bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting uncoupling of copy number and gene expression in cancers
Authors: Mohanty Vakul , Wang Fang , Mills Gordon , Chen Ken
Published: August 2020

The high degree of aneuploidy in cancer is likely tolerated via extensive uncoupling of copy number (CN) and mRNA expression (UCNE) of deleterious genes located in copy number aberrations (CNAs). To test the extent and role of UCNE in cancer, we performed integrative analysis of multiomics data across The Cancer Genome Atlas (TCGA), encompassing ∼ 5000 individual tumors. We found many genes having UCNE, the degree of which are associated with increased oncogenic signaling, proliferation and immune-suppression. The occurrence of UCNE appears to be orchestrated by complex epigenetic and regulatory changes, with transcription factors (TFs) playing a prominent role. To further dissect the regulatory mechanisms, we developed a systems-biological approach to identify candidate TFs, which upon perturbation can offset UCNE and reduce tumor fitness. Applying our approach on TCGA data, we identified 20 putative targets, 45% of which were validated by independent sources. Among them areIRF1, which plays a prominent role in anti-tumor immunity and response to immune checkpoint therapy,ETS1, TRIM21andGATA3, which are associated with anti-tumor immunity, tumor proliferation and metastasis. Together, our study indicates that UCNE is likely an important mechanism in cancer development that can be exploited therapeutically.
Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics
Authors: Benedetti Lorena , Marvin Jonathan S. , Falahati Hanieh , Guillén-Samander Andres , Looger Loren L. , De Camilli Pietro
Published: August 2020

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from theNeurospora crassaphotoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these “enhanced” Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.
Celsr1 and CAMSAP3 differently regulate intercellular and intracellular cilia orientation in oviduct multiciliated cells
Authors: Usami Fumiko Matsukawa , Arata Masaki , Shi Dongbo , Oka Sanae , Higuchi Yoko , Tissir Fadel , Takeichi Masatoshi , Fujimori Toshihiko
Published: August 2020

The molecular mechanisms by which cilia orientation is coordinated within and between multiciliated cells (MCCs) is not fully understood. By observing the orientation of basal bodies (BB) in MCCs of mouse oviducts, here, we show that Celsr1, a planar cell polarity (PCP) factor involved in tissue polarity regulation, is dispensable for determining BB orientation in individual cells, whereas CAMSAP3, a microtubule minus-end regulator, is critical for this process but not for PCP. MCCs exhibit a characteristic BB orientation and microtubule gradient along the tissue axis, and these intracellular polarities were maintained in the cells lacking Celsr1, although the intercellular coordination of the polarities was partly disrupted. On the other hand, CAMSAP3 regulated the assembly of microtubules interconnecting BBs by localizing at the BBs, and its mutation led to disruption of intracellular coordination of BB orientation, but not affecting PCP factor localization. Thus, both Celsr1 and CAMSAP3 are responsible for BB orientation but in distinct ways; and therefore, their cooperation should be critical for generating functional multiciliated tissues.
Fine-tuning Nanog expression heterogeneity by altering MicroRNA regulation
Authors: Samanta Tagari , Kar Sandip
Published: August 2020

Nanog exhibits a robust heterogeneous expression pattern within a population of embryonic stem cells (ESCs) under varied culture conditions. However, an efficient way to fine-tune the Nanog expression heterogeneity remains elusive. Herein, by employing a stochastic modeling approach, we demonstrate that Nanog expression heterogeneity can be controlled by modulating the regulatory features of a Nanog transcript specific microRNA. We demonstrate how and why the extent of origin dependent fluctuations in Nanog expression level can be altered by varying either the binding efficiency of the microRNA-mRNA complex or the expression level of the respective microRNA. Moreover, our model makes experimentally feasible and insightful predictions to maneuver the Nanog expression heterogeneity explicitly to achieve cell-type specific differentiation of ESCs.
Micro-topographical guidance of macropinocytic signaling patches
Authors: Honda Gen , Saito Nen , Fujimori Taihei , Hashimura Hidenori , Nakamura Mitsuru J. , Nakajima Akihiko , Sawai Satoshi
Published: August 2020

In fast moving cells such as amoeba and immune cells, spatio-temporal regulation of dendritic actin filaments shapes large-scale plasma membrane protrusions. Despite the importance in migration as well as in particle and liquid ingestion, how these processes are affected by the micrometer-scale surface features is poorly understood. Here, through quantitative imaging analysis ofDictyosteliumon micro-fabricated surfaces, we show that there is a distinct mode of topographically guided cell migration ‘phagotaxis’ directed by the macropinocytic Ras/PI3K signaling patches. The topography guidance was PI3K-dependent and involved nucleation of a patch at the convex curved surface and confinement at the concave surface. Due to the topography-dependence, constitutive cup formation for liquid uptake in the axenic strain is also destined to trace large surface features. Given the fact that PI3K-dependency of phagocytosis are restricted to large particles in bothDictyosteliumand immune cells, topography-dependency and the dual-use of membrane cups may be wide-spread.
MyoD is a structure organizer of 3D genome architecture in muscle cells
Authors: Chen Qian , Chen Fengling , Wang Ruiting , Shi Minglei , Chen Antony K. , Ma Zhao , Li Guohong , Wang Min , Li Hu , Zhang Xu , Ma Jinbiao , Zhong Jiayun , Chen Meihong , Zhang Michael Q. , Zhang Yong , Chen Yang , Zhu Dahai
Published: August 2020

