bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of the Viscoelastic Mechanical Properties of the Porcine Optic Nerve Head using Micromechanical Testing and Finite Element Modeling
Authors: Safa Babak N. , Read A. Thomas , Ethier C. Ross 
Published: April 2021

AbstractOptic nerve head (ONH) biomechanics is centrally involved in the pathogenesis of glaucoma, a blinding ocular condition often characterized by elevation and fluctuation of the intraocular pressure and resulting loads on the ONH. Further, tissue viscoelasticity is expected to strongly influence the mechanical response of the ONH to mechanical loading, yet the viscoelastic mechanical properties of the ONH remain unknown. To determine these properties, we conducted micromechanical testing on porcine ONH tissue samples, coupled with finite element modeling based on a mixture model consisting of a biphasic material with a viscoelastic solid matrix. Our results provide a detailed description of the viscoelastic properties of the porcine ONH at each of its four anatomical quadrants (i.e., nasal, superior, temporal, and inferior). We showed that the ONH’s viscoelastic mechanical response can be explained by a dual mechanism of fluid flow and solid matrix viscoelasticity, as is common in other soft tissues. We obtained porcine ONH properties as follows: matrix Young’s modulusE=1.895 [1.056,2 .391] kPa (median [min., max.]), Poisson’s ratioν=0.142 [0.060,0 .312], kinetic time-constantτ=214 [89,921] sec, and hydraulic permeabilityk=3.854 × 10−1[3.457 × 10−2,9.994 × 10−1] mm4/(N sec). These values can be used to design and fabricate physiologically appropriateex vivotest environments (e.g., 3D cell culture) to further understand glaucoma pathophysiology.
Copy-number intratumor heterogeneity contributes to predict relapse in chemotherapy-naive stage II colon cancer
Authors: Lahoz Sara , Archilla Iván , Asensio Elena , Hernández-Illán Eva , Ferrer Queralt , López-Prades Sandra , Nadeu Ferran , del Rey Javier , Sanz-Pamplona Rebeca , Lozano Juan José , Castells Antoni , Cuatrecasas Miriam , Camps Jordi 
Published: April 2021

ABSTRACTOptimal selection of high-risk patients with stage II colon cancer is crucial to ensure clinical benefit of adjuvant chemotherapy after surgery. Here, we investigate the prognostic and predictive value of genomic intratumor heterogeneity and aneuploidy for disease recurrence. We combined SNP arrays, targeted next-generation sequencing, fluorescence in situ hybridization and inmunohistochemistry on a retrospective cohort of 84 untreated stage II colon cancer patients. We assessed the subclonality of copy-number alterations (CNAs) and mutations, CD8+ lymphocyte infiltration and their association with time to recurrence (TTR). Prognostic factors were included in machine learning analysis to evaluate their ability to predict individual relapse risk. Tumors from recurrent patients (N = 38) exhibited greater proportion of CNAs compared with non-recurrent (N = 46) (mean 31.3% vs. 23%, respectively;P= 0.014), which was confirmed in an independent cohort. Candidate chromosome-specific aberrations included the gain of the chromosome arm 13q (P= 0.02; HR, 2.67) and loss of heterozygosity at 17q22-q24.3 (P= 0.05; HR, 2.69), both associated with shorter TTR. CNA load positively correlated with intratumor heterogeneity (R= 0.52;P< 0.0001), indicating ongoing chromosomal instability. Consistently, subclonal copy-number heterogeneity was associated with elevated risk of relapse (P= 0.028; HR, 2.20), which we did not observe for subclonal mutations. The clinico-genomic model rated an area under the curve of 0.83, achieving a 10% incremental gain compared to clinicopathological markers. In conclusion, tumor aneuploidy and copy-number heterogeneity were predictive of a poor outcome in early-stage colon cancer, and improved discriminative performance in comparison to clinicopathological data.
Oligopaint DNA FISH as a tool for investigating meiotic chromosome dynamics in the silkworm,Bombyx mori
Authors: Rosin Leah F. , Gil Jose , Drinnenberg Ines A. , Lei Elissa P. 
Published: April 2021

