bluuebunny/crossref_metadata_2025_split huggingface dataset:
Harnessing the functional diversity of plant cystatins to design inhibitor variants highly active against herbivorous arthropod digestive proteases
Authors: Tremblay Jonathan , Goulet Marie-Claire , Vorster Juan , Goulet Charles , Michaud Dominique 
Published: July 2021

AbstractProtein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases. These approaches typically involve the site-directed mutagenesis of functionally relevant amino acids, the production and selection of improved inhibitory variants by molecular phage display procedures, or the design of bi/multifunctional translational fusions integrating one or several cystatin inhibitory domains. Here, we propose a new approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Cys protease inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including herbivorous arthropod digestive proteases, to appreciate the extent of functional variability among plant cystatin protein family members. The most, and less, potent of these cystatins were then used as ‘donors’ of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model ‘recipient’ inhibitor. Our data confirm the wide variety of cystatin protease inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.
iPSC-derived Vascular Smooth Muscle Cells in a Fibronectin Functionalized Collagen Scaffold Augment Endothelial Cell Morphogenesis
Authors: Duan Kaiti , Dash Biraja C. , Sasson Daniel , Hsia Henry C. 
Published: July 2021

AbstractTissue-engineered constructs have immense potential as autologous grafts for wound healing. Despite the rapid advancement in fabrication technology, the major limitation is controlling angiogenesis within these constructs to form a vascular network. Here, we aimed to develop a 3D scaffold that can regulate angiogenesis. We tested the effect of fibronectin and vascular smooth muscle cells derived from human induced pluripotent stem cells (hiPSC-VSMC) on the morphogenesis of endothelial cells. The results demonstrate that fibronectin increases the number of endothelial networks. However, hiPSC-VSMC in the presence of fibronectin further substantiated the number and size of endothelial networks. A mechanistic study shows that blocking αvβ3 integrin signaling between hiPSC-VSMC and fibronectin impacts the endothelial network formation. Collectively, this study set forth initial design criteria in developing an improved pre-vascularized construct.
Switching Promotor Recognition of Phage RNA Polymerase in Silico Following Path along Lab Directed Evolution
Authors: Chao E , Dai Liqiang , Yu Jin 
Published: July 2021

AbstractIn this work we computationally investigated how a viral RNA polymerase (RNAP) from bacteriophage T7 evolves into RNAP variants under lab-directed evolution to switch recognition from T7 promoter to T3 promoter in transcription initiation. We first constructed a closed initiation complex for the wild-type T7 RNAP, and then for six mutant RNAPs discovered from phage assisted continuous evolution experiments. All-atom molecular dynamics (MD) simulations up to one microsecond each were conducted on these RNAPs in complex with T7/T3 promoter. Our simulations show notably that protein-DNA electrostatic interactions or stabilities at the RNAP-DNA promoter interface well dictate the promoter recognition preference of the RNAP and variants. Key residues and structural elements that contribute significantly to switching the promoter recognition were identified. Followed by a first point mutation N748D on the specificity loop to slightly disengage the RNAP from the promoter to hinder the original recognition, we found an auxiliary helix (206-225) that takes over switching the promoter recognition upon further mutations (E222K and E207K), by forming additional charge interactions with the promoter DNA and reorientating differently on the T7 and T3 promoter. Further mutations on the AT-rich loop and the specificity loop can fully switch the RNAP-promoter recognition to the T3 promoter. Overall, our studies reveal energetics and structural dynamics details along an exemplary directed evolutionary path of the phage RNAP variants for a rewired promoter recognition function. The findings demonstrate underlying physical mechanisms and are expected to assist knowledge/data learning or rational redesign of the protein enzyme structure-function.
The MKRN1-BRAF exon4-exon9 fusion is a targetable oncogenic driver
Authors: Girtman Megan A. , Richmond Craig S. , Kenny Paraic A. 
Published: July 2021

