bluuebunny/crossref_metadata_2025_split huggingface dataset:
Solution structure ofGaussiaLuciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR
Authors: Wu Nan , Kobayashi Naohiro , Tsuda Kengo , Unzai Satoru , Saotome Tomonori , Kuroda Yutaka , Yamazaki Toshio 
Published: July 2020

AbstractGaussialuciferase (GLuc) is the smallest luciferase (18.2kDa; 168 residues) reported so far and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a solubility tag. Almost perfect assignments of GLuc’s1H,13C and15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36-81, 96-145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.
Characterisation of Geometric Variance in the Epithelial Nerve Net of the CtenophorePleurobrachia pileus
Authors: Courtney Amy , Liegey Jérémy , Burke Niamh , Lowey Madeleine , Pickering Mark 
Published: July 2020

AbstractNeuroscience currently lacks a diverse repertoire of model organisms, resulting in an incomplete understanding of what principles of neural function generalise and what are species-specific. Ctenophores display many neurobiological and experimental features which make them a promising candidate to fill this gap. They possess a nerve net distributed across their outer body surface, just beneath the epithelial layer. There is a long-held assumption that nerve nets are ‘simple’ and random while lacking distinct organisational principles. We want to challenge this assumption and determine how stereotyped the structure of this network really is. We validated an approach to estimate body surface area inPleurobrachia pileususing custom Optical Projection Tomography and Light Sheet Morphometry imaging systems. We used an antibody against tyrosylatedα-tubulin to visualise the nerve net in situ. We used an automated segmentation approach to extract the morphological features of the nerve net. We characterised organisational rules of the epithelial nerve net inP. pileusin animals of different sizes and at different regions of the body. We found that specific morphological features within the nerve net are largely un-changed during growth. These properties must be essential to the functionality of the nervous system and therefore are maintained during a change in body size. We have also established the principles of organisation of the network and showed that some of the geometric properties are variable across different parts of the body. This suggests that there may be different functions occurring in regions with different structural characteristics. This is the most comprehensive structural description of a nerve net to date. This study also demonstrates the amenability of the ctenophoreP. pileusfor whole organism network analysis and shows their promise as a model organism for neuroscience, which may provide insights into the foundational principles of nervous systems.
A glycan cluster on the SARS-CoV-2 spike ectodomain is recognized by Fab-dimerized glycan-reactive antibodies
Authors: Acharya Priyamvada , Williams Wilton , Henderson Rory , Janowska Katarzyna , Manne Kartik , Parks Robert , Deyton Margaret , Sprenz Jordan , Stalls Victoria , Kopp Megan , Mansouri Katayoun , Edwards Robert J , Meyerhoff R. Ryan , Oguin Thomas , Sempowski Gregory , Saunders Kevin , Haynes Barton F. 
Published: July 2020

SummaryThe COVID-19 pandemic caused by SARS-CoV-2 has escalated into a global crisis. The spike (S) protein that mediates cell entry and membrane fusion is the current focus of vaccine and therapeutic antibody development efforts. The S protein, like many other viral fusion proteins such as HIV-1 envelope (Env) and influenza hemagglutinin, is glycosylated with both complex and high mannose glycans. Here we demonstrate binding to the SARS-CoV-2 S protein by a category of Fab-dimerized glycan-reactive (FDG) HIV-1-induced broadly neutralizing antibodies (bnAbs). A 3.1 Å resolution cryo-EM structure of the S protein ectodomain bound to glycan-dependent HIV-1 bnAb 2G12 revealed a quaternary glycan epitope on the spike S2 domain involving multiple protomers. These data reveal a new epitope on the SARS-CoV-2 spike that can be targeted for vaccine design.HighlightsFab-dimerized, glycan-reactive (FDG) HIV-1 bnAbs cross-react with SARS-CoV-2 spike.3.1 Å resolution cryo-EM structure reveals quaternary S2 epitope for HIV-1 bnAb 2G12.2G12 targets glycans, at positions 709, 717 and 801, in the SARS-CoV-2 spike.Our studies suggest a common epitope for FDG antibodies centered around glycan 709.
Prolonged mitosis causes separase deregulation and chromosome nondisjunction
Authors: Shindo Norihisa , Otsuki Makoto , Uchida Kazuhiko S.K. , Hirota Toru 
Published: July 2020

