bluuebunny/crossref_metadata_2025 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

The 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

Abstract

Proteins 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

Implementation 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

The 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

Preclinical 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.
Robust foreground detection in somatic copy number data
Authors: Deshpande Aditya , Walradt Trent , Hu Ya , Koren Amnon , Imielinski Marcin
Published: November 2019

Sensitive detection of somatic copy number alterations (SCNA) in cancer genomes is confounded by “waviness” in read depth data. We present dryclean, a signal processing algorithm to optimize SCNA detection in whole genome (WGS) and targeted sequencing platforms through foreground detection and background subtraction of read depth data. Application of dryclean to WGS demonstrates that WGS waviness is driven by replication timing. Re-analysis of thousands of tumor profiles reveals that dryclean provides superior detection of biologically relevant SCNAs relative to state-of-the-art algorithms. Applied to in silico tumor dilutions, dryclean improves the sensitivity of relapse detection 10-fold relative to current standards. dryclean is available as an R package in the GitHub repository https://github.com/mskilab/dryclean
Characterization of OTUB1 activation and inhibition by different E2 enzymes
Authors: Que Lauren T. , Morrow Marie E. , Wolberger Cynthia
Published: November 2019

OTUB1 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

CRISPR-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

Background

UP-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 rats

Methods

Local 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.

Results

Our 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.

Conclusion

Our 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

The 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

Objective

Eicosapentaenoic 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.

Methods

C57BL/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.

Results

Administration 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.

Conclusions

The 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

Drained 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

Successful 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

Proximity-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

The 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.
Unoccupied aerial system enabled functional modeling of maize (Zea mays L.) height reveals dynamic expression of loci associated to temporal growth
Authors: Anderson Steven L. , Murray Seth C. , Chen Yuanyuan , Malambo Lonesome , Chang Anjin , Popescu Sorin , Cope Dale , Jung Jinha
Published: November 2019

Unoccupied aerial systems (UAS) were used to phenotype growth trajectories of inbred maize populations under field conditions. Three recombinant inbred line populations were surveyed on a weekly basis collecting RGB images across two irrigation regimens (irrigated and non-irrigated/rain fed). Plant height, estimated by the 95th percentile (P95) height from UAS generated 3D point clouds, exceeded 70% correlation to manual ground truth measurements and 51% of experimental variance was explained by genetics. The Weibull sigmoidal function accurately modeled plant growth (R2: >99%; RMSE: < 4 cm) from P95 genetic means. The mean asymptote was strongly correlated (r2=0.66-0.77) with terminal plant height. Maximum absolute growth rates (mm d-1) were weakly correlated to height and flowering time. The average inflection point ranged from 57 to 60 days after sowing (DAS) and was correlated with flowering time (r2=0.45-0.68). Functional growth parameters (asymptote, inflection point, growth rate) alone identified 34 genetic loci, each explaining 3 to 15% of total genetic variation. Plant height was estimated at one-day intervals to 85 DAS, identifying 58 unique temporal quantitative trait loci (QTL) locations. Genomic hotspots on chromosome 1 and 3 indicated chromosomal regions associated with functional growth trajectories influencing flowering time, growth rate, and terminal growth. Temporal QTL demonstrated unique dynamic expression patterns not observable previously, no QTL were significantly expressed throughout the entire growing season. UAS technologies improved phenotypic selection accuracy and permitted monitoring traits on a temporal scale previously infeasible using manual measurements, furthering understanding of crop development and biological trajectories.

Author summary

Unoccupied aerial systems (UAS) now can provide high throughput phenotyping to functionally model plant growth and explore genetic loci underlying temporal expression of dynamic phenotypes, specifically plant height. Efficient integration of temporal phenotyping via UAS, will improve the scientific understanding of dynamic, quantitative traits and developmental trajectories of important agronomic crops, leading to new understanding of plant biology. Here we present, for the first time, the dynamic nature of quantitative trait loci (QTL) over time under field conditions. To our knowledge, this is first empirical study to expand beyond selective developmental time points, evaluating functional and temporal QTL expression in maize (Zea mays L.) throughout a growing season within a field-based environment.
Native Hydrogen/Deuterium Exchange Mass Spectrometry of Structured DNA Oligonucleotides
Authors: Largy Eric , Gabelica Valérie
Published: November 2019

