bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a flexible AAV-DTR/DT mouse model of acute epithelial lung injury
Authors: Griesser Eva , Schönberger Tanja , Stierstorfer Birgit , Wyatt Hannah , Rist Wolfgang , Lamla Thorsten , Thomas Matthew James , Lamb David , Geillinger-Kästle Kerstin 
Published: June 2021

AbstractBackground & aimRecurring epithelial injury and aberrant repair are considered as a major driver of idiopathic pulmonary fibrosis (IPF) leading to chronic inflammation, fibroblast activation and ultimately to scarring and stiffening of the lung. As decline of lung function is the first reported symptom by IPF patients and occurs once fibrosis is firmly established, animal models are required to study early disease-driving mechanisms.MethodsWe developed a novel and flexible mouse model of acute epithelial injury based on adeno-associated virus (AAV) variant 6.2 mediated expression of the human diphtheria toxin receptor (DTR). Following intratracheal administration of diphtheria toxin (DT), a cell-specific death of bronchial epithelial and alveolar epithelial type II cells can be observed.ResultsDetailed characterization of the AAV-DTR/DT mouse model revealed increasing cell numbers in bronchoalveolar lavage (BAL; macrophages, neutrophils, and atypical cells) and elevation of apoptotic cells and infiltrated leukocytes in lung tissue, which were dependent of viral genome load and DT dose. Cytokine levels in BAL fluid showed different patterns dependent of viral genome load with IFNγ, TNFα, and IP-10 increasing and IL-5 and IL-6 decreasing, while lung function was not affected. Additionally, laser-capture microdissection-based proteomics of bronchial and alveolar epithelium showed upregulated immune and inflammatory response in all epithelial cell regions and extracellular matrix deposition in infiltrated alveoli, while proteins involved in pulmonary surfactant synthesis, alveolar fluid clearance and alveolar-capillary barrier were downregulated in the parenchyma.ConclusionOur novel AAV-DTR/DT model resembles specific aspects of pulmonary diseases like IPF and acute respiratory distress syndrome.Short summary for social mediaA novel and flexible mouse model of acute epithelial lung injury based on AAV-mediated expression of the human diphtheria toxin receptor followed by intratracheal instillation of diphtheria toxin resembles specific aspects of pulmonary diseases like IPF.
Human bone marrow milieu identifies a clinically actionable driver of niche-mediated treatment resistance in leukaemia
Authors: Pal Deepali , Blair Helen , Boyd Sophie , Sharon Angel Hanmy , Nizami Salem , Isa Asmida , Beckett Melanie , Nelson Ryan , Wilson Aaron , Singh Mankaran , Sankar Shalini , Tirtakusuma Ricky , Sirintra Nakjang , Knill Carly , Fuller Andrew , McNeill Hesta , Russell Lisa , Schwab Claire , Zhous Peixun , Sinclair Paul , Coxhead Jonathan , Filby Andrew , Halsey Christina , Allan James M. , Harrison J. Christine , Moorman Anthony , Olaf Heidenreich , Vormoor Josef 
Published: June 2021

