bluuebunny/crossref_metadata_2025_split huggingface dataset:
Caenorhabditis elegans PIEZO Channel Coordinates Multiple Reproductive Tissues to Govern Ovulation
Authors: Bai Xiaofei , Bouffard Jeff , Lord Avery , Brugman Katherine , Sternberg Paul W. , Cram Erin J. , Golden Andy 
Published: November 2019

AbstractThe PIEZO proteins are involved in a wide range of developmental and physiological processes. Human PIEZO1 and PIEZO2 are newly identified excitatory mechano-sensitive proteins; they are non-selective ion channels that exhibit a preference for calcium in response to mechanical stimuli. To further understand the function of these proteins, we investigated the roles of pezo-1, the sole PIEZO ortholog in C. elegans. pezo-1 is expressed throughout development in C. elegans, with strong expression in reproductive tissues. A number of deletion alleles as well as a putative gain-of-function mutant caused severe defects in reproduction. A reduced brood size was observed in the strains depleted of PEZO-1. In vivo observations show that oocytes undergo a variety of transit defects as they enter and exit the spermatheca during ovulation. Post ovulation oocytes were frequently damaged during spermathecal contraction. Calcium signaling in the spermatheca is normal during ovulation in pezo-1 mutants, however, pezo-1 interacts genetically with known regulators of calcium signaling. Lastly, loss of PEZO-1 caused defective sperm navigation after being pushed out of the spermatheca during ovulation. Mating with males rescued these reproductive deficiencies in our pezo-1 mutants. These findings suggest that PEZO-1 may act in different reproductive tissues to promote proper ovulation and fertilization in C. elegans.
Unsupervised Graph-Based Learning Predicts Mutations That Alter Protein Dynamics
Authors: Peach Robert L. , Saman Dominik , Yaliraki Sophia N. , Klug David R. , Ying Liming , Willison Keith R. , Barahona Mauricio 
Published: November 2019

AbstractProteins exhibit complex dynamics across a vast range of time and length scales, from the atomistic to the conformational. Adenylate kinase (ADK) showcases the biological relevance of such inherently coupled dynamics across scales: single mutations can affect large-scale protein motions and enzymatic activity. Here we present a combined computational and experimental study of multiscale structure and dynamics in proteins, using ADK as our system of choice. We show how a computationally efficient method for unsupervised graph partitioning can be applied to atomistic graphs derived from protein structures to reveal intrinsic, biochemically relevant substructures at all scales, without re-parameterisation ora prioricoarse-graining. We subsequently perform full alanine and argininein silicomutagenesis scans of the protein, and score all mutations according to the disruption they induce on the large-scale organisation. We use our calculations to guide Förster Resonance Energy Transfer (FRET) experiments on ADK, and show that mutating residue D152 to alanine or residue V164 to arginine induce a large dynamical shift of the protein structure towards a closed state, in accordance with our predictions. Our computations also predict a graded effect of different mutations at the D152 site as a result of increased coherence between the core and binding domains, an effect confirmed quantitatively through a high correlation (R2= 0.93) with the FRET ratio between closed and open populations measured on six mutants.
Radiosafe micro-computed tomography for longitudinal evaluation of murine disease models
Authors: Berghen Nathalie , Dekoster Kaat , Marien Eyra , Dabin Jérémie , Hillen Amy , Wouters Jens , Deferme Jasmine , Vosselman Thibault , Tiest Eline , Lox Marleen , Vanoirbeek Jeroen , De Langhe Ellen , Bogaerts Ria , Hoylaerts Marc , Lories Rik , Vande Velde Greetje 
Published: November 2019