ABSTRACTBackgroundBRAF, when mutated at V600E, is a potent oncogenic driver in melanoma, lung and colorectal cancer with well understood signaling mechanisms and established treatment guidelines. Non-V600E mutations are less common, more functionally diverse and do not yet have clear treatment guidelines. One class of non-V600E mutations are BRAF fusion genes which typically involve the C-terminal kinase domain of BRAF joined to one of a wide repertoire of potential N-terminal fusion partners. Here, we functionally characterized an MKRN1-BRAF fusion gene which we detected in multi-gene panel sequencing of a metastatic colorectal tumor.MethodsLevels of MEK/ERK pathway activity were evaluated by western blotting in HEK293 cells ectopically expressing MKRN1-BRAF or a series of other BRAF constructs. Dependence on dimerization was evaluated by introducing a dimerization deficiency mutation and drug sensitivity was evaluated by treatment with sorafenib, dabrafenib and trametinib.ResultsMKRN1-BRAF potently activated MEK/ERK signaling and did not require dimerization for activity. Among the inhibitors evaluated, trametinib most effectively suppressed MKRN1-BRAF driven pathway activity.ConclusionOur data demonstrate that the MKRN1-BRAF fusion gene encodes an oncoprotein that strongly activates MEK/ERK signaling in a trametinib-sensitive manner.
Novel Class of Psychedelic Iboga Alkaloids Disrupts Opioid Use
Authors: Havel Václav , Kruegel Andrew C. , Bechand Benjamin , McIntosh Scot , Stallings Leia , Hodges Alana , Wulf Madalee G. , Nelson Mel , Hunkele Amanda , Ansonoff Michael , Pintar John E. , Hwu Christopher , Abi-Gerges Najah , Zaidi Saheem A. , Katritch Vsevolod , Yang Mu , Javitch Jonathan A. , Majumdar Susruta , Hemby Scott E. , Sames Dalibor 
Published: July 2021

AbstractSubstance use and related mental health epidemics are causing increasing suffering and death in diverse communities.1,2Despite extensive efforts focused on developing pharmacotherapies for treating substance use disorders, there is an urgent need for radically different therapeutic approaches.3,4Ibogaine provides an important drug prototype in this direction, as a psychoactive iboga alkaloid suggested to have the ability to interrupt opioid use in drug-dependent humans.5However, ibogaine and its major metabolite noribogaine present considerable safety risk associated with cardiac arrhythmias.6We introduce a new class of iboga alkaloids - “oxa-iboga” - defined as benzofuran-containing iboga analogs and created via structural editing of the iboga skeleton. The oxa-iboga compounds act as potent kappa opioid receptor agonistsin vitroandin vivo, but exhibit atypical behavioral features compared to standard kappa psychedelics. We show that oxa-noribogaine has greater therapeutic efficacy in rat models of opioid use, and no cardiac pro-arrhythmic potential, in contrast to noribogaine. Oxa-noribogaine induces long-lasting suppression of morphine and fentanyl intake after a single dose, persistent reduction of morphine intake and reinforcing efficacy after a short treatment regimen, and suppression of morphine and fentanyl drug seeking in relapse models. Oxa-noribogaine also induces a lasting elevation of neurotrophin proteins in the ventral tegmental area and medial prefrontal cortex, consistent with targeted neuroplasticity induction and alteration of addiction-like states. As such, oxa-iboga compounds represent candidates for a novel type of pharmacotherapy for treatment of opioid use disorder.
Phenomics data processing: Extracting dose-response curve parameters from high-resolution temperature courses and repeated field-based wheat height measurements
Authors: Roth Lukas , Piepho Hans-Peter , Hund Andreas 
Published: July 2021

AbstractTemperature is a main driver of plant growth and development. New phenotyping tools enable quantifying the temperature response of hundreds of genotypes. Yet, for field-derived data, temperature response modeling bears flaws and pitfalls concerning the interpretation of derived parameters. In this study, climate data from five growing seasons with differing temperature distributions served as starting point for a growth simulation of wheat stem elongation, based on a four-parametric temperature response function (Wang-Engel) including all cardinal temperatures. In a novel approach, we re-extracted dose-responses from the simulation by combining high-resolution (hours) temperature courses with low-resolution (days) height data. The collection of such data is common in field phenotyping platforms. To take advantage of the lack of supra-optimal temperatures during the stem elongation, simpler (linear and asymptotic) models to predict temperature-response parameters were investigated. The asymptotic model extracted the base temperature of growth and the maximum absolute growth rate with high precision, whereas simpler, linear models failed to do so. Additionally, the asymptotic model provided a proxy estimate for the optimum temperature. However, when including seasonally changing cardinal temperatures, the prediction accuracy of the asymptotic model was strongly reduced. In a field study with three winter wheat varieties, significant differences were found for all three asymptotic dose-response curve parameters. We conclude that the asymptotic model based on high-resolution temperature courses is suitable to extract meaningful parameters from field-based data.
Regulation of sedimentation rate shapes the evolution of multicellularity in a unicellular relative of animals
Authors: Dudin Omaya , Wielgoss Sébastien , New Aaron M. , Ruiz-Trillo Iñaki 
Published: July 2021

