bluuebunny/crossref_metadata_2025_split huggingface dataset:
Melanocore uptake by keratinocytes occurs through phagocytosis and involves Protease-activated receptor-2 activation
Authors: Moreiras Hugo , Neto Matilde V. , Bento-Lopes Liliana , Escrevente Cristina , Ramalho José S. , Seabra Miguel C. , Barral Duarte C. 
Published: April 2021

AbstractIn the skin epidermis, melanin is produced and stored within melanosomes in melanocytes and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred – either in a membrane-bound melanosome or as a melanosome core, i.e. melanocore. Here we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1 and CtBP1/BARS, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Furthermore, we confirmed that melanocore, but not melanosome uptake is dependent on the Protease-activated receptor-2 (PAR-2) and found that PAR-2 can be specifically activated by melanocores. As skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.
Defense by duplication: The relation between phenotypic glyphosate resistance and EPSPS gene copy number variation inAmaranthus palmeri
Authors: Yakimowski Sarah B. , Teitel Zachary , Caruso Christina M. 
Published: April 2021

AbstractGene copy number variation (CNV) has been increasingly associated with organismal responses to environmental stress, but we know little about the quantitative relation between CNV and phenotypic variation. In this study we quantify variation in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) copy number using digital drop PCR and variation in phenotypic glyphosate resistance in 22 populations ofAmaranthus palmeri(Palmer Amaranth), a range-expanding agricultural weed. Overall, we detected a significant positive relation between population mean copy number and mean resistance. The majority of populations exhibited high glyphosate resistance, yet maintained low-resistance individuals resulting in bimodality in many populations. We investigated linear and threshold models for the relation between copy number and resistance, and found evidence for a threshold of ~15 EPSPS copies: there was a steep increase in resistance before the threshold, followed by a much shallower slope. Moreover, as copy number increases, the range of variation in resistance decreases. This suggests a working hypothesis that as EPSPS copies and dosage increases, negative epistatic interactions may be compensated. We detected a quadratic relation between mean resistance and variation (s.d.) in resistance, consistent with the prediction that as phenotypic resistance increases in populations, stabilizing selection decreases variation in the trait. Finally, patterns of variation across the landscape are consistent with less variation among populations in mean copy number / resistance in Georgia where glyphosate resistance was first detected, and wider variation among populations in resistance and copy number in a more northern state where resistance evolution may be at a younger evolutionary state.
Kinetic and structural characterization of the self-labeling protein tags HaloTag7, SNAP-tag and CLIP-tag
Authors: Wilhelm Jonas , Kühn Stefanie , Tarnawski Miroslaw , Gotthard Guillaume , Tünnermann Jana , Tänzer Timo , Karpenko Julie , Mertes Nicole , Xue Lin , Uhrig Ulrike , Reinstein Jochen , Hiblot Julien , Johnsson Kai 
Published: April 2021

AbstractThe self-labeling protein tags (SLPs) HaloTag7, SNAP-tag and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study on the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rates with certain rhodamine substrates, which are more than two orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rates however are less affected by the structure of the label than those of HaloTag7, which vary over six orders of magnitude for commonly employed substrates. Solving the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.
Subthalamic nucleus stabilizes movements by reducing neural spike variability in monkey basal ganglia: chemogenetic study
Authors: Hasegawa Taku , Chiken Satomi , Kobayashi Kenta , Nambu Atsushi 
Published: April 2021

AbstractsThe subthalamic nucleus (STN) projects to the external pallidum (GPe) and internal pallidum (GPi), the relay and output nuclei of the basal ganglia (BG), respectively, and plays an indispensable role in controlling voluntary movements. To elucidate the neural mechanism by which the STN controls GPe/GPi activity and movements, we utilized a chemogenetic method to reversibly suppress the motor subregion of the STN in three macaque monkeys (Macaca fuscata, both sexes) engaged in reaching tasks. Systemic administration of chemogenetic ligands prolonged movement time and increased spike train variability in the GPe/GPi, but only slightly affected firing rate modulations. Across-trial analyses revealed that the irregular discharge activity in the GPe/GPi coincided with prolonged movement time. STN suppression also induced excessive abnormal movements in the contralateral forelimbs, which was preceded by STN and GPe/GPi phasic activity changes. Our results suggest that the STN stabilizes spike trains in the BG and achieves stable movements.
Proteome-scale amino-acid resolution footprinting of protein-binding sites in the intrinsically disordered regions of the human proteome
Authors: Benz Caroline , Ali Muhammad , Krystkowiak Izabella , Simonetti Leandro , Sayadi Ahmed , Mihalic Filip , Kliche Johanna , Andersson Eva , Jemth Per , Davey Norman E. , Ivarsson Ylva 
Published: April 2021