AbstractIntercellular karyotype diversity, or aneuploidy, is a widespread feature of cancers propagated through continuous mitotic chromosome segregation errors, the condition called chromosomal instability. The protease separase is an essential enzyme that sever cohesin between sister chromatids, and a probe for separase activity has articulated that separase undergoes abrupt activation shortly before anaphase onset, after being suppressed through metaphase; however the relevance of this robust control remains unclear. In this study, we found that separase activates precociously during prolonged metaphase, consistently in multiple types of cancer cell lines. An artificial extension of metaphase alone in chromosomally stable diploid cells was found to induce precocious activation. These kinetic changes resulted in an incomplete removal of cohesin and emergence of chromosomal bridges in anaphase. Conversely, in transformed cells, shortening back of their prolonged metaphase restored the robust activation of separase and ameliorated anaphase bridge formation. These observations suggest that retarded metaphase progression directly affects the separase activation profile, which provides a previously unanticipated etiology for chromosomal instability in cancers and underscores the significance of the swift mitotic transitions for fail-safe chromosome segregation.
The Arabidopsis V-ATPase is localized to the TGN/EE via a seed plant specific motif and acts in a partially redundant manner with the tonoplast enzyme
Authors: Lupanga Upendo , Röhrich Rachel , Askani Jana , Hilmer Stefan , Kiefer Christiane , Krebs Melanie , Kanazawa Takehiko , Ueda Takashi , Schumacher Karin 
Published: July 2020

AbstractVacuolar-type H+-ATPases (V-ATPases) are versatile proton pumps that control the pH of many intracellular compartments in all eukaryotic cells. The localization of the Arabidopsis V-ATPase was previously shown to be determined by the isoforms of subunit a (VHA-a). The incorporation of VHA-a1 targets the V-ATPase to the trans-Golgi network/early endosome (TGN/EE) whilst the incorporation of VHA-a2 or VHA-a3 targets the V-ATPase to the tonoplast. By employing chimeric proteins and site directed mutagenesis we identified a targeting domain (a1-TD) containing an acidic cluster in the N-terminus of VHA-a1 that serves as both an ER export signal and as a TGN retention motif. The a1-TD is conserved among seed plants and we confirmed experimentally that its presence is predictive of TGN/EE- localization. In contrast to many other non-seed plants, the liverwortMarchantia polymorphaencodes only a single VHA-a subunit (MpVHA-a) and we show here that it is predominantly localized at the tonoplast. In our attempts to determine if MpVHA-a can functionally replace the Arabidopsis VHA-a isoforms, we used CRISPR/Cas9 to generate null-alleles lacking VHA-a1 and discovered that its function is essential for male gametophyte development but can be replaced by VHA-a2 and VHA-a3 during vegetative growth.
Population genomics on the origin of lactase persistence in Europe and South Asia
Authors: Satta Yoko , Takahata Naoyuki 
Published: July 2020

AbstractThe C to T mutation at rs4988235 located upstream of the lactase (LCT) gene is the primary determinant for lactase persistence (LP) that is prevalent among Europeans and South Asians. Here, we review evolutionary studies of this mutation based on ancient and present-day human genomes with the following concluding remarks: the mutation arose in the Pontic Steppe somewhere between 23,000 and 5960 years ago, emigrated into Europe and South Asia in the Bronze Age via the expansion of the Steppe ancestry, and experienced local hard sweeps with their delayed onsets occurring between 5000 and 3280 years ago. We also argue that the G to A mutation at rs182549 arose earlier than 23,000 years ago, the intermediate CA haplotype ancestral to the LP-related TA haplotype is still represented by samples from Tuscans, admixed Americans and South Asians, and the great majority of G to A mutated descendants have hitchhiked since the C to T mutation was favored by local selection.
Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target
Authors: Sobala Łukasz F. , Fernandes Pearl Z , Hakki Zalihe , Thompson Andrew J , Howe Jonathon D , Hill Michelle , Zitzmann Nicole , Davies Scott , Stamataki Zania , Butters Terry D. , Alonzi Dominic S. , Williams Spencer J , Davies Gideon J 
Published: July 2020

ABSTRACTMammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the soleendo-acting glycoside hydrolase involved in N-glycan trimming and unusually is located within the Golgi, where it allows ER escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur upon substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. The structures inspired the development of new inhibitors that disrupted host protein N-glycan processing of viral glycans and reduced infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts of developing broad-spectrum antiviral agents and bring about a more detailed view of the biology of mammalian glycosylation.SIGNIFICANCE STATEMENTThe glycosylation of proteins is a major protein modification that occurs extensively in eukaryotes. Glycosidases in the secretory pathway that trim N-linked glycans play a key role in protein quality control and in the specific modifications leading to mature glycoproteins. Inhibition of glucosidases in the secretory pathway is a proven therapeutic strategy, and one with great promise in the treatment of viral disease. The enzyme endo-α-1,2-mannosidase, MANEA, provides an alternative processing pathway to evade glucosidase inhibitors. We report the 3D structure of human MANEA and complexes with enzyme inhibitors that we show act as antivirals for bovine viral diarrhea and human dengue viruses. The structure of MANEA will support inhibitor optimization and the development of more potent antivirals.
Proteus software for physics-based protein design
Authors: Mignon David , Druart Karen , Opuu Vaitea , Polydorides Savvas , Villa Francesco , Gaillard Thomas , Michael Eleni , Archontis Georgios , Simonson Thomas 
Published: July 2020