AbstractImplementation of in vivo high-resolution micro-computed tomography (μCT), a powerful tool for longitudinal analysis of murine lung disease models, is hampered by the lack of data on cumulative low-dose radiation effects on the investigated disease models. We aimed to measure radiation doses and effects of repeated μCT scans, to establish cumulative radiation levels and scan protocols without relevant toxicity.Lung metastasis, inflammation and fibrosis models and healthy mice were weekly scanned over one-month with μCT using high-resolution respiratory-gated 4D and expiration-weighted 3D protocols, comparing 5-times weekly scanned animals with controls. Radiation dose was measured by ionization chamber, optical fiberradioluminescence probe and thermoluminescent detectors in a mouse phantom. Dose effects were evaluated by in vivo μCT and bioluminescence imaging read-outs, gold standard endpoint evaluation and blood cell counts.Weekly exposure to 4D μCT, dose of 540-699 mGy/scan, did not alter lung metastatic load nor affected healthy mice. We found a disease-independent decrease in circulating blood platelets and lymphocytes after repeated 4D μCT. This effect was eliminated by optimizing a 3D protocol, reducing dose to 180-233 mGy/scan while maintaining equally high-quality images.We established μCT safety limits and protocols for weekly repeated whole-body acquisitions with proven safety for the overall health status, lung, disease process and host responses under investigation, including the radiosensitive blood cell compartment.
Integer linear programming outperforms simulated annealing for solving conservation planning problems
Authors: Schuster Richard , Hanson Jeffrey O. , Strimas-Mackey Matt , Bennett Joseph R. 
Published: November 2019

AbstractThe resources available for conserving biodiversity are limited, and so protected areas need to be established in places that will achieve objectives for minimal cost. Two of the main algorithms for solving systematic conservation planning problems are Simulated Annealing (SA) and Integer linear programming (ILP). Using a case study in British Columbia, Canada, we compare the cost-effectiveness and processing times of SA versus ILP using both commercial and open-source algorithms. Plans for expanding protected area systems based on ILP algorithms were 12 to 30% cheaper than plans using SA. The best ILP solver we examined was on average 1071 times faster than the SA algorithm tested. The performance advantages of ILP solvers were also observed when we aimed for spatially compact solutions by including a boundary penalty. One practical advantage of using ILP over SA is that the analysis does not require calibration, saving even more time. Given the performance of ILP solvers, they can be used to generate conservation plans in real-time during stakeholder meetings and can facilitate rapid sensitivity analysis, and contribute to a more transparent, inclusive, and defensible decision-making process.
Engrafted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Undergo Clonal Expansion In Vivo
Authors: El-Nachef Danny , Bugg Darrian , Beussman Kevin M. , Martinson Amy M. , Murry Charles E. , Sniadecki Nathan J. , Davis Jennifer 
Published: November 2019

AbstractPreclinical studies have suggested that transplanted human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) grafts expand due to proliferation. This knowledge came from cell cycle activity measurements that cannot discriminate between cytokinesis or DNA synthesis associated with hypertrophy. To refine our understanding of hPSC-CM cell therapy, we genetically engineered a cardiomyocyte-specific fluorescent barcoding system into an hPSC line. Since cellular progeny have the same color as parental hPSC-CMs, we could identify subsets of engrafted hPSC-CMs that clonally expanded, with the remainder being non-proliferative and hypertrophic.
Characterization of OTUB1 activation and inhibition by different E2 enzymes
Authors: Que Lauren T. , Morrow Marie E. , Wolberger Cynthia 
Published: November 2019

AbstractOTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another’s activity. OTUB1 can non-catalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2~Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. In order to assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the autoubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3.
CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing inYarrowia lipolytica
Authors: Yang Zhiliang , Edwards Harley , Xu Peng 
Published: November 2019

