bluuebunny/crossref_metadata_2025_split huggingface dataset:
Safety and exceptional immunogenicity of novel 5T4 viral vectored vaccination regimes in early stage prostate cancer: a phase I clinical trial
Authors: Cappuccini Federica , Bryant Richard , Pollock Emily , Carter Lucy , Verrill Clare , Hollidge Julianne , Poulton Ian , Baker Megan , Mitton Celia , Baines Andrea , Meier Armin , Schmidt Guenter , Harrop Richard , Protheroe Andrew , MacPherson Ruth , Kennish Steven , Morgan Susan , Vigano Selena , Romero Pedro , Evans Thomas , Catto James W.F. , Hamdy Freddie , Hill Adrian V.S. , Redchenko Irina 
Published: March 2020

AbstractProstate cancer (PCa) has been under investigation as a target for antigen-specific immunotherapies in metastatic disease settings for a decade. However, neither of the two clinically most developed prostate cancer vaccines, Sipuleucel-T and ProstVac, induce strong T cell immunity. In this first-in-man study, VANCE, we evaluated a novel vaccination platform based on two replication-deficient viruses, chimpanzee adenovirus (ChAd) and MVA (Modified Vaccinia Ankara), targeting the oncofetal self-antigen 5T4 in early stage PCa. Forty patients, either newly diagnosed with early stage prostate cancer and scheduled for radical prostatectomy or patients with stable disease on an active surveillance protocol, were recruited to the study to assess the vaccine safety and T cell immunogenicity. Secondary and exploratory endpoints included immune infiltration into the prostate, prostate specific antigen (PSA) change and assessment of phenotype and functionality of antigen-specific T cells. The vaccine had an excellent safety profile. Vaccination-induced 5T4-specific T cell responses were measured in blood by ex vivo IFN-γ ELISpot and were detected in the majority of patients with a mean level in responders of 198 spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMCs). Flow cytometry analysis demonstrated the presence of both CD8+ and CD4+ polyfunctional 5T4-specific T cells in the circulation. 5T4-reactive tumour infiltrating lymphocytes (TILs) were isolated from post-treatment prostate tissue. Some of the patients had a transient PSA rise 2-8 weeks following vaccination, possibly indicating an inflammatory response in the target organ. The potent T cell responses elicited support the evaluation of these vectored vaccine in efficacy trials.
Organ-restricted vascular delivery of nanoparticles for lung cancer therapy
Authors: Bölükbas Deniz A. , Datz Stefan , Meyer-Schwickerath Charlotte , Morrone Carmela , Doryab Ali , Gößl Dorothee , Vreka Malamati , Yang Lin , Argyo Christian , van Rijt Sabine H. , Lindner Michael , Eickelberg Oliver , Stoeger Tobias , Schmid Otmar , Lindstedt Sandra , Stathopoulos Georgios T. , Bein Thomas , Wagner Darcy E. , Meiners Silke 
Published: March 2020

AbstractNanomedicines hold immense promise for a number of devastating diseases due to the ability to custom-design both the carrier and cargo. However, their clinical implementation has been hampered by physicochemical and biological barriers and off-target deposition which impair cell specific targeting, especially in internal organs. This study reports a new delivery approach using organ-restricted vascular delivery to allow for direct administration and recirculation of stimuli-responsive nanoparticles to promote cellular uptake into an organ of interest. Using this technique, nanoparticles reach the interior of dense tumors and are selectively taken up by lung cancer cells. Importantly, this surgical approach is essential as the same nanoparticles do not reach lung tumor cells upon systemic or intratracheal administration. Organ-restricted vascular delivery thus opens up new avenues for optimized nanotherapies for cancer and other diseases.
Nascent transcript folding plays a major role in determining RNA polymerase elongation rates
Authors: Turowski Tomasz W. , Petfalski Elisabeth , Goddard Benjamin D. , French Sarah L. , Helwak Aleksandra , Tollervey David 
Published: March 2020

ABSTRACTTranscription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed thisin vivo, analyzing RNAPI inS.cerevisiae. Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5’ bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C-content.In vitroexperiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI fromS.pombewas similarly sensitive to transcript folding, as wereS.cerevisiaeRNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding.Abstract FigureHIGHLIGHTSStructures in the nascent RNA correlate with rapid elongation by RNAPIin vivoStable RNA structures limit RNAPI backtrackingin vitroGC content in the transcription bubble tunes transcription elongation rateNascent transcript folding modulates dynamics of all three RNAPsin vivo
Transient interactions and influence among bacteria in field-grown Arabidopsis thaliana tissues
Authors: Beilsmith Kathleen , Perisin Matthew , Bergelson Joy 
Published: March 2020

