bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a high-throughput γ-H2AX assay based on imaging flow cytometry
Authors: Lee Younghyun , Wang Qi , Shuryak Igor , Brenner David J. , Turner Helen C. 
Published: May 2019

AbstractBackgroundMeasurement of γ-H2AX foci formation in cells provides a sensitive and reliable method for quantitation of the radiation-induced DNA damage response. The objective of the present study was to develop a rapid, high-throughput γ-H2AX assay based on imaging flow cytometry (IFC) using the ImageStream®X Mk II (ISX MKII) platform to evaluate DNA double strand break (DSB) repair kinetics in human peripheral blood cells after exposure to ionizing irradiation.MethodsThe γ-H2AX protocol was optimized for small volumes (100 µl) of blood in Matrix™ 96-tube format and blood cell lymphocytes were identified and captured by ISX INSPIRE™ software and analyzed by Data Exploration and Analysis Software.ResultsPresented here are: 1) dose response curves based on γ-H2AX fluorescence intensity and foci number, 2) measurements of DNA repair kinetics up to 24 h after exposure to 4 Gy γ rays and, 3) a mathematical approach for modeling DNA DSB rejoining kinetics using two key parameters a) rate of γ-H2AX decay, and b) yield of residual unrepaired breaks.ConclusionsThe results indicate that the IFC-based γ-H2AX protocol may provide a practical, high-throughput and inexpensive platform for measurements of individual global DSB repair capacity and facilitate the prediction of precision medicine concepts.
Somatosensory-Motor Dysconnectivity Spans Multiple Transdiagnostic Dimensions of Psychopathology
Authors: Kebets Valeria , Holmes Avram J. , Orban Csaba , Tang Siyi , Li Jingwei , Sun Nanbo , Kong Ru , Poldrack Russell A. , Yeo B.T. Thomas 
Published: May 2019

AbstractBackgroundThere is considerable interest in a dimensional transdiagnostic approach to psychiatry. Most transdiagnostic studies have derived factors based only on clinical symptoms, which might miss possible links between psychopathology, cognitive processes and personality traits. Furthermore, many psychiatric studies focus on higher-order association brain networks, thus neglecting the potential influence of huge swaths of the brain.MethodsA multivariate data-driven approach (partial least squares; PLS) was utilized to identify latent components linking a large set of clinical, cognitive and personality measures to whole-brain resting-state functional connectivity (RSFC) patterns across 224 participants. The participants were either healthy (N=110) or diagnosed with bipolar disorder (N=40), attention-deficit/hyperactivity disorder (N=37), schizophrenia (N=29) or schizoaffective disorder (N=8). In contrast to traditional case-control analyses, the diagnostic categories were not utilized in the PLS analysis, but were helpful for interpreting the components.ResultsOur analyses revealed three latent components corresponding to general psychopathology, cognitive dysfunction and impulsivity. Each component was associated with a unique whole-brain RSFC signature and shared across all participants. The components were robust across multiple control analyses and replicated using independent task functional magnetic resonance imaging data from the same participants. Strikingly, all three components featured connectivity alterations within the somatosensory-motor network, and its connectivity with subcortical structures and cortical executive networks.ConclusionsWe identified three distinct dimensions with dissociable (but overlapping) whole-brain RSFC signatures across healthy individuals and individuals with psychiatric illness, providing potential intermediate phenotypes that span across diagnostic categories. Our results suggest expanding the focus of psychiatric neuroscience beyond higher-order brain networks.
Branched-Chain Amino Acid Metabolic Reprogramming Orchestrates Drug Resistance to EGFR Tyrosine Kinase Inhibitors
Authors: Wang Yuetong , Zhang Jian , Ren Shengxiang , Sun Dan , Huang Hsin-Yi , Wang Hua , Jin Yujuan , Li Fuming , Zheng Chao , Yang Liu , Deng Lei , Jiang Zhonglin , Jiang Tao , Han Xiangkun , Hou Shenda , Guo Chenchen , Li Fei , Gao Dong , Qin Jun , Gao Daming , Chen Luonan , Wong Kwok-Kin , Li Cheng , Hu Liang , Zhou Caicun , Ji Hongbin 
Published: May 2019

