenous genes of interest is another promising approach to study molecular pathways but is technically challenging. Many approaches have been suggested, but the proof of correct integration has often been relied on less convenient validation experiments. Within this work, we investigated homology-directed repair (HDR), non-homologous end joining (NHEJ), and PCRextension (PCRext) based approaches as three different methods to knock-in large DNA fragments (>1000 bp), and compared feasibility, cost effectiveness, and reliability. As a knock-in fragment, we used a fluorescent reporter sequence in order to directly assess successful integration by microscopy, subsequently proven by sequencing. For NHEJ and PCRext, we demonstrate that it is insufficient to rely on the fluorescent reporter due to false positive results. Both NHEJ and PCRext failed to reliably knock-in large DNA sequences, they were accompanied by massive generation of InDels driving the methodology cost-intensive and non-reliable. In contrast, combination of CRISPR/Cas9 and HDR revealed correct integration, proven by correct fluorescence of the subcellular localization and sequencing, and thus, corresponds to the method of choice for large fragment integration. Next to HEK293T, we demonstrate successful HDR based knock-in in human induced pluripotent stem cells (hiPSCs). Subsequent differentiation of gene-edited hiPSCs into cerebral organoids showed relevance of the approach to study subcellular protein localization and abundance in 3D tissue.
Thiol-based mucolytics exhibit antiviral activity against SARS-CoV-2 through allosteric disulfide disruption in the spike glycoprotein
Authors: Shi Yunlong , Zeida Ari , Edwards Caitlin E. , Mallory Michael L. , Sastre Santiago , Machado Matías R. , Pickles Raymond J. , Fu Ling , Liu Keke , Yang Jing , Baric Ralph S. , Boucher Richard C. , Radi Rafael , Carroll Kate S. 
Published: July 2021

AbstractSmall molecule therapeutics targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have lagged far behind the development of vaccines in the fight to control the COVID-19 pandemic. Here, we show that thiol-based mucolytic agents, P2119 and P2165, potently inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity of repurposed mucolytic agents to the reduction of key disulfides, specifically, by disruption of the Cys379–Cys432 and Cys391–Cys525 pairs distal to the receptor binding motif (RBM) in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol warhead pointed directly towards Cys432. These collective findings establish the vulnerability of human coronaviruses to repurposed thiol-based mucolytics and lay the groundwork for developing these compounds as a potential treatment, preventative and/or adjuvant against infection.
Biosynthetic Glycan Labeling
Authors: Marando Victoria M. , Kim Daria E. , Calabretta Phillip J. , Kraft Matthew B. , Bryson Bryan D. , Kiessling Laura L. 
Published: July 2021

ABSTRACTGlycans are ubiquitous and play important biological roles, yet chemical methods for probing their structure and function within cells remain limited. Strategies for studying other biomacromolecules, such as proteins, often exploit chemoselective reactions for covalent modification, capture, or imaging. Unlike amino acids that constitute proteins, glycan building blocks lack distinguishing reactivity because they are composed primarily of polyol isomers. Moreover, encoding glycan variants through genetic manipulation is complex. Therefore, we formulated a new, generalizable strategy for chemoselective glycan modification that directly takes advantage of cellular glycosyltransferases. Many of these enzymes are selective for the products they generate yet promiscuous in their donor preferences. Thus, we designed reagents with bioorthogonal handles that function as glycosyltransferase substrate surrogates. We validated the feasibility of this approach by synthesizing and testing probes of D-arabinofuranose (D-Araf), a monosaccharide found in bacteria and an essential component of the cell wall that protects mycobacteria, including Mycobacterium tuberculosis. The result is the first probe capable of selectively labeling arabinofuranose-containing glycans. Our studies serve as a platform for developing new chemoselective labeling agents for other privileged monosaccharides. This probe revealed an asymmetric distribution of D-Araf residues during mycobacterial cell growth and could be used to detect mycobacteria in THP1-derived macrophages.
Dendro-somatic synaptic inputs to ganglion cells violate receptive field and connectivity rules in the mammalian retina
Authors: Sedlacek Miloslav , Grimes William N. , Musgrove Morgan , Nath Amurta , Tian Hua , Hoon Mrinalini , Rieke Fred , Singer Joshua H. , Diamond Jeffrey S. 
Published: July 2021

SummaryIn retinal neurons, morphology strongly influences visual response features. Ganglion cell (GC) dendrites ramify in distinct strata of the inner plexiform layer (IPL) so that GCs responding to light increments (ON) or decrements (OFF) receive appropriate excitatory inputs. This vertical stratification prescribes response polarity and ensures consistent connectivity between cell types, whereas the lateral extent of GC dendritic arbors typically dictates receptive field (RF) size. Here, we identify circuitry in mouse retina that contradicts these conventions. A2 amacrine cells are interneurons understood to mediate “cross-over” inhibition by relaying excitatory input from the ON layer to inhibitory outputs in the OFF layer. Ultrastructural and physiological analyses show, however, that some A2s deliver powerful inhibition to OFF GC somas and proximal dendrites in the ON layer, rendering their inhibitory RFs smaller than their dendritic arbors. This OFF pathway, avoiding entirely the OFF region of the IPL, challenges several tenets of retinal circuitry.
Dopamine D1 receptor activation and cAMP/PKA signalling mediate Brd4 recruitment to chromatin to regulate gene expression in rat striatal neurons
Authors: Jones-Tabah Jace , Martin Ryan D. , Chen Jennifer J. , Tanny Jason C. , Clarke Paul B.S. , Hébert Terence E. 
Published: July 2021