Although solution hydrogen-deuterium exchange mass spectrometry (HDX/MS) is well-established for the analysis of the structure and dynamics of proteins, it is currently not exploited for nucleic acids. Here we used DNA G-quadruplex structures as model systems to demonstrate that DNA oligonucleotides are amenable to in-solution HDX/MS in native conditions. In trimethylammonium acetate solutions and in soft source conditions, the protonated phosphate groups are fully back-exchanged in the source, while the exchanged nucleobases remain labeled without detectable back-exchange. As a result, the exchange rates depend strongly on the secondary structure (hydrogen bonding status) of the oligonucleotides, but neither on their charge state nor on the presence of non-specific adducts. We show that native mass spectrometry methods can measure these exchange rates on the second to the day time scale with high precision. Such combination of HDX with native MS opens promising avenues for the analysis of the structural and biophysical properties of oligonucleotides and their complexes.
Ubiquitin transfer by a RING E3 ligase occurs from a closed E2~Ub conformation
Authors: Branigan Emma , Penedo J. Carlos , Hay Ronald T.
Published: November 2019

Ubiquitination is a eukaryotic post-translational modification that modulates a host of cellular processes1. Modification is mediated by an E1 activating enzyme (E1), an E2 conjugating enzyme (E2) and an E3 ligase (E3). The E1 catalyses formation of a highly reactive thioester linked conjugate between ubiquitin and E2 (E2~Ub)2. The largest class of ubiquitin E3 ligases, which is represented by RING E3s, bind both substrate and E2~Ub and facilitate transfer of ubiquitin from the E2 to substrate. Based on extensive structural analysis3–5 it has been proposed that RING E3s prime the E2~Ub conjugate for catalysis by locking it into a “closed” conformation where ubiquitin is folded back onto the E2 exposing the restrained thioester bond to attack by a substrate nucleophile. However the proposal that the RING dependent closed conformation of E2~Ub represents the active form that mediates ubiquitin transfer is a model that has yet to be experimentally tested. Here we use single molecule Förster Resonance Energy Transfer (smFRET) to test this hypothesis and demonstrate that ubiquitin is transferred from the closed conformation during an E3 catalysed reaction. Using Ubc13 as an E2, we designed a FRET labelled E2~Ub conjugate, which distinguishes between closed and alternative conformations. Firstly, we defined the high FRET state as the closed conformation using a stable isopeptide linked E2~Ub conjugate, while the low FRET state represents more open conformations. Secondly, we developed a real-time smFRET assay to monitor RING E3 catalysed ubiquitination with a thioester linked E2~Ub conjugate and determined the catalytically active conformation. Our results demonstrate that the reaction proceeds from the high FRET or closed conformation and confirm the hypothesis that the closed conformation is the active form of the conjugate. These findings are not only relevant to RING E3 catalysed ubiquitination but are also broadly applicable to E3 mediated ligation of other ubiquitin-like proteins (Ubls) to substrates.
Developmental trajectory of pre-hematopoietic stem cell formation from endothelium
Authors: Zhu Qin , Gao Peng , Tober Joanna , Bennett Laura , Chen Changya , Uzun Yasin , Li Yan , Mumau Melanie , Yu Wenbao , He Bing , Speck Nancy A. , Tan Kai
Published: November 2019

Hematopoietic stem and progenitor cells (HSPCs) differentiate from hemogenic endothelial (HE) cells through an endothelial to hematopoietic cell transition (EHT). Newly formed HSPCs accumulate in intra-arterial clusters (IACs) before colonizing the fetal liver. To examine the cell and molecular transitions during the EHT, and the heterogeneity of HSPCs within IACs, we profiled ∼37,000 cells from the caudal arteries of embryonic day 9.5 (E9.5) to E11.5 mouse embryos by single-cell transcriptome and chromatin accessibility sequencing. We identified an intermediate developmental stage prior to HE that we termed pre-HE, characterized by increased accessibility of chromatin enriched for SOX, FOX, GATA, and SMAD motifs. A developmental bottleneck separates pre-HE from HE, with RUNX1 dosage regulating the efficiency of the pre-HE to HE transition. Distinct developmental trajectories within IAC cells result in two populations of CD45+HSPCs; an initial wave of lympho-myeloid-biased progenitors, followed by precursors of hematopoietic stem cells (pre-HSCs).
Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling
Authors: You Inchul , Erickson Emily C. , Donovan Katherine A. , Eleuteri Nicholas A. , Fischer Eric S. , Gray Nathanael S. , Toker Alex
Published: November 2019