SUMMARYDrug resistance is a significant hindrance to effective cancer treatment. Although resistance mechanisms of epidermal growth factor receptor (EGFR)-mutant cancer cells to lethal EGFR tyrosine kinase inhibitors (TKI) treatment have been investigated intensively, how cancer cells orchestrate adaptive response under sublethal drug challenge remains largely unknown. Here we find that 2-hour sublethal TKI treatment elicits a transient drug-tolerant state in EGFR-mutant lung cancer cells. Continuous sublethal treatment reinforces this tolerance and eventually establishes long-term TKI resistance. This adaptive process involves H3K9 demethylation-mediated epigenetic upregulation of branched-chain amino acid aminotransferase 1 (BCAT1) and subsequent metabolic reprogramming, which promotes TKI resistance through attenuating reactive oxygen species (ROS) accumulation. Combinational treatment with TKI and ROS-inducing reagents overcomes this drug resistance in preclinical mouse models. Clinical information analyses support the correlation of BCAT1 expression with EGFR TKI response. Collectively, our findings reveal the importance of epigenetically regulated BCAT1-engaged metabolism reprogramming in TKI resistance in lung cancer.HIGHLIGHTSSublethal EGFR TKI treatment induces transient drug-tolerant state and long-term resistance in EGFR-mutant lung cancer cellsEpigenetically regulated BCAT1-mediated metabolic reprogramming orchestrates EGFR TKI-induced drug resistanceCombinational treatment with TKI and ROS-inducing agents overcomes the drug resistance induced by EGFR TKI treatment
A role for theSaccharomyces cerevisiaeABCF protein New1 during translation termination
Authors: Kasari Villu , Pochopien Agnieszka A. , Margus Tõnu , Murina Victoriia , Zhou Yang , Nissan Tracy , Graf Michael , Nováček Jiří , Atkinson Gemma C. , Johansson Marcus J.O. , Wilson Daniel N. , Hauryliuk Vasili 
Published: May 2019

ABSTRACTTranslation on the ribosome is controlled by numerous accessory proteins and translation factors. In the yeastSaccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related ABCF ATPase, New1, is encoded by a non-essential gene with a cold sensitivity and ribosome assembly defect knock-out phenotype. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is abona fideribosome assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed a combination of yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3’-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination.
Membrane insertion of soluble CLIC1 into active chloride channels is triggered by specific divalent cations
Authors: Varela Lorena , Hendry Alex C. , Medina-Carmona Encarnacion , Cantoni Diego , Ortega-Roldan Jose L. 
Published: May 2019

ABSTRACTThe CLIC family of proteins display the unique feature of altering their structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, can be found in the cytoplasm or in nuclear, ER and plasma membranes, with membrane overexpression linked to tumour proliferation. The molecular switch promoting CLIC1 membrane insertion has been related to environmental factors, but still remains unclear. Here, we use solution NMR studies to confirm that both the soluble and membrane bound forms are in the same oxidation state. Our data from fluorescence assays and chloride efflux assays indicate that Ca2+and Zn2+trigger association to the membrane into active chloride channels. We use fluorescence microscopy to confirm that an increase of the intracellular Ca2+leads to re-localisation of CLIC1 to both plasma and internal membranes. Finally, we show that soluble CLIC1 adopts an equilibrium of oligomeric species, and Ca2+/Zn2+mediated membrane insertion promotes the formation of a tetrameric assembly. Thus, our results identify Ca2+and Zn2+binding as the molecular switch promoting CLIC1 membrane insertion.SIGNIFICANCE STATEMENTCLIC1, a member of the CLIC family of proteins, is expressed as a soluble protein in cells but can insert in the membrane forming a chloride channel. This chloride channel form is upregulated in different types of cancers including glioblastoma and promote tumour invasiveness and metastasis. The factors promoting CLIC1 membrane insertion nor the mechanism of this process are yet understood. Here, we use a combination of solution NMR, biophysics and fluorescence microscopy to identify Ca2+and Zn2+binding as the switch to promote CLIC1 insertion into the membrane to form active chloride channels. We also provide a simple mechanism how such transition to the membrane occurs. Such understanding will enable subsequent studies on the structure of the chloride channel form and its inhibition.
Advancing functional genetics throughAgrobacterium-mediated insertional mutagenesis and CRISPR/Cas9 in the commensal and pathogenic yeastMalassezia furfur
Authors: Ianiri Giuseppe , Dagotto Gabriel , Heitman Joseph 
Published: May 2019

AbstractMalasseziaencompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals.Malasseziaspecies have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study we applied a recently-developedAgrobacterium tumefaciens-mediated transformation protocol to perform T-DNA random insertional mutagenesis inMalassezia furfur. A total of 767 transformants were screened after exposure to 10 different stresses, and the 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within the open reading frames of genes, within untranslated regions, and in intergenic regions. Some T-DNA insertions generated chromosomal rearrangements, and others could not be characterized. To validate the findings of the forward genetic screen, a novel CRISPR/Cas9 system was developed to generate targeted deletion mutants for 2 genes identified in the screen:CDC55andPDR10. This system is based on co-transformation ofM. furfurmediated byA. tumefaciensto deliver both aCAS9-gRNA construct that induces double-strand DNA breaks, and a gene replacement allele that serves as a homology directed repair template. Targeted deletion mutants for bothCDC55andPDR10were readily generated with this method. This study demonstrates the feasibility and reliability ofA. tumefaciens-mediated transformation to aid in the identification of gene functions inM. furfurthrough both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.
Role of coiled-coil registry shifts in activation of human Bicaudal D2 for dynein recruitment upon cargo-binding
Authors: Noell Crystal R. , Loh Jia Ying , Debler Erik W. , Loftus Kyle M. , Cui Heying , Russ Blaine B. , Goyal Puja , Solmaz Sozanne R. 
Published: May 2019

SUMMARYDynein adaptors such as Bicaudal D2 (BicD2) recognize cargo for dynein-dependent transport. BicD2-dependent transport pathways are important for brain and muscle development. Cargo-bound adaptors are required to activate dynein for processive transport, but the mechanism of action is elusive. Here, we report the structure of the cargo-binding domain of human BicD2 that forms a dimeric coiled-coil with homotypic registry, in which both helices are aligned. To investigate if BicD2 can switch to an asymmetric registry, where a portion of one helix is vertically shifted, we performed molecular dynamics simulations. Both registry types are stabilized by distinct conformations of F743. For the F743I variant, which increases dynein recruitment in theDrosophilahomolog, and for the human R747C variant, which causes spinal muscular atrophy, spontaneous coiled-coil registry shifts are observed, which may cause the BicD2-hyperactivation phenotype and disease. We propose that a registry shift upon cargo-binding activates auto-inhibited BicD2 for dynein recruitment.HighlightsStable, bona fide BicD2 coiled-coils with distinct registries can be formed.We provide evidence that a human disease mutation causes a coiled-coil registry shift.A coiled-coil registry shift could relieve BicD2-autoinhibition upon cargo-binding.The ability to undergo registry shifts may be an inherent property of coiled-coils.In BriefOur results support that stable coiled-coils of BicD2 with distinct registries can be formed, and suggest a molecular mechanism for such registry switches. We provide evidence that disease-causing mutations in coiled-coils may alter the equilibrium between registry-shifted conformers, which we propose as a general mechanism of pathogenesis for coiled-coils.Graphical Abstract
Cryo-EM structure of the ClpXP protein degradation machinery
Authors: Gatsogiannis C , Balogh D , Merino F , Sieber SA , Raunser S 
Published: May 2019

AbstractThe ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and eukaryotes. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the first high-resolution cryo-EM structure of ClpXP fromListeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXP’s complex mode of action and provides a structural framework for future pharmacological applications.
K+in a Kv1.2 Channel Pore: Hydration, Selectivity, and the Role of a Conserved Threonine
Authors: Kariev A. M. , Green M. E 
Published: May 2019