AbstractCRISPR-Cas9 has been widely adopted as the basic toolkit for precise genome-editing and engineering in various organisms. Alternative to Cas9, Cas12 or Cpf1 uses a simple crRNA as a guide and expands the protospacer adjacent motif (PAM) sequence to TTTN. This unique PAM sequence of Cpf1 may significantly increase the on-target editing efficiency due to lower chance of Cpf1 misreading the PAMs on a high GC genome. To demonstrate the utility of CRISPR-Cpf1, we have optimized the CRISPR-Cpf1 system and achieved high-editing efficiency for two counter-selectable markers in the industrially-relevant oleaginous yeastYarrowia lipolytica: arginine permease (93% for CAN1) and orotidine 5’-phosphate decarboxylase (∼96% for URA3). Both mutations were validated by indel mutation sequencing. For the first time, we further expanded this toolkit to edit three sulfur house-keeping genetic markers (40%–75% forMET2,MET6andMET25), which confers yeast distinct colony color changes due to the formation of PbS (lead sulfide) precipitates. Different from Cas9, we demonstrated that the crRNA transcribed from a standard type II RNA promoter was sufficient to guide Cpf1 endonuclease activity. Furthermore, modification of the crRNA with 3’ polyUs facilitates the faster maturation and folding of crRNA and improve the genome editing efficiency. We also achieved multiplexed genome editing, and the editing efficiency reached 75%–83% for duplex genomic targets (CAN1-URA3andCAN1-MET25) and 41.7% for triplex genomic targets (CAN1-URA3-MET25). Taken together, this work expands the genome-editing toolbox for oleaginous yeast species and may accelerate our ability to engineer oleaginous yeast for both biotechnological and biomedical applications.
DOWN state in the anterior cingulate and prelimbic areas in rats during immobility
Authors: Bragin Anatol , Li Lin , Masmanidis Sotiris C. , Engel Jerome 
Published: November 2019

AbstractBackgroundUP-DOWN state is considering as a dominant electrographic pattern during immobility and slow wave sleep. This study is focused on the analysis of spatial distribution and neuronal correlates of the DOWN state in ratsMethodsLocal field potentials and multiunit discharges were recorded bilaterally in the prefrontal cortex (PFC) and hippocampus of non-anesthetized, tethered rats (Sprague Dawley, males, weight 350-400g) with 256 channel, 4 shank silicon probes. We have focused our study on the analysis of the positive wave of slow oscillations (SOs), which is considered as the DOWN (silent) state of the UP-DOWN state in the anterior cingulate (AC), prelimbic (PL) areas of PFC and hippocampus during immobility.ResultsOur experiments showed that SOs occurred intermittently with a mean interval 1.4±0.8 (±SD) seconds. The SOs began with the DOWN state, and they were generated locally within AC or PL areas, or simultaneously in AC, PL and hippocampus bilaterally (generalized SOs). The DOWN state of local SOs in the AC was associated with a decreased rate of multiunit discharges. Similar waves in the PL area were associated with increased multiunit discharges. We observed high speed propagation of generalized SOs that occurred with 3-6ms delay within left and right PFC and less than 10ms delay between the PFC and CA1 area of hippocampus. All generalized SOs were associated with decreased multiunit discharges.ConclusionOur data support the hypothesis that neocortical networks are sufficient to generate focal SOs but the participation of external input is needed for occurrence of generalized SOs.
Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation
Authors: Bhattacharyya Moitrayee , Lee Young Kwang , Muratcioglu Serena , Qiu Baiyu , Nyayapati Priya , Schulman Howard , Groves Jay , Kuriyan John 
Published: November 2019

AbstractThe activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) depends on the balance between activating and inhibitory autophosphorylation (Thr 286 and Thr 305/306, respectively, in the human α isoform). Variation in the lengths of the flexible linkers that connect the kinase domains of CaMKII to a central oligomeric hub could alter transphosphorylation rates within a holoenzyme, thereby affecting the balance of autophosphorylation outcomes. Using a single-molecule assay for visualization of CaMKII phosphorylation on glass, we show that the balance of autophosphorylation is flipped between CaMKII-α and CaMKII-β, the two principal isoforms in the brain. CaMKII-α, with a ∼30 residue kinase-hub linker, readily acquires activating autophosphorylation, which we show is resistant to removal by phosphatases. CaMKII-β, with a ∼200 residue kinase-hub linker, is biased towards inhibitory autophosphorylation. Thus, the responsiveness of CaMKII to calcium signals can be tuned by varying the relative levels of the α and β isoforms.
Resolvin E1 derived from eicosapentaenoic acid prevents hyperinsulinemia and hyperglycemia in a host genetic manner
Authors: Pal Anandita , Al-Shaer Abrar E. , Guesdon William , Torres Maria J. , Armstrong Michael , Quinn Kevin , Davis Traci , Reisdorph Nichole , Neufer P. Darrell , Spangenburg Espen E. , Carroll Ian , Bazinet Richard P. , Halade Ganesh V. , Clària Joan , Shaikh Saame Raza 
Published: November 2019