AbstractLeukaemia cells re-program their microenvironment to provide proliferation support and protection from standard chemotherapy, molecularly targeted therapies, and immunotherapy. Although much is becoming known about molecules that drive niche-dependent treatment resistance; means of targeting these in the clinics has remained a key obstacle. To address this challenge, we have developed human induced pluripotent stem cell engineered nichesex vivoto reveal insights into druggable cancer-niche dependencies. We show that mesenchymal (iMSC) and vascular niche-like (iANG) cells supportex vivoproliferation of patient-derived leukaemia cells, impact dormancy and mediate therapy resistance. iMSC protected both non-cycling and cycling blasts against dexamethasone treatment while iANG protected only dormant blasts. Leukaemia proliferation and protection from dexamethasone induced-apoptosis was dependent on direct cell-cell contact and mediated by CDH2. To explore the therapeutic potential of disrupting this cell-cell interaction, we tested the CDH2 antagonist ADH-1 (previously in phase I / II for solid tumours) in a very aggressive patient-derived xenograft leukaemia mouse model. ADH-1 showed highin vivoefficacy. ADH-1/ dexamethasone combination therapy was superior to dexamethasone alone with no ADH1 conferred additional toxicity. These findings provide a proof-of-concept starting point to develop novel, potentially safer therapeutics that target niche-mediated cancer cell dependencies in haematological malignancies.SummaryCDH2 mediated niche-dependent cancer proliferation and treatment resistance is clinically targetable via ADH-1, a low toxic agent that could be potentially repurposed for future clinical trials in acute leukaemia.
Zebrafish Cre/loxregulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation
Authors: Almeida Maira P. , Kambakam Sekhar , Liu Fang , Ming Zhitao , Welker Jordan M. , Wierson Wesley A. , Schultz-Rogers Laura E. , Ekker Stephen C. , Clark Karl J. , Essner Jeffrey J. , McGrail Maura 
Published: June 2021

AbstractThe ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/loxsystem has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/loxregulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron inhdac1, rbbp4, andrb1. Active, gene off orientationhdac1-UFlip-Offandrb1-UFlip-Offintegration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientationrbbp4-UFlip-Onandrb1-UFlip-Onintegration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs ofloxPandlox2272sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstraterbbp4-UFlip-Onandrb1-UFlip-Ongene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP inrbbp4-UFlip-Off, or 2A-KalTA4 inrb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/loxresponsive conditional gene alleles to advance zebrafish Cre recombinase genetics.
Imaging transcriptomics: Convergent cellular, transcriptomic, and molecular neuroimaging signatures in the healthy adult human brain
Authors: Martins Daniel , Giacomel Alessio , Williams Steven CR , Turkheimer Federico , Dipasquale Ottavia , Veronese Mattia ,   PET templates working group
Published: June 2021

AbstractThe expansion of neuroimaging techniques over the last decades has opened a wide range of new possibilities to characterize brain dysfunction in several neurological and psychiatric disorders. However, the lack of specificity of most of these techniques, such as magnetic resonance imaging (MRI)-derived measures, to the underlying molecular and cellular properties of the brain tissue poses limitations to the amount of information one can extract to inform precise models of brain disease. The integration of transcriptomic and neuroimaging data, known as ‘imaging transcriptomics’, has recently emerged as an indirect way forward to test and/or generate hypotheses about potential cellular and transcriptomic pathways that might underly specific changes in neuroimaging MRI biomarkers. However, the validity of this approach is yet to be examined in-depth. Here, we sought to bridge this gap by performing imaging transcriptomic analyses of the regional distribution of well-known molecular markers, assessed by positron emission tomography (PET), in the healthy human brain. We focused on tracers spanning different elements of the biology of the brain, including neuroreceptors, synaptic proteins, metabolism, and glia. Using transcriptome-wide data from the Allen Brain Atlas, we applied partial least square regression to rank genes according to their level of spatial alignment with the regional distribution of these neuroimaging markers in the brain. Then, we performed gene set enrichment analyses to explore the enrichment for specific biological and cell-type pathways among the genes most strongly associated with each neuroimaging marker. Overall, our findings show that imaging transcriptomics can recover plausible transcriptomic and cellular correlates of the regional distribution of benchmark molecular imaging markers, independently of the type of parcellation used to map gene expression and neuroimaging data. Our data support the plausibility and robustness of imaging transcriptomics as an indirect approach for bridging gene expression, cells and macroscopical neuroimaging and improving our understanding of the biological pathways underlying regional variability in neuroimaging features.
Estimates of persistent inward currents are reduced in upper limb motor units of older adults
Authors: Hassan Altamash S , Fajardo Melissa E , Cummings Mark , McPherson Laura Miller , Negro Francesco , Dewald Julius P A , Heckman C J , Pearcey Gregory E P 
Published: June 2021