AbstractInteractions between bacteria are thought to play an important role in the assembly of plant microbial communities (1), yet the extent of temporal variation in these interactions is unclear. We inferred interactions from sequence-based counts of bacteria in a series of Arabidopsis thaliana tissue samples spanning major developmental transitions in the plant life cycle (2). Bacterial interactions were transient, even among variants found together at consecutive developmental stages. The overwhelming majority of these interactions were positive, indicating that competition for the plant niche might be a less important driver of bacterial abundances than cooperation or common responses to host and environmental factors. Over time, interaction networks diverged from an initial scale-free structure and became increasingly modular. In all networks, we found evidence of a hierarchical structure in which hub bacteria bridged network modules. However, the identities of bacteria in these influential roles also varied during plant growth.
Potassium Glutamate and Glycine Betaine Induce Self-Assembly of Sliding Clamps into Higher Order Oligomers
Authors: Purohit Anirban , Douma Lauren G. , Bloom Linda B. , Levitus Marcia 
Published: March 2020

ABSTRACTSliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of theEscherichia coliβ-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of β is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. Replacing NaCl by KGlu, the primary cytoplasmic salt inE. coli, results in the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with theS. cerevisiaePCNA sliding clamp, suggesting that KGlu effects are not specific to β. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly whenE. coliis exposed to high concentrations of extracellular solute. Possible biological implications are discussed.
High throughput functional variant screens via in-vivo production of single-stranded DNA
Authors: Schubert Max G. , Goodman Daniel B. , Wannier Timothy M. , Kaur Divjot , Farzadfard Fahim , Lu Timothy K. , Shipman Seth L. , Church George M. 
Published: March 2020

AbstractTremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, we optimize efficient and continuous generation of precise genomic edits inEscherichia coli, via in-vivo production of single-stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method, enabling multiplexed applications. We introduce Retron Library Recombineering (RLR), a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome.
Comprehensive in virio structure probing analysis of the influenza A virus identifies a functional RNA structure involved in replication and segment interactions
Authors: Takizawa Naoki , Higashi Koichi , Kawaguchi Risa Karakida , Gotoh Yasuhiro , Suzuki Yutaka , Hayashi Tetsuya , Kurokawa Ken 
Published: March 2020

AbstractThe influenza A virus genome is segmented into eight viral RNAs (vRNA). Secondary structures on vRNA are thought to be involved in the viral proliferation process, such as intersegment interactions that are necessary for segment bundling. However, the functional RNA structure on vRNA is not well known because the secondary structure of vRNA in virion was partially unwound by binding viral non-specific RNA binding proteins in a sequence-independent manner. Here, we establish the global map of the vRNA secondary structure in virion using the combination of dimethyl sulfate (DMS)-seq and selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)-seq. By integrating DMS-seq and SHAPE-seq analyses with robust statistical analysis, we inferred quite a few bases paired regions including a pseudoknot structure on segment 5. Notably, when cells were infected with the recombinant virus which had mutations in the pseudoknot structure, the impairment of replication and packaging was observed on the other specific segment. Moreover, we analyzed the comprehensive intersegment RNA interactions in virion by ligation of interacting RNA followed by high-throughput sequencing (LIGR-seq). Our LIGR-seq analysis revealed that the intersegment interactions of the specific segment became less frequent and rearranged in the recombinant virus in concordance with the strength of genome packaging impairment. Our data provide evidence that the functional RNA structure motif on the influenza A virus genome can affect the efficiency of replication and segment bundling through the segment interactions.
Differentiation drives widespread rewiring of the neural stem cell chaperone network
Authors: Vonk Willianne I. M. , Rainbolt T. Kelly , Dolan Patrick T. , Webb Ashley E. , Brunet Anne , Frydman Judith 
Published: March 2020