The genome is not a linear molecule of DNA randomly folded in the nucleus, but exists as an organized, three-dimensional (3D) dynamic architecture. Intriguingly, it is now clear that each cell type has a unique and characteristic 3D genome organization that functions in determining cell identity during development. A currently challenging basic question is how cell-type specific 3D genome structures are established during development. Herein, we analyzed 3D genome structures in primary myoblasts and myocytes from MyoD knockout (MKO) and wild type (WT) mice and discovered that MyoD, a pioneer transcription factor (TF), can function as a “genome organizer” that specifies the proper 3D genome architecture unique to muscle cell development. Importantly, we genetically demonstrate that H3K27ac is insufficient for establishing MyoD-induced chromatin loops in muscle cells. The establishment of MyoD’s “architectural role” should have profound impacts on advancing understanding of other pioneer transcription factors in orchestrating lineage specific 3D genome organization during development in a potentially very large number of cell types in diverse organisms.
TLR4 signaling and macrophage inflammatory responses are dampened by GIV/Girdin
Authors: Swanson Lee , Katkar Gajanan D. , Tam Julian , Pranadinata Rama F. , Chareddy Yogitha , Coates Jane , Amandachar Mahitha Shree , Castillo Vanessa , Olson Joshua , Nizet Victor , Kufareva Irina , Das Soumita , Ghosh Pradipta
Published: August 2020

Sensing of pathogens by Toll-like receptor 4 (TLR4) induces an inflammatory response; controlled responses confer immunity but uncontrolled responses cause harm. Here we define how a multi-modular scaffold, GIV (a.k.a Girdin) titrates such inflammatory response in macrophages. Upon challenge with either live microbes or microbe-derived lipopolysaccharides (LPS, a ligand for TLR4), macrophages with GIV mount a more tolerant (hypo-reactive) transcriptional response and suppress pro-inflammatory cytokines and signaling pathways (i.e., NFkB and CREB) downstream of TLR4 compared to their GIV-depleted counterparts. Myeloid-specific gene depletion studies confirmed that the presence of GIV ameliorates DSS-induced colitis and sepsis-induced death. The anti-inflammatory actions of GIV are mediatedviaits C-terminally located TIR-like BB-loop (TILL)-motif which binds the cytoplasmic TIR-modules of TLR4 in a manner that precludes receptor dimerization; the latter is a pre-requisite for pro-inflammatory signaling. Binding of GIV’s TILL motif to other TIR modules inhibits pro-inflammatory signalingviaother TLRs, suggesting a convergent paradigm for fine-tuning macrophage inflammatory responses.

Significance

To ensure immunity, and yet limit pathology, inflammatory responses must be confined within the proverbial ‘Goldilockszone’. TLR4 is the prototypical sensor that orchestrates inflammatory responses through a series of well characterized downstream cascades. How TLR4 signals are confined remain incompletely understood. Using trans-scale approaches ranging from disease modeling in live animals, through cell-based interventional studies, to structure-guided biochemical studies that offer an atomic-level resolution, this study unravels the existence of a ‘brake’ within the TLR4 signaling cascade, i.e., GIV; the latter is a prototypical member of an emerging class of scaffold proteins. By showing that GIV uses conserved mechanisms to impact multi-TLR signaling, this work unravels a multi-scale point of convergence of immune signaling of broader impact beyond TLR4.
Size Effect on Structure and Stiffness of Viral DNA during Temperature Variation
Authors: Zhang Cheng-Yin , Zhang Neng-Hui
Published: August 2020

Structure and stiffness of nucleic acids are closely related to viral infectivity. The article is aimed to clarify a recent controversy on structure variation of viral DNA in temperature-dependent experiments. A multi-scale correlation between microscopic structure of viral DNA and its bulk stiffness is formulated by presenting a two-zone mesoscopic free energy model. Results reveal that the increasing temperature promotes DNA structure transformation from order to disorder, and leads to size effect on temperature-dependency of stiffness and structure of viral DNA.
Live imaging and biophysical modeling support a button-based mechanism of somatic homolog pairing inDrosophila
Authors: Child Myron , Bateman Jack R. , Jahangiri Amir , Reimer Armando , Lammers Nicholas C. , Sabouni Nica , Villamarin Diego , McKenzie-Smith Grace C. , Johnson Justine E. , Jost Daniel , Garcia Hernan G.
Published: August 2020