AbstractAccurate chromosome segregation during meiosis is essential for reproductive success. Yet, many fundamental aspects of meiosis remain unclear, including the mechanisms regulating homolog pairing across species. This gap is partially due to our inability to visualize individual chromosomes during meiosis. Here, we employ Oligopaint FISH to investigate homolog pairing and compaction of meiotic chromosomes in a classical model system, the silkwormBombyx mori. Our Oligopaint design combines multiplexed barcoding with secondary oligo labeling for high flexibility and low cost. These studies illustrate that Oligopaints are highly specific in whole-mount gonads and on meiotic chromosome spreads. We show that meiotic pairing is robust in both males and female meiosis. Additionally, we show that meiotic bivalent formation inB. morimales is highly similar to bivalent formation inC. elegans, with both of these pathways ultimately resulting in the pairing of chromosome ends with non-paired ends facing the spindle pole and microtubule recruitment independent of the centromere-specifying factor CENP-A.Author’s SummaryMeiosis is the specialized cell division occurring exclusively in ovaries and testes to produce egg and sperm cells, respectively. The accurate distribution of chromosomes (the genetic material) during this process is essential to prevent infertility/sterility and developmental disorders in offspring. As researchers are specifically unable to study the mechanisms regulating meiosis in depth in humans, identifying broadly conserved aspects of meiotic chromosome segregation is essential for making accurate inferences about human biology. Here, we use a sophisticated chromosome painting approach called Oligopaints to visualize and study chromosomes during meiosis in the silkworm,Bombyx mori. We illustrate that Oligopaints are highly specific inB. moriand demonstrate how Oligopaints can be used to study the dynamics of meiotic chromosomes in diverse species.
High-resolution structure and dynamics of mitochondrial complex I – insights into the proton pumping mechanism
Authors: Parey Kristian , Lasham Jonathan , Mills Deryck J. , Djurabekova Amina , Haapanen Outi , Yoga Etienne Galemou , Xie Hao , Kühlbrandt Werner , Sharma Vivek , Vonck Janet , Zickermann Volker 
Published: April 2021

SummaryMitochondrial NADH:ubiquinone oxidoreductase (complex I) is a 1 MDa membrane protein complex with a central role in energy metabolism. Redox-driven proton translocation by complex I contributes substantially to the proton motive force that drives ATP synthase. Several structures of complex I from bacteria and mitochondria have been determined but its catalytic mechanism has remained controversial. We here present the cryo-EM structure of complex I fromYarrowia lipolyticaat 2.1 Å resolution, which reveals the positions of more than 1600 protein-bound water molecules, of which ∼100 are located in putative proton translocation pathways. Another structure of the same complex under steady-state activity conditions at 3.4 Å resolution indicates conformational transitions that we associate with proton injection into the central hydrophilic axis. By combining high-resolution structural data with site-directed mutagenesis and large-scale molecular dynamics simulations, we define details of the proton translocation pathways, and offer new insights into the redox-coupled proton pumping mechanism of complex I.
Generation of photocaged nanobodies forin vivoapplications using genetic code expansion and computationally guided protein engineering
Authors: O’Shea Jack M. , Goutou Angeliki , Sethna Cyrus , Wood Christopher W. , Greiss Sebastian 
Published: April 2021

AbstractNanobodies are becoming increasingly popular as tools for manipulating and visualising proteinsin vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode wormC. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting forin vivoeffects on protein-protein interactions.
Deletion of the AMPylase mFICD alters cytokine secretion and affects cognitive plasticity in vivo
Authors: McCaul Nicholas , Porter Corey M , Becker Anouk , Antony Tang Chih-Hang , Wijne Charlotte , Chatterjee Bhaskar , Bousbaine Djenet , Bilate Angelina M , Hu Chih-Chi Andrew , Ploegh Hidde L , Truttmann Matthias C 
Published: April 2021

AbstractFic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This post-translational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mFICD AMPylase knock-out mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well-tolerated in unstressed mice. We show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM and IL-1β, without affecting the unfolded protein response. Finally, we demonstrate that older mFICD-/- mice show improved cognitive plasticity. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo.
SARS-CoV-2 spike protein expressing epithelial cells promotes senescence associated secretory phenotype in endothelial cells and increased inflammatory response
Authors: Meyer Keith , Patra Tapas , Mahantesh Vijay , Ray Ranjit 
Published: April 2021