SummaryNeural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Life-long maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPCs chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.
A missense in HSF2BP causing Primary Ovarian Insufficiency affects meiotic recombination by its novel interactor C19ORF57/MIDAP
Authors: Felipe-Medina Natalia , Caburet Sandrine , Sánchez-Sáez Fernando , Condezo Yazmine B. , de Rooij Dirk , Gómez-H Laura , García-Valiente Rodrigo , Todeschini Anne-Laure , Duque Paloma , Sánchez-Martín Manuel , Shalev Stavit A. , Llano Elena , Veitia Reiner , Pendás Alberto M. 
Published: March 2020

AbstractPrimary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with 3 cases of POI, we identified the candidate missense variant S167L inHSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse, compared to a newHSF2BPknock-out mouse showed that it behaves as a hypomorphic allele. HSF2BP-S167L females show reduced fertility with small litter sizes. To obtain mechanistic insights, we identified C19ORF57/MIDAP as a strong interactor and stabilizer of HSF2BP by forming a higher-order macromolecular structure involving BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L mutation showed a strongly decreased expression of both MIDAP and HSF2BP at the recombination nodules. Although HSF2BP-S167L does not affect heterodimerization between HSF2BP and MIDAP, it promotes a lower expression of both proteins and a less proficient activity in replacing RPA by the recombinases RAD51/DMC1, thus leading to a lower frequency of cross-overs. Our results provide insights into the molecular mechanism of two novel actors of meiosis underlying non-syndromic ovarian insufficiency.SummaryFelipe-Medina et al. describe a missense variant in the meiotic geneHSF2BPin a consanguineous family with Premature Ovarian Insufficiency, and characterize it as an hypormorphic allele, thatin vivoimpairs its dimerization with a novel meiotic actor, MIDAP/ C19ORF57, and affect recombination at double-strand DNA breaks.
An Artificial Cofactor catalyzing the Baylis-Hillman Reaction using Designed Streptavidin as Protein Host
Authors: Lechner Horst , Emann Vincent R. , Breuning M. , Höcker Birte 
Published: March 2020

AbstractAn artificial cofactor based on an organocatalyst embedded in a protein was used to conduct the Baylis-Hillman reaction in a buffered system. As protein host we chose streptavidin, since it can be easily crystallized and thereby supports the design process. The protein host around the cofactor was rationally designed based on high-resolution crystal structures obtained after each variation of the amino acid sequence. Additionally, DFT-calculated intermediates and transition states were used to rationalize activity. Finally, repeated cycles of structure determination and redesign led to a system with 24 to 35-fold increased activity over the bare cofactor and to the most active proteinogenic catalyst for the Baylis-Hillman reaction known today.
Global Profiling of 2-hydroxyisobutyrylome in Common Wheat
Authors: Zhang Ning , Zhang Lingran , Li Linjie , Geng Junyou , Zhao Lei , Ren Yan , Dong Zhongdong , Chen Feng 
Published: March 2020

AbstractAs a novel post-translational modification (PTM), lysine 2-hydroxyisobutyrylation (Khib) has been found to play a role in active gene transcription in mammalian cells and yeast, but the function of Khib proteins in plants remains unknown. In this study, we used western blot to demonstrate that Khib is an evolutionarily-conserved PTM in wheat and its donators, with the highest Khib abundance occurring in hexaploidy wheat. Additionally, global profiling using affinity purification and mass spectroscopy of 2-hydroxyisobutyrylome revealed that there were 3348 lysine modification sites from 1074 proteins in common wheat (Triticum aestivumL.). Moreover, bioinformatic data indicated that Khib proteins participate in a wide variety of biological and metabolic pathways. Immunoprecipitation and western blot confirmed that Khib proteins had anin vivoorigin. A comparison of Khib and other major PTMs revealed that Khib proteins were simultaneously modified by multiple PTMs. Using mutagenesis experiments and Co-IP, we demonstrated that Khib on K206 is a key regulatory modification of phosphoglycerate kinase enzymatic activity and found that de-Khib on K206 affects protein interactions. Furthermore, Khib production of low-molecular-weight proteins was a response to the deacetylase inhibitors nicotinamide and trichostatin A. This study provides evidence that enhances our current understanding of Khib in wheat plants, including the cooperation between this PTM and metabolic regulation.
Photoperiod stress alters the cellular redox status and is associated with an increased peroxidase and decreased catalase activity
Authors: Abuelsoud Walid , Cortleven Anne , Schmülling Thomas 
Published: March 2020