The spatial configuration of the eukaryotic genome is organized and dynamic, providing the structural basis for regulated gene expression in living cells. InDrosophila melanogaster, 3D genome organization is characterized by somatic homolog pairing, where homologous chromosomes are intimately paired from end to end; however, the process by which homologs identify one another and pair has remained mysterious. A recent model proposed that specifically interacting “buttons” encoded along the lengths of homologous chromosomes drive somatic homolog pairing. Here, we turned this hypothesis into a precise biophysical model to demonstrate that a button-based mechanism can lead to chromosome-wide pairing. We tested our model and constrained its free parameters using live-imaging measurements of chromosomal loci tagged with the MS2 and PP7 nascent RNA labeling systems. Our analysis showed strong agreement between model predictions and experiments in the separation dynamics of tagged homologous loci as they transition from unpaired to paired states, and in the percentage of nuclei that become paired as embryonic development proceeds. In sum, as a result of this dialogue between theory and experiment, our data strongly support a button-based mechanism of somatic homolog pairing inDrosophilaand provide a theoretical framework for revealing the molecular identity and regulation of buttons.
The Impact of Pf Bacteriophages on the Fitness ofPseudomonas aeruginosa; A Mathematical Modeling Approach
Authors: Pourtois Julie D. , Kratochvil Michael J. , Chen Qingquan , Haddock Naomi L. , Burgener Elizabeth B. , De Leo Giulio A. , Bollyky Paul L.
Published: August 2020

Pseudomonas aeruginosa(Pa) is a major bacterial pathogen responsible for chronic lung infections in cystic fibrosis patients. Recent work by ourselves and others has implicated Pf bacteriophages, non-lytic filamentous viruses produced byPa, in the chronicity and severity ofPainfections. Pf phages act as structural elements inPabiofilms and sequester aerosolized antibiotics, thereby contributing to antibiotic tolerance. Consistent with a selective advantage in this setting, the prevalence of Pf+ bacteria increases over time in these patients. However, the production of Pf phages comes at a metabolic cost to bacteria, such that Pf+ strains grow more slowly than Pf- strains in vitro. Here, we use a mathematical model to investigate how these competing pressures might influence the relative abundance of Pf+ versus Pf- strains in different settings. Our model predicts that Pf+ strains ofPacan only outcompete Pf- strains if the benefits of phage production falls solely onto Pf+ strains and not onto the overall bacterial community in the lung. Further, phage production only leads to a net positive gain in fitness at antibiotic concentrations slightly above the minimum inhibitory concentration (i.e., concentrations for which the benefits of antibiotic sequestration outweigh the metabolic cost of phage production), but which are not lethal for Pf+ strains. As a result, our model predicts that frequent administration of intermediate doses of antibiotics with low decay rates favors Pf+ over Pf- strains. These models inform our understanding of the ecology of Pf phages and suggest potential treatment strategies for Pf+Painfections.

Importance

Filamentous phages are a frontier in bacterial pathogenesis, but the impact of these phages on bacterial fitness is unclear. In particular, Pf phages produced byPapromote antibiotic tolerance but are metabolically expensive to produce, suggesting that competing pressures may influence the prevalence of Pf+ versus Pf- strains ofPain different settings. Our results identify conditions likely to favor Pf+ strains and thus antibiotic tolerance. This study contributes to a better understanding of the unique ecology of filamentous phages and may facilitate improved treatment strategies for combating antibiotic tolerance.
Sequence-encoded and Composition-dependent Protein-RNA Interactions Control Multiphasic Condensate Morphologies
Authors: Kaur Taranpreet , Raju Muralikrishna , Alshareedah Ibraheem , Davis Richoo B. , Potoyan Davit A. , Banerjee Priya R.
Published: August 2020

Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases−homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.
Structural classification of neutralizing antibodies against the SARS-CoV-2 spike receptor-binding domain suggests vaccine and therapeutic strategies
Authors: Barnes Christopher O. , Jette Claudia A. , Abernathy Morgan E. , Dam Kim-Marie A. , Esswein Shannon R. , Gristick Harry B. , Malyutin Andrey G. , Sharaf Naima G. , Huey-Tubman Kathryn E. , Lee Yu E. , Robbiani Davide F. , Nussenzweig Michel C. , West Anthony P. , Bjorkman Pamela J.
Published: August 2020