The PI3K/AKT signaling cascade is one of the most commonly dysregulated pathways in cancer, with over half of tumors exhibiting aberrant AKT activation. Although potent small molecule AKT inhibitors have entered clinical trials, robust and durable therapeutic responses have not been observed. As an alternative strategy to target AKT, we report the development of INY-03-041, a pan-AKT degrader consisting of the ATP-competitive AKT inhibitor GDC-0068 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon (CRBN). INY-03-041 induced potent degradation of all three AKT isoforms and displayed enhanced anti-proliferative effects relative to GDC-0068. Notably, INY-03-041 promoted sustained AKT degradation and inhibition of downstream signaling effects for up to 96 hours, even after compound washout. Our findings indicate that AKT degradation may confer prolonged pharmacological effects compared to inhibition, and highlight the potential advantages of AKT-targeted degradation.
MEIS1 down-regulation by MYC mediates prostate cancer development through elevated HOXB13 expression and AR activity
Authors: Whitlock Nichelle C. , Trostel Shana Y. , Wilkinson Scott , Terrigino Nicholas T. , Hennigan S. Thomas , Lake Ross , Carrabba Nicole V. , Atway Rayann , Walton Elizabeth D. , Gryder Berkley E. , Capaldo Brian J. , Ye Huihui , Sowalsky Adam G.
Published: November 2019

Localized prostate cancer develops very slowly in most men, with the androgen receptor (AR) and MYC transcription factors amongst the most well-characterized drivers of prostate tumorigenesis. Canonically, MYC up-regulation in luminal prostate cancer cells functions to oppose the terminally differentiating effects of AR. However, the effects of MYC up-regulation are pleiotropic and inconsistent with a poorly proliferative phenotype. Here we show that increased MYC expression and activity are associated with the down-regulation ofMEIS1, a HOX-family transcription factor. Using RNA-seq to profile a series of human prostate cancer specimens laser capture microdissected on the basis of MYC immunohistochemistry, MYC activity andMEIS1expression were inversely correlated. Knockdown ofMYCexpression in prostate cancer cells increased expression ofMEIS1and increased occupancy of MYC at theMEIS1locus. Finally, we show in laser capture microdissected human prostate cancer samples and the prostate TCGA cohort thatMEIS1expression is inversely proportional to AR activity as well asHOXB13, a known interacting protein of both AR and MEIS1. Collectively, our data demonstrate that elevated MYC in a subset of primary prostate cancers functions in a negative role in regulatingMEIS1expression, and that this down-regulation may contribute to MYC-driven development and progression.
Targeting RNA Polymerase I transcription synergises with TOP1 inhibition in potentiating the DNA damage response in high-grade serous ovarian cancer
Authors: Yan Shunfei , Madhamshettiwar Piyush B. , Simpson Kaylene J. , Ellis Sarah , Kang Jian , Cullinane Carleen , Sheppard Karen E. , Hannan Katherine M. , Hannan Ross D. , Sanij Elaine , Pearson Richard B. , Chan Keefe T.
Published: November 2019

Limited effective therapeutic options are available for patients with recurrent high-grade serous carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. We have shown efficacy in poly-ADP ribose polymerase (PARP) inhibitor-resistant HGSC for the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 through its ability to activate a nucleolar-associated DNA damage response (DDR). Here, we screen the protein-coding genome to identify potential targets whose inhibition enhances the efficacy of CX-5461. We identify a network of cooperating inhibitory interactions, including components of homologous recombination (HR) DNA repair and DNA topoisomerase 1 (TOP1). We highlight that CX-5461 combined with topotecan, a TOP1 inhibitor used as salvage therapy in HGSC, induces robust cell cycle arrest and cell death in a panel of HR-proficient HGSC cell lines. The combination potentiates a nucleolar-associated DDR via recruitment of phosphorylated replication protein A (RPA) and ataxia telangiectasia and Rad3 related protein (ATR). CX-5461 plus low-dose topotecan cooperate to potently inhibit xenograft tumour growth, indicating the potential for this strategy to improve salvage therapeutic regimens to treat HGSC.
An early warning system to identify house price bubbles
Authors: Hagemann Daniel , Wohlmann Monika
Published: November 2019

Purpose

The global financial and economic crisis resulting from the US housing crisis has shown that house prices can have far-reaching consequences for the real economy. For macroprudential supervision, it is, therefore, necessary to identify house price bubbles at an early stage to counteract speculative price developments and to ensure financial market stability. This paper aims to develop an early warning system to signal speculative price bubbles.

Design/methodology/approach

The results of explosivity tests are used to identify periods of excessive price increases in 18 industrialized countries. The early warning system is then based on a logit and an ordered logit regression, in which monetary, macroeconomic, regulatory, demographic and private factors are used as explanatory variables.

Findings

The empirical results show that monetary developments have the highest explanatory power for the existence of house price bubbles. Further, the study reveals currently emerging house price bubbles in Norway, Sweden and Switzerland.