AbstractPeriodic changes of light and dark regulate numerous processes in plants. Recently, a novel type of stress caused by an extended light period has been discovered inArabidopsisand was named photoperiod stress. Photoperiod stress causes the induction of numerous stress response genes during the night following the extended light period of which many are indicators of oxidative stress. The next day, stress-sensitive genotypes display reduced photosynthetic efficiency and programmed cell death in leaves. Here, we have analysed further the consequences of photoperiod stress and report that it causes changes of the cellular redox status. A prolonged light period caused a strong reduction of the AsA redox during the following night indicating that it induces an oxidizing cellular environment. Further, photoperiod stress was associated with an increased activity of peroxidases and a decreased activity of catalases. Increased peroxidase activity was localized to the apoplast and might be causal for the oxidative stress induced by photoperiod stress.
The effect of ethanol concentration on the morphological and molecular preservation of insects for biodiversity studies
Authors: Marquina Daniel , Buczek Mateusz , Ronquist Fredrik , Łukasik Piotr 
Published: March 2020

Abstract1.Traditionally, insects collected for scientific purposes have been dried and pinned, or preserved in 70 % ethanol. Both methods preserve taxonomically informative exoskeletal structures well but are suboptimal for preserving DNA. Highly concentrated ethanol (95 – 100 %), preferred as a DNA preservative, has generally been assumed to make specimens brittle and prone to breaking. However, systematic studies on the correlation between ethanol concentration and specimen preservation are lacking.2.We tested how preservative ethanol concentration in combination with different sample handling regimes affect the integrity of seven insect species representing four orders, and differing substantially in the level of sclerotization. After preservation and treatments (various levels of disturbance), we counted the number of appendages (legs, wings, antennae, heads) that specimens had lost. Additionally, we assessed the preservation of DNA after long-term storage by comparing the ratio of PCR amplicon copy numbers to an added artificial standard.3.We found that high ethanol concentrations indeed induce brittleness in insects. However, the magnitude and nature of the effect varied strikingly among species. In general, ethanol concentrations at or above 90 % made the insects more brittle, but for species with robust, thicker exoskeletons, this did not translate to an increased loss of appendages. Neither freezing nor drying the insects after immersion in ethanol had a negative effect on the retention of appendages. We also found that DNA preserves less well at lower ethanol concentrations when stored at room temperature for an extended period. However, the magnitude of the effect varies among species; the concentrations at which the number of COI amplicon copies relative to the standard was significantly decreased compared to 95 % ethanol ranged from 90 % to as low as 50 %.4.While higher ethanol concentrations positively affect long-term DNA preservation, there is a clear trade-off between preserving insects for morphological examination and genetic analysis. The optimal ethanol concentration for the latter is detrimental for the former, and vice versa. These trade-offs need to be considered in large insect biodiversity surveys and other projects aiming to combine molecular work with traditional morphology-based characterization of collected specimens.
FliI6-FliJ molecular motor assists with unfolding in the type III secretion export apparatus
Authors: Kucera Jiri , Terentjev Eugene M. 
Published: March 2020

AbstractThe role of rotational molecular motors of the ATP synthase class is integral to the metabolism of cells. Yet the function of FliI6-FliJ complex - a homolog of theF1ATPase motor - within the flagellar export apparatus remains unclear. We use a simple two-state model adapted from studies of linear molecular motors to identify key features of this motor. The two states are the ‘locked’ ground state where the FliJ coiled coil filament experiences fluctuations in an asymmetric torsional potential, and a ‘free’ excited state in which FliJ undergoes rotational diffusion. Michaelis-Menten kinetics was used to treat transitions between these two states, and obtain the average angular velocity of the FliJ filament within the FliI6stator:ωmax≈ 9.0 rps. The motor was then studied under external counter torque conditions in order to ascertain its maximal power output:Pmax≈ 42 kBT/s, and the stall torque:Gstall≈ 3 kBT/rad. Two modes of action within the flagellar export apparatus are proposed, in which the motor performs useful work either by continuously ‘grinding’ through the resistive environment, or by exerting equal and opposite stall force on it. In both cases, the resistance is provided by flagellin subunits entering the flagellar export channel prior to their unfolding. We therefore propose that the function of the FliI6-FliJ complex is to lower the energy barrier and therefore assist in unfolding of the flagellar proteins before feeding them into the transport channel.