The COVID-19 pandemic presents an urgent health crisis. Human neutralizing antibodies (hNAbs) that target the host ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike1–5 show therapeutic promise and are being evaluated clincally6–8. To determine structural correlates of SARS-CoV-2 neutralization, we solved 8 new structures of distinct COVID-19 hNAbs5 in complex with SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed classification into categories: (1) VH3-53 hNAbs with short CDRH3s that block ACE2 and bind only to “up” RBDs, (2) ACE2-blocking hNAbs that bind both “up” and “down” RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize “up” and “down” RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only “up” RBDs9. Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a VH3-53 hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent “down” RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally-occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects, suggesting combinations for clinical use, and providing insight into immune responses against SARS-CoV-2.
Protein Phosphatase 1 in association with Bud14 inhibits mitotic exit inSaccharomyces cerevisiae
Authors: Kocakaplan Dilara , Karabürk Hüseyin , Dilege Cansu , Kirdok Idil , Bektaş Şeyma Nur , Caydasi Ayse Koca
Published: August 2020

Mitotic exit in budding yeast is dependent on correct orientation of the mitotic spindle along the cell polarity axis. When accurate positioning of the spindle fails, a surveillance mechanism named the Spindle Position Checkpoint (SPOC) prevents cells from exiting mitosis. Mutants with a defective SPOC become multinucleated and lose their genomic integrity. Yet, a comprehensive understanding of the SPOC mechanism is missing. In this study, we identified the type 1 protein phosphatase, Glc7, in association with its regulatory protein Bud14 as a novel checkpoint component. We further showed that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results suggest a model in which two mechanisms act in parallel for a robust checkpoint response: first, the SPOC kinase Kin4 isolates Bfa1 away from the inhibitory kinase Cdc5 and second, Glc7-Bud14 dephosphorylates Bfa1 to fully activate the checkpoint effector.
FlyWire: Online community for whole-brain connectomics
Authors: Dorkenwald Sven , McKellar Claire , Macrina Thomas , Kemnitz Nico , Lee Kisuk , Lu Ran , Wu Jingpeng , Popovych Sergiy , Mitchell Eric , Nehoran Barak , Jia Zhen , Bae J. Alexander , Mu Shang , Ih Dodam , Castro Manuel , Ogedengbe Oluwaseun , Halageri Akhilesh , Ashwood Zoe , Zung Jonathan , Brittain Derrick , Collman Forrest , Schneider-Mizell Casey , Jordan Chris , Silversmith William , Baker Christa , Deutsch David , Encarnacion-Rivera Lucas , Kumar Sandeep , Burke Austin , Gager Jay , Hebditch James , Koolman Selden , Moore Merlin , Morejohn Sarah , Silverman Ben , Willie Kyle , Willie Ryan , Yu Szi-chieh , Murthy Mala , Seung H. Sebastian
Published: August 2020

Due to advances in automated image acquisition and analysis, new whole-brain connectomes beyondC. elegansare finally on the horizon. Proofreading of whole-brain automated reconstructions will require many person-years of effort, due to the huge volumes of data involved. Here we present FlyWire, an online community for proofreading neural circuits in a fly brain, and explain how its computational and social structures are organized to scale up to whole-brain connectomics. Browser-based 3D interactive segmentation by collaborative editing of a spatially chunked supervoxel graph makes it possible to distribute proofreading to individuals located virtually anywhere in the world. Information in the edit history is programmatically accessible for a variety of uses such as estimating proofreading accuracy or building incentive systems. An open community accelerates proofreading by recruiting more participants, and accelerates scientific discovery by requiring information sharing. We demonstrate how FlyWire enables circuit analysis by reconstructing and analysing the connectome of mechanosensory neurons.
Branching and extinction in evolutionary public goods games
Authors: Johnson Brian , Altrock Philipp M. , Kimmel Gregory J.
Published: August 2020

Public goods games (PGGs) describe situations in which individuals contribute to a good at a private cost, but others can free-ride by receiving their share of the public benefit at no cost. PGGs can be nonlinear, as often observed in nature, whereby either benefit, cost, or both are nonlinear functions of the available public good (PG): at low levels of PG there can be synergy whereas at high levels, the added benefit of additional PG diminishes. PGGs can be local such that the benefits and costs are relevant only in a local neighborhood or subset of the larger population in which producers (cooperators) and free-riders (defectors) co-evolve. Cooperation and defection can be seen as two extremes of a continuous spectrum of traits. The level of public good production, and similarly, the neighborhood size can vary across individuals. To better understand how distinct strategies in the nonlinear public goods game emerge and persist, we study the adaptive dynamics of production rate and neighborhood size. We explain how an initially monomorphic population, in which individuals have the same trait values, could evolve into a dimorphic population by evolutionary branching, in which we see distinct cooperators and defectors emerge, respectively characterized by high production and low neighborhood sizes, and low production and high neighborhood sizes. We find that population size plays a crucial role in determining the final state of the population, as smaller populations may not branch, or may observe extinction of a subpopulation after branching. Our work elucidates the evolutionary origins of cooperation and defection in nonlinear local public goods games, and highlights the importance of small population size effects on the process and outcome of evolutionary branching.
SARS-CoV-2 infects human pluripotent stem cell-derived cardiomyocytes, impairing electrical and mechanical function
Authors: Marchiano Silvia , Hsiang Tien-Ying , Higashi Ty , Khanna Akshita , Reinecke Hans , Yang Xiulan , Pabon Lil , Sniadecki Nathan J. , Bertero Alessandro , Gale Michael , Murry Charles E.
Published: August 2020