ABSTRACTQuantum calculations describing transport of K+through a Kv1.2 channel cavity, plus the lower half of the selectivity filter (SF), show hydration in the pore and cosolvation by threonine at the entrance to the SF. Comparison to calculations on Na+ions gives the probable selectivity mechanism. A single K+ion is calculated at five positions in its course through the cavity, and two ions calculated at three positions at the entrance to the SF. Three Na+pairs of ions were also calculated, and one shows how an ion is trapped asymmetrically, tightly held by two threonine −OH, and with a water tightly bound ahead of it, so that overall it has a major barrier to advancing, while K+advances with minimal barriers. In the cavity below the SF, the ion passes in a hydrated state through pore water, between the intracellular gate and the SF, until it is cosolvated by the threonines at the selectivity filter entrance. These calculations show how the ion associates with the water, and enters the SF. A characteristic arrangement of four water molecules adjacent to the SF in the KcsA channel, shown in earlier work, is now found in Kv1.2. A single ion passing through the channel cavity is found to have an energy minimum within 1 Å of the K+ion position in the 3Lut pdb structure of this channel. Properties (e.g. dipole moment) of the system are calculated. Charge transfer to the ion produces K+charge 0.74 ≤ q(ion) ≤ 0.87e, in different conditions. The calculations of pairs of Na+and K+ions at the SF entrance include the threonine, valine, and glycine of the conserved SF TVGYG sequence. The Na+/K+difference shows a reason for the conservation of the threonine in producing selectivity, as the –OH groups trap Na+but not K+.STATEMENT OF SIGNIFICANCEPotassium channels are found in all cells, and have a characteristic selectivity filter that blocks the passage of Na+while allowing K+to pass. These channels are implicated in many diseases. We use quantum calculations to show how the K+ion passes from the intracellular gate of the channel, entering the channel pore, to the selectivity filter at the extracellular end of the channel; at the selectivity filter, we use comparable calculations of K+and Na+to show how the channel selects K+over Na+, as well as the probable reason for the conservation of a key residue (threonine) at the base of the selectivity filter. We find properties (e.g., charge transfer, bond order) that require quantum calculations.
Rad9/53BP1 promotes crossover recombination DNA repair by limiting the Sgs1 and Mph1 helicases
Authors: Ferrari Matteo , Rawal Chetan C. , Lodovichi Samuele , Pellicioli Achille 
Published: May 2019

AbstractA DNA double strand break (DSB) is primed for homologous recombination (HR) repair through the nucleolytic processing (resection) of its ends, leading to the formation of a 3′ single-stranded DNA (ssDNA). Generation of the ssDNA is accompanied by the loading of several repair factors, including the ssDNA binding factor RPA and the recombinase Rad51. Then, depending upon the availability and location of a homologous sequence, different types of HR mechanisms can occur. Inefficient or slow HR repair results in the activation of the DNA damage checkpoint (DDC)1. In budding yeast, the 53BP1 ortholog Rad9 acts as a scaffold, mediating signal from upstream kinases Mec1 and Tel1 (ATR and ATM in human) to downstream effectors kinases Rad53 and Chk1 (CHK2 and CHK1 in human). In addition to its role in DDC, Rad9 limits DSB resection 2. Remarkably, this function is conserved in 53BP1, also being implicated in cancer biology in human cells 3,4.Here we show that Rad9 limits the recruitment of the helicases Sgs1 and Mph1 on to a DSB, promoting Rad51-dependent recombination with long track DNA conversions, crossovers and break-induced replication (BIR). This regulation couples the DDC with the choice and effectiveness of HR sub-pathways, and might be critical to limit genome instability with implication for cancer research.
Mental Chronometry in the Pocket? Timing Accuracy of Web Applications on Touchscreen and Keyboard Devices
Authors: Pronk Thomas , Wiers Reinout W. , Molenkamp Bert , Murre Jaap 
Published: May 2019