AbstractSpecific protein-protein interactions are central to all processes that underlie cell physiology. Numerous studies using a wide range of experimental approaches have identified tens of thousands of human protein-protein interactions. However, many interactions remain to be discovered, and low affinity, conditional and cell type-specific interactions are likely to be disproportionately under-represented. Moreover, for most known protein-protein interactions the binding regions remain uncharacterized. We previously developed proteomic peptide phage display (ProP-PD), a method for simultaneous proteome-scale identification of short linear motif (SLiM)-mediated interactions and footprinting of the binding region with amino acid resolution. Here, we describe the second-generation human disorderome (HD2), an optimized ProP-PD library that tiles all disordered regions of the human proteome and allows the screening of ~1,000,000 overlapping peptides in a single binding assay. We define guidelines for how to process, filter and rank the results and provide PepTools, a toolkit for annotation and analysis of identified hits. We uncovered 2,161 interaction pairs for 35 known SLiM-binding domains and confirmed a subset of 38 interactions by biophysical or cell-based assays. Finally, we show how the amino acid resolution binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the human proteome. The HD2 ProP-PD library paired with PepTools represents a powerful pipeline for unbiased proteome-wide discovery of SLiM-based interactions.
Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing
Authors: Chen Kun , Yan Rui , Xiang Limin , Xu Ke 
Published: April 2021

AbstractThe multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lampviaan acousto-optic tunable filter (AOTF), up to 6 subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (∼1%) crosstalks and high temporal resolutions (down to ∼10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and FRET (Förster resonance energy transfer) fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.
Engineering Insulin Cold Chain Resilience to Improve Global Access
Authors: Maikawa Caitlin L. , Mann Joseph L. , Kannan Aadithya , Meis Catherine M. , Grosskopf Abigail K. , Ou Ben S. , Smith Anton A. A. , Fuller Gerald G. , Maahs David M. , Appel Eric A. 
Published: April 2021

ABSTRACTThere are 150 million people with diabetes worldwide who require insulin replacement therapy and the prevalence of diabetes is rising fastest in middle and low-income countries. Current formulations require costly refrigerated transport and storage to prevent loss of insulin integrity. This study shows the development of simple “drop-in” amphiphilic copolymer excipients to maintain formulation integrity, bioactivity, pharmacokinetics and pharmacodynamics for over 6 months when subjected to severe stressed aging conditions that cause current commercial formulation to fail in under 2 weeks. Further, when these copolymers are added to Humulin R (Eli Lilly) in original commercial packaging they prevent insulin aggregation for up to 4 days at 50 °C compared to less than 1 day for Humulin R alone. These copolymers demonstrate promise as simple formulation additives to increase the cold chain resilience of commercial insulin formulations, thereby expanding global access to these critical drugs for treatment of diabetes.
Data-driven and interpretable machine-learning modeling to explore the fine-scale environmental determinants of malaria vectors biting rates in rural Burkina Faso
Authors: Taconet Paul , Porciani Angélique , Soma Dieudonné Diloma , Mouline Karine , Simard Frédéric , Koffi Alphonsine Amanan , Pennetier Cedric , Dabiré Roch Kounbobr , Mangeas Morgan , Moiroux Nicolas 
Published: April 2021

AbstractBackgroundImproving the knowledge and understanding of the environmental determinants of malaria vectors abundances at fine spatiotemporal scales is essential to design locally tailored vector control intervention. This work aimed at exploring the environmental tenets of human-biting activity in the main malaria vectors (Anopheles gambiae s.s.,Anopheles coluzzii andAnopheles funestus)in the health district of Diébougou, rural Burkina Faso.MethodsAnopheleshuman-biting activity was monitored in 27 villages during 15 months (in 2017-2018), and environmental variables (meteorological and landscape) were extracted from high resolution satellite imagery. A two-step data-driven modeling study was then carried-out. Correlation coefficients between the biting rates of each vector species and the environmental variables taken at various temporal lags and spatial distances from the biting events were first calculated. Then, multivariate machine-learning models were generated and interpreted to i) pinpoint primary and secondary environmental drivers of variation in the biting rates of each species and ii) identify complex associations between the environmental conditions and the biting rates.ResultsMeteorological and landscape variables were often significantly correlated with the vectors’ biting rates. Many nonlinear associations and thresholds were unveiled by the multivariate models, both for meteorological and landscape variables. From these results, several aspects of the bio-ecology of the main malaria vectors were precised or hypothesized for the Diébougou area, including breeding sites typologies, development and survival rates in relation to weather, flight ranges from breeding sites, dispersal related to landscape openness.ConclusionsUsing high resolution data in an interpretable machine-learning modeling framework proved to be an efficient way to enhance the knowledge of the complex links between the environment and the malaria vectors at a local scale. More broadly, the emerging field of interpretable machine-learning has significant potential to help improving our understanding of the complex processes leading to malaria transmission.
The ubiquitin ligase HOIL-1L regulates immune responses by interacting with linear ubiquitin chains
Authors: Gomez-Diaz Carlos , Jonsson Gustav , Schodl Katrin , Deszcz Luiza , Bestehorn Annika , Eislmayr Kevin , Almagro Jorge , Kavirayani Anoop , Fennell Lilian M , Hagelkruys Astrid , Kovarik Pavel , Penninger Josef M , Ikeda Fumiyo 
Published: April 2021