AbstractWe describe methods and software for physics-based protein design. The folded state energy combines molecular mechanics with Generalized Born solvent. Sequence and conformation space are sampled with Replica Exchange Monte Carlo, assuming one or a few fixed protein backbone structures and discrete side chain rotamers. Whole protein design and enzyme design are presented as illustrations. Full redesign of three PDZ domains was done using a simple, empirical, unfolded state model. Designed sequences were very similar to natural ones. Enzyme redesign exploited a powerful, adaptive, importance sampling approach that allows the design to directly target substrate binding, reaction rate, catalytic efficiency, or the specificity of these properties. Redesign of tyrosyl-tRNA synthetase stereospecificity is reported as an example.
Genetic variants in TMPRSS2 and Structure of SARS-CoV-2 spike glycoprotein and TMPRSS2 complex
Authors: Vishnubhotla Ravikanth , Vankadari Naveen , Ketavarapu Vijayasarathy , Amanchy Ramars , Avanthi Steffie , Bale Govardhan , Reddy Duvvur Nageshwar , Sasikala Mitnala 
Published: July 2020

AbstractSARS-CoV-2, a highly transmittable pathogen has infected over 3.8 million people around the globe. The spike glycoprotein of SARS-CoV-2 engages host ACE2 for adhesion, TMPRSS2 for activation and entry. With the aid of whole-exome sequencing, we report a variant rs12329760 inTMPRSS2gene and its mutant V160M, which might impede viral entry. Furthermore, we identified TMPRSS2 cleavage sites in S2 domain of spike glycoprotein and report the structure of TMPRSS2 in complex with spike glycoprotein. We also report the structures of protease inhibitors in complex with TMPRSS2, which could hamper the interaction with spike protein. These findings advance our understanding on the role of TMPRSS2 and in the development of potential therapeutics.
Capturing limbal epithelial stem cell population dynamics, signature, and their niche
Authors: Altshuler Anna , Amitai-Lange Aya , Tarazi Noam , Dey Sunanda , Strinkovsky Lior , Bhattacharya Swarnabh , Hadad-Porat Shira , Nasser Waseem , Imeri Jusuf , Ben-David Gil , Tiosano Beatrice , Berkowitz Eran , Karin Nathan , Savir Yonatan , Shalom-Feuerstein Ruby 
Published: July 2020

AbstractStem cells (SCs) are traditionally viewed as rare, slow-cycling cells that follow deterministic rules dictating their self-renewal or differentiation. It was several decades ago, when limbal epithelial SCs (LSCs) that regenerate the corneal epithelium were one of the first sporadic, quiescent SCs ever discovered. However, LSC dynamics, heterogeneity and genetic signature are largely unknown. Moreover, recent accumulating evidence strongly suggested that epithelial SCs are actually abundant, frequently dividing cells that display stochastic behavior.In this work, we performed an in-depth analysis of the murine limbal epithelium by single-cell RNA sequencing and quantitative lineage tracing. The generated data provided an atlas of cell states of the corneal epithelial lineage, and particularly, revealed the co-existence of two novel LSC populations that reside in separate and well-defined sub-compartments. In the “outer” limbus, we identified a primitive widespread population of quiescent LSCs (qLSCs) that uniformly express Krt15/Gpha2/Ifitm3/Cd63 proteins, while the “inner” limbus host prevalent active LSCs (aLSCs) co-expressing Krt15-GFP/Atf3/Mt1-2/Socs3. Analysis of LSC population dynamics suggests that while qLSCs and aLSCs possess different proliferation rates, they both follow similar stochastic rules that dictate their self-renewal and differentiation. Finally, T cells were distributed in close proximity to qLSCs. Indeed, their absence or inhibition resulted in the loss of quiescence and delayed wound healing. Taken together, we propose that divergent regenerative strategies are tailored to properly support tissue-specific physiological constraints. The present study suggests that in the case of the cornea, quiescent epithelial SCs are abundant, follow stochastic rules and neutral drift dynamics.
Structure of Full Length Plasmodium Myosin A and its light chain PfELC, dual targets against malaria parasite pathogenesis
Authors: Moussaoui Dihia , Robblee James P. , Auguin Daniel , Krementsova Elena B. , Haase Silvia , Blake Thomas C. A. , Baum Jake , Robert-Paganin Julien , Trybus Kathleen M. , Houdusse Anne 
Published: July 2020