Global health has been threatened by the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2)1. Although considered primarily a respiratory infection, many COVID-19 patients also suffer severe cardiovascular disease2–4. Improving patient care critically relies on understanding if cardiovascular pathology is caused directly by viral infection of cardiac cells or indirectly via systemic inflammation and/or coagulation abnormalities3,5–9. Here we examine the cardiac tropism of SARS-CoV-2 using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and three-dimensional engineered heart tissues (3D-EHTs). We observe that hPSC-CMs express the viral receptor ACE2 and other viral processing factors, and that SARS-CoV-2 readily infects and replicates within hPSC-CMs, resulting in rapid cell death. Moreover, infected hPSC-CMs show a progressive impairment in both electrophysiological and contractile properties. Thus, COVID-19-related cardiac symptoms likely result from a direct cardiotoxic effect of SARS-CoV-2. Long-term cardiac complications might be possible sequelae in patients who recover from this illness.
Ribosome inhibition byC9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM
Authors: Loveland Anna B. , Svidritskiy Egor , Susorov Denis , Lee Soojin , Park Alexander , Demo Gabriel , Gao Fen-Biao , Korostelev Andrei A.
Published: August 2020

Toxic dipeptide repeat (DPR) proteins are produced from expanded G4C2hexanucleotide repeats in theC9ORF72gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥ 20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with the DPR proteins and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity inC9ORF72-ALS/FTD.
Quantitative imaging of RNA polymerase II activity in plants reveals the single-cell basis of tissue-wide transcriptional dynamics
Authors: Alamos Simon , Reimer Armando , Niyogi Krishna K. , Garcia Hernan G.
Published: August 2020

The responses of plants to their environment often hinge on the spatiotemporal dynamics of transcriptional regulation. While live-imaging tools have been used extensively to quantitatively capture rapid transcriptional dynamics in living animal cells, lack of implementation of these technologies in plants has limited concomitant quantitative studies. Here, we applied the PP7 and MS2 RNA-labeling technologies for the quantitative imaging of RNA polymerase II activity dynamics in single cells of living plants as they respond to experimental treatments. Using this technology, we count nascent RNA transcripts in real-time inNicotiana benthamiana(tobacco) andArabidopsis thaliana(Arabidopsis). Examination of heat shock reporters revealed that plant tissues respond to external signals by modulating the number of cells engaged in transcription rather than the transcription rate of active cells. This switch-like behavior, combined with cell-to-cell variability in transcription rate, results in mRNA production variability spanning three orders of magnitude. We determined that cellular heterogeneity stems mainly from the stochasticity intrinsic to individual alleles. Taken together, our results demonstrate that it is now possible to quantitatively study the dynamics of transcriptional programs in single cells of living plants.
Plant NLR targets P-type ATPase for executing plasma membrane depolarization leading to calcium influx and cell death
Authors: Lee Hye-Young , Seo Ye-Eun , Lee Joo Hyun , Lee So Eui , Oh Soohyun , Kim Jihyun , Jung Seungmee , Kim Haeun , Park Hyojeong , Kim Sejun , Mang Hyunggon , Choi Doil
Published: August 2020

Hypersensitive response (HR) is a robust immune response mediated by plant nucleotide-binding and leucine-rich repeat receptor (NLR). However, the early molecular event linking NLR to cell death is obscure. Here we demonstrate that NLR targets plasma membrane H+-ATPases (PMA) generating electrochemical potential across the membrane. CCA309, an autoactive N-terminal domain of pepper coiled-coil NLR (CNL), associates with PMAs and its autoactivity is affected by silencing or overexpression of PMA. CCA309-induced extracellular alkalization accompanied with membrane depolarization is followed by calcium influx and cell death. CCA309 interacts with C-terminal regulatory domain of PMA and 14-3-3 negatively affects CCA309-induced cell death. Moreover, pharmacological experiments with fusicoccin, an irreversible PMA activator, confirmed that CC- and CNL-mediated cell death occurred through inhibiting PMA. We propose PMAs as the primary target of plasma membrane-associated CNL to disrupt electrochemical homeostasis leading to HR cell death.
Murine intrarectal instillation of purified recombinantC. difficiletoxins enables mechanistic studies of pathogenesis
Authors: Markham Nicholas O. , Bloch Sarah C. , Shupe John A. , Laubacher Erin N. , Washington M. Kay , Coffey Robert J. , Lacy D. Borden
Published: August 2020