AbstractThe Linear Ubiquitin Assembly Complex (LUBAC), composed of HOIP, HOIL-1L and SHARPIN, promotes Tumor Necrosis Factor (TNF)-dependent NF-κB signaling in diverse cell types. HOIL-1L contains an Npl4 Zinc Finger (NZF) domain that specifically recognizes linear ubiquitin chains, but its physiological rolein vivohas remained unclear. Here, we demonstrate that the HOIL-1L NZF domain has important regulatory functions in inflammation and immune responses in mice. We generated knockin mice (Hoil-1lT20;A;R208A/T201A;R208A) expressing a HOIL-1L NZF mutant, and observed attenuated responses to TNF- and LPS-induced shock, including prolonged survival, stabilized body temperature, reduced cytokine production and liver damage markers. Cells derived from the HOIL-1L knockin mice show reduced TNF-dependent NF-κB activation and incomplete recruitment of HOIL-1L into TNF Receptor (TNFR) Complex I. We further show that the HOIL-1L-NZF domain cooperates with SHARPIN to prevent TNFR-dependent skin inflammation. Collectively, our data suggest that linear ubiquitin-chain binding by HOIL-1L regulates immune responses and inflammationin vivo.
Pch2 orchestrates the meiotic recombination checkpoint from the cytoplasm
Authors: Herruzo Esther , Lago-Maciel Ana , Baztán Sara , Santos Beatriz , Carballo Jesús A. , San-Segundo Pedro A. 
Published: April 2021

ABSTRACTDuring meiosis, defects in critical events trigger checkpoint activation and restrict cell cycle progression. The budding yeast Pch2 AAA+ ATPase orchestrates the checkpoint response launched by synapsis deficiency; deletion ofPCH2or mutation of the ATPase catalytic sites suppress the meiotic block of thezip1Δ mutant lacking the central region of the synaptonemal complex. Pch2 action enables adequate levels of phosphorylation of the Hop1 axial component at threonine 318, which in turn promotes activation of the Mek1 effector kinase and the ensuing checkpoint response. Inzip1Δ chromosomes, Pch2 is exclusively associated to the rDNA region, but this nucleolar fraction is not required for checkpoint activation, implying that another yet uncharacterized Pch2 population must be responsible for this function. Here, we have artificially redirected Pch2 to different subcellular compartments by adding ectopic NES or NLS sequences or by trapping Pch2 in an immobile extranuclear domain, and we have evaluated the effect on Hop1 chromosomal distribution and checkpoint activity. We have also deciphered the spatial and functional impact of Pch2 regulators including Orc1, Dot1 and Nup2. We conclude that the cytoplasmic pool of Pch2 is sufficient to support the meiotic recombination checkpoint involving the subsequent Hop1-Mek1 activation on chromosomes, whereas the nuclear accumulation of Pch2 has pathological consequences. We propose that cytoplasmic Pch2 provokes a conformational change in Hop1 that poises it for its chromosomal incorporation and phosphorylation. Our discoveries shed light into the intricate regulatory network controlling the accurate balance of Pch2 distribution among different cellular compartments, which is essential for proper meiotic outcomes.
Perineuronal nets affect memory and learning after synapse withdrawal
Authors: Ruzicka Jiri , Dalecka Marketa , Safrankova Kristyna , Peretti Diego , Mallucci Giovanna , Jendelova Pavla , Kwok Jessica CF , Fawcett James W 
Published: April 2021