AbstractParasites from the genusPlasmodiumare the causative agents of malaria. The mobility, infectivity and ultimately pathogenesis of this parasite relies on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor’s mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future antimalarials targeting both the glideosome motor and its regulatory elements.HighlightsThe first structures of the full length PfMyoA motor in two states of its motor cycle.A unique priming of the PfMyoA lever arm results from specific lever arm/motor domain interactions, which allows for a larger powerstroke to enhance speed.Sequence adaptations within the motor domain and degenerate IQ motifs in the lever arm dictate PfMyoA motor properties.PfELC is essential for blood cell invasion and is a weak link in the assembly of a fully functional motor, providing a second novel target for antimalarial drug design.
Local Field Potentials in a Pre-motor Region Predict Learned Vocal Sequences
Authors: Brown Daril E. , Chavez Jairo I. , Nguyen Derek H. , Kadwory Adam , Voytek Bradley , Arneodo Ezequiel , Gentner Timothy Q. , Gilja Vikash 
Published: July 2020

AbstractNeuronal activity within the premotor region HVC is tightly synchronized to, and crucial for, the articulate production of learned song in birds. Characterizations of this neural activity typically focuses on patterns of sequential bursting in small carefully identified subsets of single neurons in the HVC population. Much less is known about population dynamics beyond the scale of individual neurons. There is a rich history of using local field potentials (LFP), to extract information about behavior that extends beyond the contribution of individual cells. These signals have the advantage of being stable over longer periods of time and have been used to study and decode complex motor behaviors, such as human speech. Here we characterize LFP signals in the putative HVC of freely behaving male zebra finches during song production, to determine if population activity may yield similar insights into the mechanisms underlying complex motor-vocal behavior. Following an initial observation that structured changes in the LFP were distinct to all vocalizations during song, we show that it is possible to extract time varying features from multiple frequency bands to decode the identity of specific vocalization elements (syllables) and to predict their temporal onsets within the motif. This demonstrates that LFP is a useful signal for studying motor control in songbirds. Surprisingly, the time frequency structure of putative HVC LFP is qualitatively similar to well established oscillations found in both human and non-human mammalian motor areas. This physiological similarity, despite distinct anatomical structures, may give insight to common computational principles for learning and/or generating complex motor-vocal behaviors.Author SummaryVocalizations, such as speech and song, are a motor process that requires the coordination of several muscle groups receiving instructions from specific brain regions. In songbirds, HVC is a premotor brain region required for singing and it is populated by a set of neurons that fire sparsely during song. How HVC enables song generation is not well understood. Here we describe network activity in putative HVC that precedes the initiation of each vocal element during singing. This network activity can be used to predict both the identity of each vocal element (syllable) and when it will occur during song. In addition, this network activity is similar to activity that has been documented in human, non-human primate, and mammalian premotor regions tied to muscle movements. These similarities add to a growing body of literature that finds parallels between songbirds and humans in respect to the motor control of vocal organs. Given the similarities of the songbird and human motor-vocal systems these results suggest that the songbird model could be leveraged to accelerate the development of clinically translatable speech prosthesis.
Comprehensive Live-cell Imaging Analysis of Cryptotanshinone and Synergistic Drug-Screening Effects in Various Human and Canine Cancer Cell Lines
Authors: Bittner Michael L. , Lopes Rosana , Hua Jianping , Sima Chao , Datta Aniruddha , Wilson-Robles Heather M. 
Published: July 2020

ABSTRACTBackgroundSeveral studies have highlighted both the extreme anticancer effects of Cryptotanshinone (CT), a Stat3 crippling component fromSalvia miltiorrhiza, as well as other STAT3 inhibitors to fight cancer.MethodsData presented in this experiment incorporates 2 years ofin vitrostudies applying a comprehensive live-cell drug-screening analysis of human and canine cancer cells exposed to CT at 20 μM concentration, as well as to other drug combinations. As previously observed in other studies, dogs are natural cancer models, given to their similarity in cancer genetics, epidemiology and disease progression compared to humans.ResultsResults obtained from several types of human and canine cancer cells exposed to CT and varied drug combinations, verified CT efficacy at combating cancer by achieving an extremely high percentage of apoptosis within 24 hours of drug exposure.ConclusionsCT anticancer efficacy in various human and canine cancer cell lines denotes its ability to interact across different biological processes and cancer regulatory cell networks, driving inhibition of cancer cell survival.