Clostridioides difficileis linked to nearly 225,000 antibiotic-associated diarrheal infections and almost 13,000 deaths per year in the United States. Pathogenic strains ofC. difficileproduce toxin A (TcdA) and toxin B (TcdB), which can directly kill cells and induce an inflammatory response in the colonic mucosa. Hirota,et al. first introduced the intrarectal instillation model of intoxication using TcdA and TcdB purified from VPI 10463 and 630C. difficilestrains. Here, we expand this technique by instilling purified, recombinant TcdA and TcdB, which allows for the interrogation of how specifically mutated toxins affect tissue. Mouse colons were processed and stained with hematoxylin and eosin (H&E) for blinded evaluation and scoring by a board-certified gastrointestinal pathologist. The amount of TcdA or TcdB needed to produce damage was lower than previously reportedin vivoandex vivo. Furthermore, TcdB mutants lacking either endosomal pore-formation or glucosyltransferase activity resemble sham negative controls. Immunofluorescent staining revealed how TcdB initially damages colonic tissue by altering the epithelial architecture closest to the lumen. Tissue sections were also immunostained for markers of acute inflammatory infiltration. These staining patterns were compared with slides from a humanC. difficileinfection (CDI). The intrarectal instillation mouse model with purified recombinant TcdA and/or TcdB provides the flexibility needed to better understand structure/function relationships across different stages of CDI pathogenesis.
Capitalizing on paradoxical activation of the MAPK pathway for treatment of Imatinib-resistant mast cell leukemia
Authors: Wilhelm Thomas , Toledo Marcelo A. S. , Simons Ilka , Stuth Christian , Mohta Vrinda , Mülfarth Ronja , Nitsche Marcus , Maschke-Neuß Karin , Schmitz Susanne , Kaiser Anne , Arock Michel , Zenke Martin , Huber Michael
Published: August 2020

Prevention of fatal side effects during cancer therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called “paradoxical activation”. Here we re-analyzing the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KITV560G,D816V-expressing human mast cell (MC) leukemia (MCL) cell line HMC-1.2. We observed that low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. The combination of Ponatinib with the MEK inhibitor Trametinib, at nanomolar concentrations, effectively suppressed HMC-1.2 proliferation, metabolic activity, and induced apoptotic cell death. Effectiveness of this drug combination was recapitulated in the humanKITD816V MC line ROSA KITD816Vand inKITD816V hematopoietic progenitors obtained from in patient-derived induced pluripotent stem cells (iPS cells). In conclusion, mutated KIT-driven Imatinib resistance can be efficiently bypassed by a low concentration combination of the TKI Ponatinib and the MEK inhibitor Trametinib, potentially reducing the negative side effects associated to MCL therapy.
ZNF410 uniquely activates the NuRD component CHD4 to silence fetal hemoglobin expression
Authors: Lan Xianjiang , Ren Ren , Feng Ruopeng , Ly Lana C. , Lan Yemin , Zhang Zhe , Aboreden Nicholas , Qin Kunhua , Horton John R. , Grevet Jeremy D. , Mayuranathan Thiyagaraj , Abdulmalik Osheiza , Keller Cheryl A. , Giardine Belinda , Hardison Ross C. , Crossley Merlin , Weiss Mitchell J , Cheng Xiaodong , Shi Junwei , Blobel Gerd A.
Published: August 2020

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA binding protein that in human erythroid cells directly and measurably activates only one gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplant settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells whose perturbation might offer new therapeutic opportunities in the treatment of hemoglobinopathies.

Highlights

A CRISPR screen implicates ZNF410 in fetal globin gene repression

The CHD4 gene is the singular direct ZNF410 target in erythroid cells

The fetal globin genes are exquisitely sensitive to CHD4 levels

Five C2H2 zinc fingers of ZNF410 recognize the major groove of a 14 base pair sequence
BAP1 and YY1 regulate expression of death receptors in malignant pleural mesothelioma
Authors: Ishii Y , Kolluri KK , Pennycuick A , Nigro E , Alrifai D , Borg E , Falzon M , Shah K , Kumar N , Janes SM
Published: August 2020

Malignant pleural mesothelioma (MPM) is a rare, aggressive, and incurable cancer arising from the mesothelial lining of the lungs with few treatment options. We recently reported loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1), a frequent event in MPM, is associated with sensitivity to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). As a potential underlying mechanism, here we report that BAP1 negatively regulates the expression of TRAIL receptors: death receptors 4 (DR4) and 5 (DR5). Using tissue microarray (TMAs) of tumour samples from MPM patients, we found a strong inverse correlation between BAP1 and TRAIL receptors.BAP1knockdown increased DR4 and DR5 expression, whereas overexpression of BAP1 had the opposite effect. Reporter assays confirmed wild-typeBAP1, but not catalytically-inactive mutantBAP1, reduced promoter activities ofDR4andDR5, suggesting deubiquinase activity plays an important role in the regulation of gene expression. Co-IP studies demonstrated direct binding of BAP1 and the transcription factor Ying Yang 1 (YY1) and ChIP assays revealed BAP1 and YY1 to be enriched in the promoter regions ofDR4andDR5. Notably, shRNA knockdown ofYY1also increased DR4 and DR5 expression, and sensitivity to TRAIL. These results demonstrate that BAP1 and YY1 together negatively regulate transcriptional activity of TRAIL receptors. BAP1 and YY1 may both therefore be strong therapeutic targets to enhance the efficacy of TRAIL-induced apoptosis.