AbstractPerineuronal nets (PNNs) enwrap mature neurons, playing a role in the control of plasticity and synapse dynamics. PNNs have been shown to have effects on memory formation, retention and extinction in a variety of animal models. It has been proposed that the cavities in PNNs which contain synapses can act as a memory store, which remains stable after events that cause synaptic withdrawal such as anoxia or hibernation. We examine this idea by monitoring positional memory before and after synaptic withdrawal caused by acute hibernation-like state (HLS). Animals lacking hippocampal PNNs due to enzymatic digestion by chondroitinase ABC or knockout of the PNN component aggrecan were compared with wild type controls. HLS-induced synapse withdrawal caused a memory deficit, but not to the level of naïve animals and not worsened by PNN attenuation. After HLS, animals lacking PNNs showed faster relearning. Absence of PNNs affected the restoration of inhibitory and excitatory synapses on PNN-bearing neurons. The results support a role for hippocampal PNNs in learning, but not in long-term memory storage.
A3D 2.0 update for the prediction and optimization of protein solubility
Authors: Pujols Jordi , Iglesias Valentín , Santos Jaime , Kuriata Aleksander , Kmiecik Sebastian , Ventura Salvador 
Published: April 2021

AbstractProtein aggregation propensity is a property imprinted in protein sequences and structures, being associated with the onset of human diseases and limiting the implementation of protein-based biotherapies. Computational approaches stand as cost-effective alternatives for reducing protein aggregation and increasing protein solubility. AGGRESCAN 3D (A3D) is a structure-based predictor of aggregation that takes into account the conformational context of a protein, aiming to identify aggregation-prone regions exposed in protein surfaces. Here we inspect the updated 2.0 version of the algorithm, which extends the application of A3D to previously inaccessible proteins and incorporates new modules to assist protein redesign. Among these features, the new server includes stability calculations and the possibility to optimize protein solubility using an experimentally validated computational pipeline. Finally, we employ defined examples to navigate the A3D RESTful service, a routine to handle extensive protein collections. Altogether, this work is conceived to train and assist A3D non-experts in the study of aggregation-prone regions and protein solubility redesign.
Rapid auxin-mediated phosphorylation of Myosin regulates trafficking and polarity in Arabidopsis
Authors: Han Huibin , Verstraeten Inge , Roosjen Mark , Mazur Ewa , Rýdza Nikola , Hajný Jakub , Ötvös Krisztina , Weijers Dolf , Friml Jiří 
Published: April 2021

AbstractThe signaling molecule auxin controls plant development through a well-known transcriptional mechanism that regulates many genes. However, auxin also triggers cellular responses within seconds or minutes, and mechanisms mediating such fast responses have remained elusive. Here, we identified an ultrafast auxin-mediated protein phosphorylation response in Arabidopsis roots that is largely independent of the canonical TIR1/AFB receptors. Among targets of this novel response are Myosin XI and its adaptor protein MadB2. We show that their auxin-mediated phosphorylation regulates trafficking and polar, subcellular distribution of PIN auxin transporters. This phosphorylation-based auxin signaling module is indispensable during developmental processes that rely on auxin-mediated PIN repolarization, such as termination of shoot gravitropic bending or vasculature formation and regeneration. Hence, we identified a fast, non-canonical auxin response targeting multiple cellular processes and revealed auxin-triggered phosphorylation of a myosin complex as the mechanism for feedback regulation of directional auxin transport, a central component of auxin canalization, which underlies self-organizing plant development.
The effect of different exercise training modes on dentate gyrus neurodegeneration and synaptic plasticity in morphine-dependent rats
Authors: Ranjbar Kamal , Zarrinkalam Ebrahim , Asl Sara Soleimani , Salehi Iraj , Taheri Masoumeh , Komaki Alireza 
Published: April 2021

AbstractVarious impacts of exercise on brain performance have been documented following morphine dependence induction; however, the underlying neuronal mechanisms remain unclear. The present research was done to investigate the impact of different exercise training modes on neuronal maturation, and synaptic plasticity in the perforant pathway (PP)-dentate gyrus (DG) synapse in the morphine-dependent rats. Five groups, including a control group (Con, ten healthy rats) and forty morphine-dependent rats were considered as follows (n=10/group): 1) sedentary-dependent (Sed-D); 2) endurance exercise-dependent (En-D); 3) strength exercise-dependent (St-D); and 4) concurrent exercise-dependent (Co-D). The exercise training groups were subjected to endurance, strength, and concurrent training 5 days per week for 10 weeks. After training sessions, the field excitatory postsynaptic potential (fEPSP) slope and population spike (PS) amplitude in DG were determined in response to high-frequency stimulation (HFS) of the PP. For assessing neurogenesis NeuroD level was evaluated after performing all experiments. Concurrent training increased PS amplitude and EPSP than the control group. NeuroD in the morphine-dependent rats significantly decreased, but concurrent training returned the NeuroD to the healthy rat level. Concurrent training can ameliorate synaptic plasticity impairment in morphine-dependent rats through neurogenesis promotion. According to the results, concurrent training can be an appropriate novel candidate for treating opioid addiction.