ABSTRACTObjectiveEicosapentaenoic acid (EPA) has recently garnered strong attention given the success of the REDUCE-IT trial, which overturned previous conclusions on EPA and led to its FDA approval for lowering cardiovascular disease risk. Therefore, there is a need to study EPA for cardiometabolic risk factors. Here we focused on EPA’s preventative role on hyperglycemia and hyperinsulinemia.MethodsC57BL/6J male mice were fed a high fat diet in the absence or presence of pure EPA. Mass spectrometry was used to identify how EPA prevents hyperinsulinemia and hyperglycemia that drove subsequent experiments with resolvin E1 (RvE1) across inbred and outbred models.ResultsAdministration of EPA to C57BL/6J mice prevented obesity-induced glucose intolerance, hyperinsulinemia, and hyperglycemia. Supporting analyses of National Health and Nutrition Examination Survey data showed fasting glucose levels of obese adults were inversely related to EPA intake in a sex-specific manner. We next investigated how EPA improved murine hyperinsulinemia and hyperglycemia. Mass spectrometry revealed EPA overturned the obesity-driven decrement in the concentration of 18-hydroxyeicosapentaenoic acid (18-HEPE) in white adipose tissue and liver. Treatment of obese mice with RvE1, the immunoresolvant metabolite of 18-HEPE, reversed hyperinsulinemia and hyperglycemia through the G-protein coupled receptor ERV1/ChemR23. RvE1’s effects were not mediated by macrophage enrichment in white adipose tissue. Finally, we determined if the metabolic effects of RvE1 were dependent on host genetics. RvE1’s effects on hyperinsulinemia and hyperglycemia were divergent in diversity outbred mice that model human genetic variation. Secondary SNP analyses further revealed extensive genetic variation in human RvE1- and EPA- metabolizing genes.ConclusionsThe data suggest EPA prevents hyperinsulinemia and hyperglycemia through the endogenous bioactive metabolite RvE1 that activates ERV1/ChemR23. Importantly, the studies reveal host genetics are an overlooked but critical factor in the metabolic response to RvE1. These results underscore the need for personalized administration of EPA-derived RvE1 based on genetic/metabolic enzyme profiles.
Rewetting of Three Drained Peatlands Drives Congruent Compositional Changes in Pro- and Eukaryotic Soil Microbiomes Through Environmental Filtering
Authors: Weil Micha , Wang Haitao , Bengtsson Mia , Köhn Daniel , Günther Anke , Jurasinski Gerald , Couwenberg John , Negassa Wakene , Zak Dominik , Urich Tim 
Published: November 2019

AbstractDrained peatlands are significant sources of the greenhouse gas (GHG) carbon dioxide. Rewetting is a proven strategy to protect carbon stocks; however, it can lead to increased emissions of the potent GHG methane. The response to rewetting of soil microbiomes as drivers of these processes is poorly understood, as are biotic and abiotic factors that control community composition.We analyzed the pro- and eukaryotic microbiomes of three contrasting pairs of minerotrophic fens subject to decade-long drainage and subsequent rewetting. Also, abiotic soil properties including moisture, dissolved organic matter, methane fluxes and ecosystem respiration rates.The composition of the microbiomes was fen-type-specific, but all rewetted sites showed higher abundance of anaerobic taxa compared to drained sites. Based on multi-variate statistics and network analyses we identified soil moisture as major driver of community composition. Furthermore, salinity drove the separation between coastal and freshwater fen communities. Methanogens were more than tenfold more abundant in rewetted than in drained sites, while their abundance was lowest in the coastal fen, likely due to competition with sulfate reducers. The microbiome compositions were reflected in methane fluxes from the sites. Our results shed light on the factors that structure fen microbiomes via environmental filtering.
Barley RIC157 is involved in RACB-mediated susceptibility to powdery mildew
Authors: Engelhardt Stefan , Trutzenberg Adriana , Probst Katja , Hofer Johanna , McCollum Christopher , Kopischke Michaela , Hückelhoven Ralph 
Published: November 2019