Statement of significance

We describe how the most-frequently mutated tumour suppressor gene in mesothelioma regulates the response to TNF-related apoptosis-inducing ligand (TRAIL). These findings will accelerate a biomarker-driven cancer therapy.
Perturbations of theAMI1IAM-amidohydrolase expression trigger plant stress responses inArabidopsis thaliana
Authors: Pérez-Alonso Marta-Marina , Ortiz-García Paloma , Moya-Cuevas José , Lehmann Thomas , Sánchez-Parra Beatriz , Björk Robert G. , Karim Sazzad , Amirjani Mohammad R. , Aronsson Henrik , Wilkinson Mark D. , Pollmann Stephan
Published: August 2020

The evolutionary success of plants relies to a large extent on their extraordinary ability to adapt to changes in their environment. These adaptations require that plants balance their growth with their stress responses. Plant hormones are crucial mediators orchestrating the underlying adaptive processes. However, whether and how the growth-related hormone auxin and the stress-related hormones jasmonic acid (JA), salicylic acid, and abscisic acid (ABA) are coordinated remains largely elusive. Here, we analyze the physiological role of AMIDASE 1 (AMI1) in plant growth and its possible connection to plant adaptations to abiotic stresses. AMI1 contributes to cellular auxin homeostasis by catalyzing the conversion of indole-acetamide into the major plant auxin indole-3-acetic acid. Functional impairment of AMI1 increases the plants’ stress status rendering mutant plants more susceptible to abiotic stresses. Transcriptomic analysis ofami1mutants disclosed the reprogramming of a considerable number of stress-related genes, including JA and ABA biosynthesis genes. Theami1mutants exhibit only moderately repressed growth, but an enhanced ABA accumulation, which suggests a role for AMI1 in the crosstalk between auxin and ABA. Altogether, our results suggest that AMI1 is involved in coordinating the trade-off between plant growth and stress responses, balancing auxin with ABA homeostasis.

HIGHLIGHT

The IAM amidohydrolase AMI1 catalyzes the conversion of IAM into IAAin vivo. Expression ofAMI1is specifically repressed by osmotic stress conditions, which triggers ABA biosynthesis through the induction ofNCED3, thereby linking auxin homeostasis with plant stress responses.
Dynamic reliability and variance-based global sensitivity analysis of pipes conveying fluid with both random and convex variables
Authors: Chen Bingqian , Wang Anqiang , Guo Qing , Dai Jiayin , Liu Yongshou
Published: August 2020

Purpose

This paper aims to solve the problem that pipes conveying fluid are faced with severe reliability failures under the complicated working environment.

Design/methodology/approach

This paper proposes a dynamic reliability and variance-based global sensitivity analysis (GSA) strategy with non-probabilistic convex model for pipes conveying fluid based on the first passage principle failure mechanism. To illustrate the influence of input uncertainty on output uncertainty of non-probability, the main index and the total index of variance-based GSA analysis are used. Furthermore, considering the efficiency of traditional simulation method, an active learning Kriging surrogate model is introduced to estimate the dynamic reliability and GSA indices of the structure system under random vibration.

Findings

The variance-based GSA analysis can measure the effect of input variables of convex model on the dynamic reliability, which provides useful reference and guidance for the design and optimization of pipes conveying fluid. For designers, the rankings and values of main and total indices have essential guiding role in engineering practice.

Originality/value

The effectiveness of the proposed method to calculate the dynamic reliability and sensitivity of pipes conveying fluid while ensuring the calculation accuracy and efficiency in the meantime.
Products labeled as “made in domestic country”: the brand matters
Authors: Bernard Yohan , Collange Véronique , Ingarao Aurore , Zarrouk-Karoui Sarra
Published: August 2020

Purpose

The purpose of this paper is to better understand an increasingly widespread practice consisting, of a brand, in signaling the domestic origin of its products aimed at domestic consumers, that is, the “made in the domestic country” (MIDC) strategy. To this end, it is proposed to analyze the MIDC label as a cue interacting with the brand’s characteristics (brand equity and country of origin of the brand).