AbstractSignificant increases in sedimentation rate accompany the evolution of multicellularity. These increases should lead to rapid changes in ecological distribution, thereby affecting the costs and benefits of multicellularity and its likelihood to evolve. However, how genetic and cellular traits control this process, their likelihood of emergence over evolutionary timescales, and the variation in these traits as multicellularity evolves, are still poorly understood. Here, using isolates of the ichthyosporean genus Sphaeroforma - close unicellular relatives of animals with brief transient multicellular life stages - we demonstrate that sedimentation rate is a highly variable and evolvable trait affected by at least two distinct physical mechanisms. First, we find extensive (>300x) variation in sedimentation rates for different Sphaeroforma species, mainly driven by size and density during the unicellular-to-multicellular life cycle transition. Second, using experimental evolution with sedimentation rate as a focal trait, we readily obtained, for the first time, fast settling and multicellular S. arctica isolates. Quantitative microscopy showed that increased sedimentation rates most often arose by incomplete cellular separation after cell division, leading to clonal “clumping” multicellular variants with increased size and density. Strikingly, density increases also arose by an acceleration of the nuclear doubling time relative to cell size. Similar size- and density-affecting phenotypes were observed in four additional species from the Sphaeroforma genus, suggesting variation in these traits might be widespread in the marine habitat. By resequencing evolved isolates to high genomic coverage, we identified mutations in regulators of cytokinesis, plasma membrane remodelling, and chromatin condensation that may contribute to both clump formation and the increase in the nuclear number-to-volume ratio. Taken together, this study illustrates how extensive cellular control of density and size drive sedimentation rate variation, likely shaping the onset and further evolution of multicellularity.
Orphan cytochrome P450 20A1 CRISPR/Cas 9 mutants and neurobehavioral phenotypes in zebrafish
Authors: Brun Nadja R. , Salanga Matthew C. , Mora-Zamorano Francisco X. , Lamb David C. , Goldstone Jared V. , Stegeman John J. 
Published: July 2021

ABSTRACTOrphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish CYP20A1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1−/− mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.
Intrinsically disordered N-terminal domain (NTD) of p53 interacts with mitochondrial PTP regulator Cyclophilin D
Authors: Zhao Jing , Liu Xinyue , Blayney Alan , Zhang Yumeng , Gandy Lauren , Zhang Fuming , Linhardt Robert J. , Chen Jianhan , Baines Christopher , Loh Stewart N. , Wang Chunyu 
Published: July 2021

AbstractMitochondrial permeability transition pore (mPTP) plays crucial roles in cell death in a variety of diseases, including ischemia/reperfusion injury in heart attack and stroke, neurodegenerative conditions, and cancer. To date, cyclophilin D is the only confirmed component of mPTP. Under stress, p53 can translocate into mitochondria and interact with CypD, triggering necrosis and cell growth arrest. However, the molecular details of p53/CypD interaction are still poorly understood. Previously, several studies reported that p53 interacts with CypD through its DNA-binding domain (DBD). However, using surface plasmon resonance (SPR), we found that full-length p53 (FLp53) binds to CypD withKDof ~1 μM, while both NTD-DBD and NTD bind to CypD at ~10 μMKD(Fig. 1C and 1D). Thus, instead of DBD, NTD is the major CypD binding site on p53. NMR titration and MD simulation revealed that NTD binds CypD with broad and dynamic interfaces dominated by electrostatic interactions. NTD 20-70 was further identified as the minimal binding region for CypD interaction, and two NTD fragments, D1 (residues 22-44) and D2 (58-70), can each bind CypD with mM affinity. Our detailed biophysical characterization of the dynamic interface between NTD and CypD provides novel insights on the p53-dependent mPTP opening and drug discovery targeting NTD/CypD interface in diseases.
Nematode Small RNA Pathways in the Absence of piRNAs
Authors: Zagoskin Maxim , Wang Jianbin , Neff Ashley T. , Veronezi Giovana M. B. , Davis Richard E. 
Published: July 2021