AbstractSuccessful obligate pathogens benefit from host cellular processes. For the biotrophic ascomycete fungus Blumeria graminis f.sp. hordei (Bgh) it has been shown that barley RACB, a small monomeric G-protein (ROP, RHO of plants), is required for full susceptibility to fungal penetration. The susceptibility function of RACB probably lies in its role in cell polarisation, which may be co-opted by the pathogen for invasive ingrowth of its haustorium. However, the actual mechanism of how RACB supports the fungal penetration success is little understood. RIC proteins (ROP-Interactive and CRIB-(Cdc42/Rac Interactive Binding) motif-containing) are considered scaffold proteins which can interact directly with ROPs via a conserved CRIB motif. Here we describe a yet uncharacterised RIC protein, RIC157, which can interact directly with RACB in planta. We show that RIC157 undergoes a recruitment from the cytoplasm to the cell periphery in the presence of activated RACB. During fungal infection, RIC157 and activated RACB colocalise at the penetration site, particularly at the haustorial neck. In a RACB-dependent manner, transiently overexpressed RIC157 renders barley epidermal cells more susceptible to fungal penetration. This suggests that RIC157 promotes fungal penetration into barley epidermal cells via its function downstream of RACB.
TurboID-mediated proximity labelling of cytoophidium proteome inDrosophila
Authors: Zhang Bo , Zhang Yuanbing , Liu Ji-Long 
Published: November 2019

AbstractProximity-based biotinylation combined with mass spectrometry has emerged as a powerful approach to study protein interaction networks and protein subcellular compartmentation. However, low kinetics and the requirement of toxic chemicals limit the broad utilisation of current proximity labelling methods in living organisms. TurboID, the newly engineered promiscuous ligase, has been reported to label bait proteins effectively in various species. Here, we systematically demonstrated the application of TurboID-mediated biotinylation in a wide range of developmental stages and tissues, and we also verified the feasibility of TurboID-mediated labelling in desired cells via cell-type-specific GAL4 driver inDrosophila. Furthermore, using TurboID-mediated biotinylation coupled with mass spectrometry, we characterized the proximate proteome of the cytoophidium, a newly identified filamentous structure containing the metabolic enzyme CTP synthase (CTPS) inDrosophila. Our study demonstrates a referable tool and resource for research in subcellular compartments of metabolic enzymes in vivo.
TRA2A-induced upregulation of LINC00662 regulates blood-brain barrier permeability by affecting ELK4 mRNA stability in Alzheimer’s microenvironment
Authors: Liu Qianshuo , Zhu Lu , Liu Xiaobai , Zheng Jian , Liu Yunhui , Ruan Xuelei , Cao Shuo , Cai Heng , Li Zhen , Xue Yixue 
Published: November 2019

AbstractThe blood-brain barrier (BBB) has an important significance in maintenance and regulation of the neural microenvironment. The occurrence of BBB disruption is the pathological change of early Alzheimer’s disease (AD). RNA-binding proteins and long non-coding RNAs are closely related to the regulation of BBB permeability. Our study was performed to demonstrate TRA2A/LINC00662/ELK4 axis that regulates BBB permeability in AD microenvironment. In Aβ1-42-incubated microvascular endothelial cells (ECs) of BBB model in vitro, TRA2A and LINC00662 were enriched. TRA2A increased the stability of LINC00662 by binding with it. The knockdown of either TRA2A or LINC00662 decreased the BBB permeability via upregulating the levels of tight junction-related proteins. ELK4 was downregulated in BBB model in vitro in AD microenvironment. LINC00662 mediated the degradation of ELK4 mRNA by SMD pathway. The downregulated ELK4 increased the permeability of BTB by inducing the tight junction-related proteins. TRA2A/LINC00662/ELK4 axis is important in the regulation of BBB permeability in AD microenvironment, which would be a new molecular target for AD treatment.