AbstractAging is a natural process that causes alterations in the neuromuscular system, which contribute to weakness and reduced quality of life. Reduced firing rates of individual motor units (MUs) likely contribute to weakness, but the mechanisms underlying reduced firing rates are not clear. Persistent inward currents (PICs) are crucial for the initiation, gain control, and maintenance of motoneuron firing, and are directly proportional to the level of monoaminergic input. Since the concentration of monoamines (i.e. serotonin and norepinephrine) are reduced with age, we sought to determine if estimates of PICs are reduced in older (>60 years old) compared to younger adults (<35 years old). We decomposed MU spike trains from high-density surface electromyography over the biceps brachii and triceps brachii during isometric ramp contractions to 20% of maximum. Estimates of PICs (i.e. ΔF) were computed using the paired MU analysis technique. Regardless of the muscle, peak firing rates of older adults were reduced by ~1.6 pulses per second (pps) (P = 0.0292), and ΔF was reduced by ~1.9 pps (P < 0.0001), compared to younger adults. We further found that age predicted ΔF in older adults (P = 0.0261), resulting in a reduction of ~1pps per decade, but there was no relationship in younger adults (P = 0.9637). These findings suggest that PICs are reduced in older adults, and, further, age is a significant predictor of estimates of PICs in older adults. Reduced PIC magnitude represents one plausible mechanism for reduced firing rates and weakness in older individuals.KEY POINTSPersistent inward currents play an important role in the neural control of human movement and are influenced by neuromodulation via monoamines originating in the brainstem.During aging, motor unit firing rates are reduced, and there is deterioration of brainstem nuclei, which may reduce persistent inward currents in alpha motoneurons.Here we show that estimates of persistent inward currents (ΔF) of both elbow flexor and extensor motor units are reduced in older adults.Estimates of persistent inward currents have a negative relationship with age in the older adults, but not young.This novel mechanism may play a role in alteration motor firing rates that occurs with aging, which may have consequences for motor control.
Nanoscale surface topography reduces focal adhesions and cell stiffness by enhancing integrin endocytosis
Authors: Li Xiao , Klausen Lasse H. , Zhang Wei , Jahed Zeinab , Tsai Ching-Ting , Li Thomas L. , Cui Bianxiao 
Published: June 2021

AbstractBoth substrate stiffness and surface topography regulate cell behavior through mechanotransduction signaling pathways. Such intertwined effects suggest that engineered surface topographies might substitute or cancel the effects of substrate stiffness in biomedical applications. However, the mechanisms by which cells recognize topographical features are not fully understood. Here we demonstrate that the presence of nanotopography drastically alters cell behavior such that neurons and stem cells cultured on rigid glass substrates behave as if they were on soft hydrogels. We further show that rigid nanotopography resembles the effect of soft hydrogels in reducing cell stiffness and membrane tension as measured by atomic force microscopy. Finally, we demonstrate that nanotopography reduces focal adhesions and cell stiffness by enhancing the endocytosis and the subsequent removal of integrin receptors. This mechanistic understanding will support the rational design of nanotopography that directs cells on rigid materials to behave as if they were on soft ones.TOC graphic
Structural basis for PoxtA-mediated resistance to Phenicol and Oxazolidinone antibiotics
Authors: Crowe-McAuliffe Caillan , Murina Victoriia , Kasari Marje , Takada Hiraku , Turnbull Kathryn Jane , Polikanov Yury S. , Sundsfjord Arnfinn , Hegstad Kristin , Atkinson Gemma C. , Wilson Daniel N. , Hauryliuk Vasili 
Published: June 2021

AbstractPoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF) that confer resistance to oxazolidinone, such as linezolid, and phenicol antibiotics, such as chloramphenicol. PoxtA/OptrA are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria. These target protection proteins are thought to confer resistance by binding to the ribosome and dislodging the antibiotics from their binding sites. However, a structural basis for their mechanism of action has been lacking. Here we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9–3.1 Å, as well as the complete E. faecalis 70S ribosome at 2.2–2.5 Å. The structures reveal that PoxtA binds within the ribosomal E-site with its antibiotic resistance domain (ARD) extending towards the peptidyltransferase center (PTC) on the large ribosomal subunit. At its closest point, the ARD of PoxtA is still located >15 Å from the linezolid and chloramphenicol binding sites, suggesting that drug release is elicited indirectly. Instead, we observe that the ARD of PoxtA perturbs the CCA-end of the P-site tRNA causing it to shift by ∼4 Å out of the PTC, which correlates with a register shift of one amino acid for the attached nascent polypeptide chain. Given that linezolid and chloramphenicol are context-specific translation elongation inhibitors, we postulate that PoxtA/OptrA confer resistance to oxazolidinones and phenicols indirectly by perturbing the P-site tRNA and thereby altering the conformation of the attached nascent chain to disrupt the drug binding site.
External fertilization is orchestrated by a pH-regulated soluble adenylyl cyclase controlling sperm motility and chemotaxis
Authors: Körschen H.G. , Hamzeh H. , Pascal R. , Alvarez L. , Bönigk W. , Kaur N. , Levin L.R. , Buck J. , Kambach C. , Michino M. , Jennings A. , Sato A. , Seifert R. , Strünker T. , Steegborn C. , Kaupp U.B. 
Published: June 2021

ABSTRACTThe reaction of CO2with H2O to form HCO3-and H+is one of the most important chemical equilibria in cells. In mammalian sperm, a soluble adenylyl cyclase (sAC) serves as cellular HCO3-sensor that conveys the equilibrium state via cAMP synthesis to cAMP-signaling molecules. The function of sAC and cAMP in non-mammalian sperm is largely unknown. Here, we identify sAC orthologs in sea urchin and salmon sperm that, surprisingly, are activated by alkaline pH rather than HCO3-. Two amino-acid residues required for HCO3-binding of mammalian sAC are lacking in pH-regulated sAC. Orthologs identified in ten other phyla are also lacking either one of these key residues, suggesting that pH control is widespread among non-mammalian metazoan. The pH-sensitive sAC controls several functions of sperm from external fertilizers. Upon spawning, alkalization triggers cAMP synthesis and, thereby, activates motility of quiescent sperm. Egg-derived chemoattractants also alkalize sperm and elevate cAMP, which then-modulates pacemaker HCN channels to trigger a chemotactic Ca2+response. Finally, the sAC and the voltage- and cAMP-activated Na+/H+exchanger sNHE mutually control each other. A picture of evolutionary significance is emerging: motility and sensory signaling of sperm from both internal and external fertilizers rely on cAMP, yet, their sAC is regulated by HCO3-or pHi, respectively. Acidification of aquatic habitats due to climate change may adversely affect pH-sensing by sAC and thereby sexual reproduction in the sea.Statement of significanceAdenylyl cyclases synthesize cAMP, a prominent cellular messenger. A bicarbonate-sensitive AC family member, soluble AC (sAC), is tied to the chemical equilibrium: H2O + CO2↔ HCO3-(bicarbonate) + H+. The sAC is required for fertilization: Mammals lacking sAC are infertile and sperm immotile. We now identify a new sAC form in sperm of non-mammalian animals that reproduce in the sea. This novel sAC is activated at alkaline pH rather than bicarbonate. It controls sperm motility and chemotaxis. The switch from HCO3-to pH rests on substitution of two amino-acids, which represents an adaptation to aquatic environments low in bicarbonate. Acidification of aquatic habitats due to climate change may adversely affect sAC activity and, thereby, fertilization.
Concealed identity information detection with pupillometry in rapid serial visual presentation
Authors: Chen Ivory Y. , Karabay Aytaç , Mathot Sebastiaan , Bowman Howard , Akyürek Elkan G. 
Published: June 2021