AbstractThe activity of striatal medium-spiny projection neurons is regulated by dopamine acting principally at D1 and D2 dopamine receptors. The dopamine D1 receptor (D1R) is a Gαs/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Transcriptional regulation downstream of the D1R involves the activation of PKA, which can translocate to the nucleus to phosphorylate various targets. The chromatin reader Brd4 regulates transcription induced by neurotrophic factors in cortical neurons and has also been implicated in dopamine-dependent striatal functions. Brd4 is activated by phosphorylation; this facilitates its binding to acetylated histones at promoters and enhancers. In non-neuronal cells, Brd4 is recruited to chromatin in response to PKA signalling. However, it is unknown whether Brd4 is involved in transcriptional activation by the D1R in striatal neurons. Here, we demonstrate that cAMP/PKA signalling increases Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown or inhibition of Brd4 modulated D1R-induced gene expression. Specifically, inhibition of Brd4 with the bromodomain inhibitor JQ1 suppressed the expression of ∼25% of D1R-upregulated genes, while increasing the expression of a subset of immediate-early genes, includingFosandJun. This pro-transcriptional effect of JQ1 was P-TEFb-dependent, and mediated through inhibition of the BD1 bromodomain of Brd4. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify Brd4 as a novel regulator of D1R-dependent transcription and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription.
Prolonging coagulant activity of factor Xa under hemophilic conditions by site-specific N-glycosylation of the surface-exposed autolysis loop
Authors: Bonde Amalie Carnbring , Lund Jacob , Hansen Jens Jacob , Winther Jakob Rahr , Zahn Stefan , Tiainen Peter , Olsen Ole Hvilsted , Petersen Helle Heibroch , Bjelke Jais Rose 
Published: July 2021

AbstractThe regulation of Factor X (FX) is critical to maintain hemostasis. To gain insights to the regulation of the active and zymogen form of coagulation FX, we probed specific molecular interactions by introducing novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. Introduction of N-glycans in the autolysis loop of these FX variants decreased Factor VIIa (FVIIa)-mediated activation ~3-fold and prothrombin activation 2- to 10-fold presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor Factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). Ki for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, while ATIII inhibition in the presence of the heparin-analogue Fondaparinux was modestly increased by 2- to 15-fold compared to wild type. To probe the in vitro hemostatic effect of the FX variants, the thrombin generation potential in FX-depleted plasma was evaluated. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FX variants with N-glycosylation at positions 148-150. These results indicate that residues of the surface-exposed autolysis loop and residues close by participate in FX activation, proteolytic activity and inhibition of FXa by TFPI and ATIII. In plasma-based assays, a modest decrease in FX-activation rate appeared to compensate for the collective reduction in inhibitor interactions.
Size Matters: A Mechanistic Model of Nanoparticle Curvature Effects on Amyloid Fibril Formation
Authors: John Torsten , Adler Juliane , Elsner Christian , Petzold Johannes , Krueger Martin , Martin Lisandra L. , Huster Daniel , Risselada Herre Jelger , Abel Bernd 
Published: July 2021

AbstractThe aggregation of peptides into amyloid fibrils is linked to ageing-related diseases, such as Alzheimer’s disease and type 2 diabetes. Interfaces, particularly those with large nanostructured surface areas, can affect the kinetics of peptide aggregation, ranging from a complete inhibition to strong acceleration. While a number of physiochemical parameters determine interface effects, we here focus on the role of nanoparticle curvature for the aggregation of the amyloidogenic peptides Aβ40, NNFGAIL, GNNQQNY and VQIYVK. Nanoparticles (NPs) provided a surface for peptide monomers to adsorb, enabling the nucleation into oligomers and fibril formation. High surface curvature, however, destabilized prefibrillar structures, providing an explanation for inhibitory effects on fibril growth. Thioflavin T (ThT) fluorescence assays as well as dynamic light scattering (DLS), atomic force microscopy (AFM) and electron microscopy experiments revealed NP size-dependent effects on amyloid fibril formation, with differences between the peptides. While 5 nm gold NPs (AuNP-5) retarded or inhibited the aggregation of most peptides, larger 20 nm gold NPs (AuNP-20) tended to accelerate peptide aggregation. Molecular dynamics (MD) studies demonstrated that NPs’ ability to catalyze or inhibit oligomer formation was influenced by the oligomer stability at curved interfaces which was lower at more highly curved surfaces. Differences in the NP effects for the peptides resulted from the peptide properties (size, aggregation propensity) and concomitant surface binding affinities. The results can be applied to the design of future nanostructured materials for defined applications.
Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6
Authors: Saijo-Hamano Yumiko , Sherif Aalaa Alrahman , Pradipta Ariel , Sasai Miwa , Sakai Naoki , Sakihama Yoshiaki , Yamamoto Masahiro , Standley Daron M , Nitta Ryo 
Published: July 2021

AbstractThe p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. It is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive due to lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP-binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.SummaryUpon Toxoplasma gondii infection, Irgb6 is recruited to the parasitophorous vacuole membrane (PVM) where it disrupts it. We solved the atomic structures of Irgb6 in two distinct nucleotide states, revealing a unique PVM binding interface sensitive to the GTPase cycling.