Design/methodology/approach

A between-subjects experiment is conducted among 293 French consumers on four different brands of pasta. The overall design is a 2 (with/without the MIDC label) × 2 (high/low brand equity) × 2 (domestic/foreign brand) mixed design.

Findings

The results show that intention to buy the product increases significantly with the presence of the MIDC label, but not so willing to pay. The positive effect on buying intention is greater when: the product has rather low brand equity, consumer ethnocentrism is high and/or consumers are strongly attached to their national identity.

Research limitations/implications

The present research extends the literature on country-of-origin effects by taking into account the role of the brand equity of the product. However, the study focused on only one low-involvement product category (pasta) and one country (France).

Practical implications

This study shows that adding an MIDC label to the product is empirically justified.

Originality/value

While moderate or high scores on “patriotic” variables reinforce the positive impact of the MIDC label, low scores reverse the trend, that is, cause rejection.
Liberal and vocational education: the Gordian encounter
Authors: Burns Caroline J. , Natale Samuel M.
Published: August 2020

Purpose

The purpose of this paper is to discuss how liberal higher education can strengthen vocational higher education.

Design/methodology/approach

The paper uses Shay's (2013) framework of curriculum differentiation to articulate how the strengths and shortcomings of liberal education differ from those of vocational education and to allow the differences highlighted to inform a resolution to each other's shortcomings.

Findings

There is nothing new in the findings that liberal education differs from vocational education and that both have shortcomings. What the paper presents is a viewpoint that the differences are not confirmation that these two approaches to education are in opposition but rather that they complement each other. The strength of one is the weakness of the other.

Originality/value

The perspective taken in this paper is developed using the language of semantic density (SD) and semantic gravity (SG). Using Shay's semantic field of recontextualized knowledge, this paper suggests that liberal and vocational education inhabit two sides of contexts and concepts continua. The paper further proposes that both are alike in a meaningful way because both have unsuccessfully managed the role of context in their curricula.
Readability of Korean-Language advertising disclosures moderates knowledge effects
Authors: Lee Taejun (David) , Huhmann Bruce A. , Yun TaiWoong
Published: August 2020

Purpose

Government policy mandates information disclosure in financial communications to protect consumer welfare. Unfortunately, low readability can hamper information disclosures’ meaningful benefits to financial decision making. Thus, this experiment tests the product evaluation and decision satisfaction of Korean consumers with less or more subjective knowledge and with or without personal finance education.

Design/methodology/approach

A between-subjects experiment examined responses of a nationally representative sample of 400 Korean consumers toward a Korean-language credit card advertisement.

Findings

Financial knowledge improves financial product evaluation and decision satisfaction. More readable disclosures improved evaluation and satisfaction among less knowledgeable consumers. Less readable disclosures did not. Consumers without financial education exhibited lower evaluations and decision satisfaction regardless of readability. More knowledgeable consumers and those with financial education performed equally well regardless of disclosure readability.

Practical implications

Financial service providers seeking more accurate evaluations and better decision satisfaction among their customers should use easier-to-read disclosures when targeting consumers with less prior financial knowledge.

Social implications

One-size-fits-all financial communications are unlikely to achieve public policy or consumer well-being goals. Government-mandated information should be complemented by augmenting financial knowledge and providing personal finance training.

Originality/value

Although almost a quarter of the world’s population lives in East Asia, this is the first examination of readability in disclosures written in East Asian characters rather than a Western alphabet. Previous readability research on Asian-originating financial disclosures has been conducted on English-language texts. This study extends knowledge of readability effects to growing East Asian markets.
Finite element simulation on the relationship between pressure and displacement for the waist of elastic pantyhose
Authors: Dan Rui , Shi Zhen
Published: August 2020

Purpose

Objective appraisal of pressure comfort is the key point of optimal designing of clothing. The purpose of this paper is to study a new method to provide pressure comfort for the waist of elastic pantyhose through the relationship between pressure and displacement using the finite element method (FEM).

Design/methodology/approach

This paper presented a simulation model of the waist cross section consisting of three parts, namely skin, soft tissue and lumbar vertebrae, respectively, according to CT scan. The finite element the model of waist cross-section was established using Mimics software. The pressure–displacement quadratic equation can be obtained using ANSYS software and fitting curves. Meanwhile, we divide the waist cross-section into 12 equal regions according to angle, and then the area shrinkage mass of the waist cross-section can be calculated, respectively.

Findings

In this research work, we got the displacement distribution trend of elastic pantyhose at the waist cross section according to the area shrinkage mass of 12 regions, and this displacement could be used as an objective evaluation index for pressure comfort. All these solutions supply a theoretical reference for optimal design of the women's elastic pantyhose.

Originality/value

The paper analyzed the relationship between pressure and displacement for the waist of elastic pantyhose using FEM, and then got the displacement distribution trend of elastic pantyhose at the waist cross section according to the area shrinkage mass of different regions. It can supply a new method to appraise pressure comfort.