AbstractWeb applications can implement procedures for studying the speed of mental processes (mental chronometry). As web applications, these procedures can be administered via web-browsers on most commodity desktops, laptops, smartphones, and tablets. This approach to conducting mental chronometry offers various opportunities, such as increased scale, ease of data collection, and access to specific samples. However, validity and reliability may be threatened due to web applications on commodity devices having less accurate timing than specialized software and hardware. We have examined how accurately web applications time stimuli and register response times on commodity touchscreen and keyboard devices running a range of popular web-browsers. Additionally, we have explored the accuracy of a range of technical innovations for timing stimuli, presenting stimuli, and estimating stimulus duration. Results offer some guidelines as to what kind of methods may be most accurate, and what kind of mental chronometry paradigms may suitably be administered via web applications. In controlled circumstances, as can be realized in a lab setting, very accurate stimulus timing and moderately accurate Reaction Time (RT) measurements could be achieved on both touchscreen and keyboard devices. In uncontrolled circumstances, as may be encountered online, short stimulus durations (of up to 100 ms) may be inaccurate, and RT measurement may be affected by the occurrence of bi-modally distributed RT overestimations.
The effect of light vs dark coat color on thermal status in Labrador Retriever dogs
Authors: Neander Caitlin , Baker Janice , Kelsey Kathleen , Feugang Jean , Perry Erin 
Published: May 2019

AbstractAlthough dark coat color in dogs has been theorized as a risk factor for thermal stress, there is little evidence in the scientific literature to support that position. We utilized 16 non-conditioned Labradors (8 black and 8 yellow) in a three-phase test to examine effects of coat color on thermal status of the dog. Rectal, gastrointestinal (GI), surface temperature, and respiration rate measured in breaths per minute (bpm), were collected prior to (Baseline — phase 1) and immediately after a controlled 30-minute walk in an open-air environment on a sunny day (Sunlight — phase 2). Follow up measurements were taken 15 minutes after walking (Cool down – phase 3) to determine post-exposure return to baseline. No effect of coat color was measured for rectal, gastrointestinal or surface temperature, or respiration (P > 0.05) in dogs following their 30-minute walk. Temperatures increased similarly across both coat colors (rectal 1.88 °C and 1.83 °C; GI 1.89 °C and 1.94 °C; eye 1.89 °C and 1.94 °C; abdominal 2.93 °C and 2.35 °C) for black and yellow dogs respectively during the sunlight phase (P > 0.05). All temperatures and respiration rates decreased similarly across coat colors for rectal (0.9°C and 1.0°C) and GI (1.5 °C and 1.3°C) for black and yellow dogs respectively (P > 0.05). Similarly, sex did not impact thermal status across rectal, gastrointestinal or surface temperature or respiration rates measured (P > 0.05). These data contradict the commonly held theory that dogs with darker coat color may experience a greater thermal change when exposed to direct sunlight compared to dogs with a lighter coat color.
A spatial analysis of childhood cancer and industrial air pollution in a metropolitan area of Colombia
Authors: Valbuena-Garcia Ana Maria , Rodriguez-Villamizar Laura Andrea , Uribe Pérez Claudia Janeth , Moreno Corzo Feisar Enrique , Ortiz Martinez Rafael Gustavo 
Published: May 2019

AbstractBackgroundAir pollutants are considered carcinogenic to humans. In some European countries, an association with childhood cancer in children has been established. This phenomenon has not been addressed in Latin America, despite the spatial variability of air pollutants that may limit the extrapolation of the results to other geographical areas.ObjectiveTo conduct a spatial analysis of the relationship between childhood cancer and air pollution from industrial sources in a metropolitan area of Colombia.MethodsIncident cases of childhood cancers were obtained from the Population-based Cancer Registry of the Bucaramanga Metropolitan Area (2000-2015). Local and focused cluster tests were used for the detection of spatial clusters and the Poisson multivariable model was used to evaluate the combined effects of spatial variables.ResultsThe Kulldorff’s focused test found a significant spatial cluster (p=0.001) around one industrial agglomerate and the multivariable model results suggests that the distance effect is modified by the directional effect of the wind.ConclusionA spatial cluster of incident cases of childhood cancer occurred in the municipality of Bucaramanga. Our finding supports the hypothesis that childhood cancer might be related with industrial air pollution exposure in a Latin American city.