AbstractIncreased mortality in COVID-19 often associates with thrombotic and microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes inflammatory cytokine IL-6/IL-6R induced trans-signaling responses which modulate MCP-1 expression in human endothelial cells. MCP-1 is secreted as a major component of the senescence associated secretory phenotype (SASP). Virus infected or Spike transfected human pulmonary epithelial cells (A549) exhibited an increase in senescence related marker proteins. TMNK; as a representative human endothelial cell line, when exposed to cell culture supernatant derived from A549 cells expressing SARS-CoV-2 spike protein (Spike CM) exhibited a senescence phenotype with enhanced p16, p21, and SA-β-galactosidase expression. Inhibition of IL-6 trans-signaling by Tocilizumab, prior to exposure of supernatant to endothelial cells, inhibited p16 and p21 induction. Likewise, inhibition of receptor signaling by Zanabrutinib or Brd4 function by AZD5153 also led to limited induction of p16 expression. Senescence lead to an enhanced level of adhesion molecule, ICAM-1 and VCAM-1 in human endothelial cells, and TPH1 attachment byin vitroassay. Inhibition of senescence or SASP function prevented ICAM/VCAM expression and leukocyte attachment. We also observed an increase in oxidative stress in A549 spike transfected and endothelial cells exposed to Spike CM. ROS generation in TMNK was reduced after treatment with the IL-6 specific inhibitor Tociliximab, and with the specific inhibitors Zanabrutinib and AZD5153. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers leading to enhanced leukocyte adhesion with coronary blockade potential.
Human adipose-derived mesenchymal stromal cells from face and abdomen undergo replicative senescence and loss of genetic integrity after long-term culture
Authors: Delben Priscilla Barros , Zomer Helena Debiazi , da Silva Camila Acordi , Gomes Rogério Schutzler , Melo Fernanda Rosene , Dillenburg-Pilla Patricia , Trentin Andrea Gonçalves 
Published: April 2021

ABSTRACTBody fat depots are heterogeneous concerning their embryonic origin, structure, exposure to environmental stressors, and availability. Thus, investigating adipose-derived mesenchymal stromal cells (ASCs) from different sources is essential to standardization for future therapies.In vitroamplification is also critical because it may predispose cell senescence and mutations, reducing regenerative properties and safety. Here, we evaluated long-term culture of human facial ASCs (fASCs) and abdominal ASCs (aASCs) and showed that both met the criteria for MSCs characterization, but presented differences in their immunophenotypic profile, and differentiation and clonogenic potentials. The abdominal tissue yielded more ASCs, but facial cells displayed fewer mitotic errors at higher passages. However, both cell types reduced clonal efficiency over time and entered replicative senescence around P12, as evaluated by progressive morphological alterations, reduced proliferative capacity, and SA-β-galactosidase expression. Loss of genetic integrity was detected by a higher proportion of cells showing nuclear alterations and γ-H2AX expression. Our findings indicate that the source of ASCs can substantially influence their phenotype and therefore should be carefully considered in future cell therapies, avoiding, however, long-term culture to ensure genetic stability.
Single-cell analysis of localized low- and high-grade prostate cancers
Authors: Eksi Sebnem Ece , Chitsazan Alex , Sayar Zeynep , Thomas George V. , Fields Andrew , Kopp Ryan P. , Spellman Paul T. , Adey Andrew 
Published: April 2021

SUMMARYApproximately, 30% of early-stage localized prostate cancer cases reoccur within 5 to 10 years [1, 2]. However, identifying precise molecular subtypes attributable to specific stages of prostate cancer has proven difficult due to high heterogeneity within localized tumors [3–5]. Bulk assays represent a population average, which is a result of the heterogeneity that exists at the individual prostate cancer cell level [6]. Here, we sequenced the accessible chromatin regions of 14,424 single-cells collected from 18 fresh-frozen prostate tumors using sci-ATAC-seq [7, 8]. We observed that shared chromatin features among low-grade prostate cancer epithelial cells were lost in high-grade tumors. Despite this loss, all high-grade tumors exhibited an enrichment for FOXA1, HOXB13 and CDX2 transcription factor binding sites within their accessible chromatin regions, indicating a shared trans-regulatory program. Single-cell analysis of the differentially accessible regions in high- versus low-grade prostate tumors identified two unique genes encoding neuronal adhesion molecules, NRXN1 and NLGN1. We found that NRXN1 and NLGN1 are expressed in the epithelial luminal, basal and neuroendocrine cells, as well as the immune, endothelial and neuronal cell types in all prostate tumors. Overall, these results provide a deeper understanding of the active gene regulatory networks in low- and high-grade prostate tumors at a striking resolution and provide critical insights for molecular stratification of the disease.
Protein visualization and manipulation inDrosophilathrough the use of epitope tags recognized by nanobodies
Authors: Xu Jun , Kim Ah-Ram , Cheloha Ross W. , Fischer Fabian A. , Shun Li Joshua Shing , Feng Yuan , Stoneburner Emily , Binari Richard , Mohr Stephanie E. , Zirin Jonathan , Ploegh Hidde , Perrimon Norbert 
Published: April 2021

AbstractExpansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteinsin vivo. Here, we characterize two short (<15 aa) NanoTag epitopes, 127D01 and VHH05) and their corresponding high-affinity nanobodies. We demonstrate their use inDrosophilaforin vivoprotein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies inDrosophilaand beyond.
A genome-scale antibiotic screen inSerratia marcescensidentifies YdgH as a conserved modifier of cephalosporin and detergent susceptibility
Authors: Lazarus Jacob E. , Warr Alyson R. , Westervelt Kathleen A. , Hooper David C. , Waldor Matthew K. 
Published: April 2021

AbstractSerratia marcescens,a member of the order Enterobacterales, is adept at colonizing healthcare environments and an important cause of invasive infections. Antibiotic resistance is a daunting problem inS. marcescensbecause in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility inS. marcescens,a high-density transposon insertion library was subjected to sub-minimal inhibitory concentrations of two cephalosporins, cefoxitin and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and choseydgH, a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of ydgHinS. marcescensresulted in decreased susceptibility to multiple 3rdgeneration cephalosporins, and in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes inEscherichia coliO157:H7 andEnterobacter cloacaemutants. YdgH is predicted to localize to the periplasm and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance inS. marcescensand will serve as a resource for further investigations of this important pathogen.
Phytophthora capsici sterol reductase PcDHCR7 has a role in mycelium development and pathogenicity
Authors: Wang Weizhen , Zhang Fan , Zhang Sicong , Xue Zhaolin , Xie Linfang , Govers Francine , Liu Xili 
Published: April 2021

AbstractThe de novo biosynthesis of sterols is critical for eukaryotes, however, some organisms lack this pathway including most oomycetes. Phytophthora spp. are sterol auxotroph but remarkably, have retained a few genes encoding enzymes in the sterol biosynthesis pathway. Here we investigated the function of PcDHCR7, a gene in Phytophthora capsici predicted to encode the △7-sterol reductase. When expressed in Saccharomyces cerevisiae, PcDHCR7 showed a △7-sterol reductase activity. Knocking out PcDHCR7 in P. capsici resulted in loss of the capacity to transform ergosterol into brassicasterol, which means PcDHCR7 has a △7-sterol reductase activity in P. capsici itself. This enables P. capsici to transform sterols recruited from the environment for better use. Biological characteristics were compared between wild-type isolate and PcDHCR7 knock-out transformants. The results indicated that PcDHCR7 plays a key role in mycelium development and pathogenicity of zoospores in P. capsici.
Time-resolved single-cell sequencing identifies multiple waves of mRNA decay during mitotic exit
Authors: Krenning Lenno , Sonneveld Stijn , Tanenbaum Marvin E. 
Published: April 2021

AbstractAccurate control of the cell cycle is critical for development and tissue homeostasis and requires precisely-timed expression of many genes. Cell cycle gene expression is regulated through transcriptional and translational control, as well as through regulated protein degradation. Here, we show that widespread and temporally-controlled mRNA decay acts as an additional mechanism for gene expression regulation during the cell cycle. We find that two waves of mRNA decay occur sequentially during the mitosis-to-G1 phase transition, and identify the deadenylase CNOT1 as a factor that contributes to mRNA decay during this cell cycle transition. Collectively, our data show that, akin to protein degradation, scheduled mRNA decay helps to reshape cell cycle gene expression as cells move from mitosis into G1 phase.
Responsible research and innovation key performance indicators in industry
Authors: Yaghmaei Emad 
Published: April 2021

Purpose
Responsible research and innovation (RRI) is taking a role in assisting all types of stakeholders, including industry members, in moving their research and innovation (R&I) initiatives to tackle grand challenges. The literature on RRI, however, focuses little on how industry can implement RRI principles. To solve this gap, the purpose of this study is to construct a conceptual framework for managing and assessing RRI principles in the industry.

Design/methodology/approach
Qualitative research was used to build the RRI key performance indicator list; 30 interviews were conducted to design a framework which was pilot tested in a company to identify how to align technology outcomes to the values, needs and expectations of the society.

Findings
This study depicts five successive RRI implementation levels and exhibits RRI key performance indicators. Drawing on extant models, this study develops RRI levels and indicators to discuss why industry should become engaged in RRI, how it can embed RRI principles into R&I processes and how RRI indicators can be managed systematically.

Originality/value
The connection between RRI key performance indicators and RRI levels determines how industry can integrate principles and methodologies of RRI into R&I processes. The model in the study shows how companies move from one RRI stage to another and this study aims to exhibit an ideal stage of RRI for industry.