AbstractSmall RNA pathways play diverse regulatory roles in the nematodeC. elegans.However, our understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited. Here, we analyzed small RNA pathways in the parasitic nematodeAscaris. Ascarishas ten Argonautes with five worm-specific Argonautes (WAGOs) that are associated with secondary 5’-triphosphate small RNAs (22-24G-RNAs). TheseAscarisWAGOs and their small RNAs target repetitive sequences (WAGO-1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar to theC. elegansmutator, nuclear, and CSR-1 small RNA pathways.AscarisCSR-1 likely functions to “license” gene expression in the absence of anAscarispiRNA pathway.AscarisALG-4 and its associated 26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably,AscarisWAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study provides key insights into the conservation and divergence of nematode small RNA pathways.
Coordination of di-acetylated histone ligands by the ATAD2 bromodomain
Authors: Evans Chiara M. , Phillips Margaret , Malone Kiera L. , Tonelli Marco , Cornilescu Gabriel , Cornilescu Claudia , Holton Simon J. , Gorjánácz Mátyás , Wang Liping , Carlson Samuel , Gay Jamie C. , Nix Jay C. , Demeler Borries , Markley John L. , Glass Karen C. 
Published: July 2021

AbstractThe ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four α-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the “RVF” shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket, but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.
Transfer Learning Compensates Limited Data, Batch-Effects, And Technical Heterogeneity In Single-Cell Sequencing
Authors: Park Youngjun , Hauschild Anne-Christin , Heider Dominik 
Published: July 2021

AbstractTremendous advances in next-generation sequencing technology have enabled the accumulation of large amounts of omics data in various research areas over the past decade. However, study limitations due to small sample sizes, especially in rare disease clinical research, technological heterogeneity, and batch effects limit the applicability of traditional statistics and machine learning analysis. Here, we present a meta-learning approach to transfer knowledge from big data and reduce the search space in data with small sample sizes. Few-shot learning algorithms integrate meta-learning to overcome data scarcity and data heterogeneity by transferring molecular pattern recognition models from datasets of unrelated domains. We explore few-shot learning models with large scale public dataset, TCGA (The Cancer Genome Atlas) and GTEx dataset, and demonstrate their potential as meta-learning dataset in other molecular pattern recognition tasks. Our results show that transfer learning is very effective for datasets with a limited sample size. Furthermore, we show that our approach can transfer knowledge across technological heterogeneity, e.g., from bulk cell to single-cell data. Our approach can overcome study size constraints, batch effects, and technological limitations in analyzing single-cell data by leveraging existing bulk-cell sequencing data.
Using Deep Learning to Count Monarch Butterflies in Dense Clusters
Authors: Patel Shruti , Kulkarni Amogh , Mukhopadhyay Ayan , Gujar Karuna , de Roode Jaap 
Published: July 2021

AbstractMonarch butterflies display one of the most fascinating migration patterns of all species, traveling over 3000 miles from their North American breeding grounds to reach overwintering sites in Central Mexico. Recent studies have suggested that monarchs have experienced an alarming decline in population size due to a combination of deforestation, loss of native milkweed and nectaring plants, and climate change. An issue that conservation efforts face is the lack of principled mechanisms to accurately estimate and count the population size of monarchs. This difficulty occurs due to their small size and existence in dense overwintering clusters in forests. We create an open-source tool to aid conservationists estimate the count of monarch butterflies from images automatically. To the best of our knowledge, our approach, based on deep convolutional neural networks, is the first automated application that can count small insects like monarch butterflies in dense clusters. We demonstrate that our approach achieves high accuracy in counting the number of butterflies even in the presence of occlusion. We also release an open-source dataset containing high resolution images of monarch butterflies along with human annotations for each butterfly’s position. Our open-source implementation can be readily used by scientists to estimate monarch numbers in overwintering clusters and could also be adapted for use in other clustering species.
Microtubule-based mitotic spindles contain a micron-sized mixed-nucleotide zone
Authors: Castrogiovanni Cédric , Inchingolo Alessio , Harrison Jonathan U. , Dudka Damian , Sen Onur , Burroughs Nigel , McAinsh Andrew D. , Meraldi Patrick 
Published: July 2021

AbstractCurrent models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores (K-fibres). Here we reveal that K-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled EBs, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro. In living cells HURP accumulates on the ends of depolymerising K-fibres, whilst avoiding recruitment to nascent polymerising K-fibres. This gives rise to a growing “HURP-gap” which we can recapitulate in a minimal computational simulation. We therefore postulate that the K-fibre lattice contains a dynamic, micron-sized mixed-nucleotide zone.One Sentence SummaryWe reveal that the microtubules of the mitotic spindle contain a third, uncharacterized domain, a mixed nucleotide zone that resides between the GTP-cap and the GDP-tubulin lattice.