Practical implications

The results implicate a new global housing boom, particularly in those countries that did not experience a major price correction during the global financial crisis.

Originality/value

The ordered logit model is an advanced approach that offers the advantage of being able to differentiate between different phases of a house price bubble, thereby allowing a multi-level assessment of the risk of speculative excesses in the housing market.
The search and matching process in the housing market
Authors: Lisi Gaetano
Published: November 2019

Purpose

This paper aims to study the relationship between the rental and selling prices, a very important topic that forms the fundamentals of real estate markets.

Design/methodology/approach

This theoretical paper makes use of a search and matching model of the housing market. The search and matching models are the benchmark models of the “matching” markets, such as the labour market and the housing market, where trade is a decentralised, uncoordinated and time-consuming economic activity.

Findings

Unlike the previous related literature, where this relation is usually analysed in the context of the present value equation, this paper shows the existence of a “dual” relation between rental and selling prices as follows: one in the homeownership market and another one in the rental market. This “dual” relation connects the rental and homeownership markets and allows to get equilibrium in both markets with positive house prices.

Research limitations/implications

Several topics could be deepened for making the paper richer and more interesting, although at the cost of much more mathematics. First of all, the introduction of specific functional forms for both the rent function and the sale price function, so as to calculate both the elasticity of rent with respect to sale price and the elasticity of sale price with respect to rent. In this way, it would be possible to understand how each market (rental and homeownership) reacts to shock and policies that affect the other market.

Practical implications

In general, this framework could help policymakers to design housing policy reforms that take into consideration the effects on both markets. Indeed, some policies could have positive effects on rental markets but perverse effects on homeownership markets and vice versa.

Originality/value

None of the existing and related works of research have considered how to take advantage of the search and matching approach to derive both a “rent function” and a “sale price function” that connect closely the rental and homeownership markets.
Sharp Force Homicide in Denmark 1992–2016
Authors: Thomsen Asser H. , Hougen Hans Petter , Villesen Palle , Brink Ole , Leth Peter M.
Published: November 2019

Sharp force trauma is a common homicide method. The weapon is typically a knife, which is easily accessible and does not require special skills. We have analyzed all 471 sharp force homicides in Denmark during 1992–2016 with special focus on aspects that are relevant to forensic pathologists, including the distribution of wounds and organ injuries. Most homicides were committed inside with a kitchen knife. The front left thorax was the most common area to be affected by sharp force trauma. In 18.9% of the victims, there was only one sharp injury, the majority on the thorax. The most common trajectory for stab wounds was directly posterior with no deviation to the sides or up/down followed by directly anterior. The heart (including pericardium) and lungs (including hemo‐ and pneumothorax) had injuries in more than 75% of the victims. 67% of victims were males. Female victims had more sharp force injuries and defense wounds than male victims. Most females were killed in domestic homicides (73.7%), most commonly in partner killings (56.4%). In contrast, many male victims were killed in a setting of nightlife/intoxication (34.0%) most by a friend/acquaintance delivering a few stab wounds. The results clearly show strong sex differences in both victims and offenders. This could be useful for shaping policies and public opinion, and as a route for understanding the developments in interpersonal violence. In the narrow setting of death investigation, our results will provide an evidence‐based approach to understanding the injury patterns in sharp force homicide.
The formation of hybrid complexes between isoenzymes of glyceraldehyde‐3‐phosphate dehydrogenase regulates its aggregation state, the glycolytic activity and sphingolipid status in Saccharomyces cerevisiae
Authors: Randez‐Gil Francisca , Sánchez‐Adriá Isabel E. , Estruch Francisco , Prieto Jose A.
Published: November 2019

The glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has been traditionally considered a housekeeping protein involved in energy generation. However, evidence indicates that GAPDHs from different origins are tightly regulated and that this regulation may be on the basis of glycolysis‐related and glycolysis‐unrelated functions. In Saccharomyces cerevisiae, Tdh3 is the main GAPDH, although two other isoenzymes encoded by TDH1 and TDH2 have been identified. Like other GAPDHs, Tdh3 exists predominantly as a tetramer, although dimeric and monomeric forms have also been isolated. Mechanisms of Tdh3 regulation may thus imply changes in its oligomeric state or be based in its ability to interact with Tdh1 and/or Tdh2 to form hybrid complexes. However, no direct evidence of the existence of these interactions has been provided and the exact function of Tdh1,2 is unknown. Here, we show that Tdh1,2 immunopurified with a GFP‐tagged version of Tdh3 and that lack of this interaction stimulates the Tdh3’s aggregation. Furthermore, we found that the combined knockout of TDH1 and TDH2 promotes the loss of cell’s viability and increases the growing rate, glucose consumption and CO2 production, suggesting a higher glycolytic flux in the mutant cells. Consistent with this, the tdh3 strain, which displays impaired in vitro GAPDH activity, exhibited the opposite phenotypes. Quite remarkably, tdh1 tdh2 mutant cells show increased sensitivity to aureobasidin A, an inhibitor of the inositolphosphoryl ceramide synthase, while cells lacking Tdh3 showed improved tolerance. The results are in agreement with a link between glycolysis and sphingolipid (SLs) metabolism. Engineering Tdh activity could be thus exploited to alter the SLs status with consequences in different aspects of yeast biotechnology.
Estimating Potential Climate Change Effects on the Upper Neuse Watershed Water Balance Using the SWAT Model
Authors: Ercan Mehmet B. , Maghami Iman , Bowes Benjamin D. , Morsy Mohamed M. , Goodall Jonathan L.
Published: November 2019

Climate change poses water resource challenges for many already water stressed watersheds throughout the world. One such watershed is the Upper Neuse Watershed in North Carolina, which serves as a water source for the large and growing Research Triangle Park region. The aim of this study was to quantify possible changes in the watershed’s water balance due to climate change. To do this, we used the Soil and Water Assessment Tool (SWAT) model forced with different climate scenarios for baseline, mid‐century, and end‐century time periods using five different downscaled General Circulation Models. Before running these scenarios, the SWAT model was calibrated and validated using daily streamflow records within the watershed. The study results suggest that, even under a mitigation scenario, precipitation will increase by 7.7% from the baseline to mid‐century time period and by 9.8% between the baseline and end‐century time period. Over the same periods, evapotranspiration (ET) would decrease by 5.5 and 7.6%, water yield would increase by 25.1% and 33.2%, and soil water would increase by 1.4% and 1.9%. Perhaps most importantly, the model results show, under a high emission scenario, large seasonal differences with ET estimated to decrease by up to 42% and water yield to increase by up to 157% in late summer and fall. Planning for the wetter predicted future and corresponding seasonal changes will be critical for mitigating the impacts of climate change on water resources.
Cross‐reactivities of non‐homologous allergens
Authors: Bublin Merima , Breiteneder Heimo
Published: November 2019

Allergen cross‐reactivities occur when IgE antibodies, originally raised against a specific allergen, bind to identical or highly similar surface areas of another related allergen. It is a commonly held view that cross‐reactivity requires more than 70% sequence identity, while proteins that share <50% sequence identity are rarely cross‐reactive. This also implies that cross‐reactive proteins have a similar 3D fold and belong to the same protein family. At first, the existence of cross‐reactivity between non‐homologous allergens was not expected because it was contrary to the above described concepts. Now, several lines of evidence demonstrate that IgE cross‐reactivity also exists between unrelated allergens. In this Editorial, we aim to summarize the existing literature on IgE cross‐reactivity between non‐homologous allergens.
Unpasteurised maternal breast milk is positively associated with growth outcomes in extremely preterm infants
Authors: Lund Anna‐My , Löfqvist Chatarina , Pivodic Aldina , Lundgren Pia , Hård Anna‐Lena , Hellström Ann , Hansen‐Pupp Ingrid
Published: November 2019

Aim

Extrauterine growth restriction is common among extremely preterm infants. We explored whether intake of unpasteurised maternal milk (MM) and pasteurised donor milk (DM) was associated with longitudinal growth outcomes and neonatal morbidities in extremely preterm infants.

Methods

Observational study of 90 preterm infants born between 2013 and 2015 in Gothenburg, Sweden. Data were prospectively collected on nutritional and breast milk intakes during the first 28 days.

Results

Ninety infants (39 girls and 51 boys) with a median gestational age of 25.3 (22.7‐27.9) weeks were evaluated. MM intake (mL/kg/d) correlated positively with almost all z‐scores for weight, length and head circumference at 28 postnatal days and at postmenstrual age (PMA) 32 and 36 weeks. After multivariable adjustment, MM intake and weight z‐score at 28 postnatal days and at PMA 32 and 36 weeks remained significantly associated. Infants consuming ≥80% MM had more favourable weight z‐scores at PMA 32 and 36 weeks. Intake of DM did not correlate with any growth outcomes. Infants without retinopathy of prematurity had a significantly higher intake of MM (mL/kg/d).

Conclusion

Unpasteurised MM was positively associated with longitudinal growth outcomes. Motivating mothers to provide their infants with their own milk after preterm birth should be emphasised.