AbstractThe concealed information test (CIT) relies on bodily reactions to stimuli that are hidden in mind. However, people can use countermeasures, such as purposely focusing on irrelevant things, to confound the CIT. A new method designed to prevent countermeasures uses rapid serial visual presentation (RSVP) to present stimuli on the fringe of awareness. Previous studies that used RSVP in combination with electroencephalography (EEG) showed that participants exhibit a clear reaction to their real first name, even when they try to prevent such a reaction (i.e., when their name is concealed information). Since EEG is not easily applicable outside the laboratory, we investigated here whether pupil size, which is easier to measure, can also be used to detect concealed identity information. In our first study, participants adopted a fake name, and searched for this name in an RSVP task, while their pupil sizes were recorded. Apart from this fake name, their real name and a control name also appeared in the task. We found pupil dilation in response to the task-irrelevant real name, as compared to control names. However, while most participants showed this effect qualitatively, it was not statistically significant for most participants individually. In a second study, we preregistered the proof-of-concept methodology and replicated the original findings. Taken together, our results show that the current RSVP task with pupillometry can detect concealed identity information at a group level. Further development of the method is needed to create a valid and reliable concealed identity information detector at the individual level.
Notch1mutation drives clonal expansion in normal esophageal epithelium but impairs tumor growth
Authors: Abby Emilie , Dentro Stefan C , Hall Michael W J , Fowler Joanna C , Ong Swee Hoe , Sood Roshan , Siebel Christian W , Gerstung Moritz , Hall Benjamin A , Jones Philip H 
Published: June 2021

SummaryNOTCH1mutant clones occupy the majority of normal human esophagus by middle age, but are comparatively rare in esophageal cancers, suggestingNOTCH1mutations may promote clonal expansion but impede carcinogenesis1–3. Here we test this hypothesis. Visualizing and sequencingNOTCH1mutant clones in aging normal human esophagus reveals frequent biallelic mutations that block NOTCH1 signaling. In mouse esophagus, heterozygousNotch1mutation confers a competitive advantage over wild type cells, an effect enhanced by loss of the second allele.Notch1loss alters transcription but has minimal effects on epithelial structure and cell dynamics. In a carcinogenesis model,Notch1mutations were less prevalent in tumors than normal epithelium. Deletion ofNotch1reduced tumor growth, an effect recapitulated by anti-NOTCH1 antibody treatment. We conclude thatNotch1mutations in normal epithelium are beneficial as wild typeNotch1promotes tumor expansion. NOTCH1 blockade may have therapeutic potential in preventing esophageal squamous cancer.
Switching an active site helix in dihydrofolate reductase reveals limits to sub-domain modularity
Authors: Zhao Victor Y. , Rodrigues Joao V. , Lozovsky Elena R. , Hartl Daniel L. , Shakhnovich Eugene I. 
Published: June 2021

AbstractTo what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study sub-domain modularity by creating 20 chimeras of an enzyme, E. coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue α-helical sequence is replaced by α-helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this α-helix are under selection pressure to maintain catalytic activity in DHFR. We performed molecular dynamics simulations using replica exchange with solute-tempering. Chimeras with low catalytic activity exhibit non-helical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although α-helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.Statement of Significanceα-helices are ubiquitous components of protein structure that exhibit a degree of independent folding behavior, making them plausible structural modules within proteins. Here, we assess the effects of switching the sequence of an α-helix in an essential enzyme for α-helical sequences from evolutionarily unrelated proteins. The resultant chimeric proteins can still fold but enzymatic activity, stability, and cellular growth rates are negatively affected. Computational investigations reveal how residues in an α-helix have been shaped by selection pressure to maintain catalytic activity and a specific, helical conformation of the protein. More broadly, we illustrate how molecular and fitness effects of switching protein segments depend on the protein and cellular context.