bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inclusion of dimethyl sulfoxide- dissolved protease inhibitor cocktail in the lysis buffer cuts down the number of proteins resolved by two dimensional gel electrophoresis
Authors: Mahesh P.P , Mundayoor Sathish 
Published: May 2020

AbstractTwo dimensional gel electrophoresis (2DE) resolves a mixture of proteins based on both isoelectric point and molecular weight of the individual proteins. Even when we followed a standard protocol for 2DE we got lesser number of proteins focused especially in the basic region of the IPG strip. Since the common troubleshooting measures did not solve the problem we replaced the protease inhibitor cocktail in the lysis buffer with PMSF which resulted in an ideal protein map following 2DE. We also found that the presence of the cocktail results in skewing of the mass spectra of a purified protein which eventually resulted in incorrect identification of the protein by MASCOT search. Later we found that dimethyl sulfoxide, the solvent of protease inhibitor cocktail also resulted in focusing of lesser number of proteins. Addition of 1% dimethyl sulfoxide to bovine serum albumin resulted in lesser sequence coverage for the protein by LC-MS/MS. Also, dimethyl sulfoxide was found to decrease the intensity value of the dominant peptide in a fraction when MALDI-TOF-TOF was done. Even though we do not provide a reason behind our observations, we guess dimethyl sulfoxide might have changed the charge distribution of peptides or proteins resulting in the peculiar observations we had.
Comparative physiology of five tropical montane songbirds reveals differential seasonal acclimatisation and cold adaption
Authors: Jones Samuel E.I. , Suanjak Martin , Tobias Joseph A. , Freeman Robin , Portugal Steven J. 
Published: May 2020

AbstractThe physiology of tropical birds is poorly understood, particularly in how it relates to local climate and changes between seasons. This is particularly true of tropical montane species, which may have sensitive thermal tolerances to local microclimates. We studied metabolic rates (using open flow respirometry), body mass and haemoglobin concentrations of five sedentary Mesoamerican songbirds between the summer and winter at two elevations (1550 m and 1950 m, respectively). We asked whether there were uniform seasonal shifts in physiological traits across species, and whether higher elevation species displayed evidence for cold tolerance. Seasonal shifts in metabolic rates differed between the three species for which data were collected. Basal metabolic rates in one species – black-headed nightingalethrushesCatharus mexicanus– were up-regulated in summer (~19% increase of winter metabolism), however two other species displayed no seasonal regulation. No species exhibited shifts in haemoglobin concentrations across season or across elevation, whereas body mass in two species was significantly higher in the summer. One species restricted to higher elevations – ruddy-capped nightingale-thrushesC. frantzii– displayed physiological traits indicative of cold-tolerance. Although only summer data were available for this species (C. frantzii), metabolic rates were constant across temperatures tested (5-34°C) and haemoglobin concentrations were significantly higher compared to the other four species. Our results suggest that seasonal acclimatisation in physiological traits is variable between species and appear unrelated to changes in local climate. As such, the distinct physiological traits observed in ruddy-capped nightingale-thrushes likely relate to historic isolation and conserved physiological traits rather than contemporary climatic adaption.
Explication of CB1receptor contributions to the hypothermic effects of Δ9-tetrahydrocannabinol (THC) when delivered by vapor inhalation or parenteral injection in rats
Authors: Nguyen Jacques D. , Creehan K. M. , Grant Yanabel , Vandewater Sophia A. , Kerr Tony M. , Taffe Michael A. 
Published: May 2020

AbstractThe use of Δ9-tetrahydrocannabinol (THC) by inhalation using e-cigarette technology grows increasingly popular for medical and recreational purposes. This has led to development of e-cigarette based techniques to study the delivery of THC by inhalation in laboratory rodents. Inhaled THC reliably produces hypothermic and antinociceptive effects in rats, similar to effects of parenteral injection of THC. This study was conducted to determine the extent to which the hypothermic response depends on interactions with the CB1receptor, using pharmacological antagonist (SR141716, AM-251) approaches. Groups of rats were implanted with radiotelemetry devices capable of reporting activity and body temperature, which were assessed after THC inhalation or injection. SR141716 (4 mg/kg, i.p.) blocked or attenuated antinociceptive effects of acute THC inhalation in male and female rats. SR141716 was unable to block the initial hypothermia caused by THC inhalation, but temperature was restored to normal more quickly. Alterations in antagonist pre-treatment time, dose and the use of a rat strain with less sensitivity to THC-induced hypothermia did not change this pattern. Pre-treatment with SR141716 (4 mg/kg, i.p.) blocked hypothermia induced by i.v. THC and reversed hypothermia when administered 45 or 90 minutes after THC (i.p.). SR141716 and AM-251 (4 mg/kg, i.p.) sped recovery from, but did not block, hypothermia caused by vapor THC in female rats made tolerant by prior repeated THC vapor inhalation. The CB2antagonist AM-630, had no effect. These results suggest that hypothermia consequent to THC inhalation is induced by other mechanisms in addition to CB1receptor activation.
Pairing two growth-based, high-throughput selections to fine tune conformational dynamics in oxygenase engineering
Authors: Maxel Sarah , Zhang Linyue , King Edward , Aspacio Derek , Acosta Ana Paula , Luo Ray , Li Han 
Published: May 2020

ABSTRACTCyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor thermostability and stringent specificity for NADPH. Efforts to engineer CHMO have been limited by the sensitivity of the enzyme to perturbations in conformational dynamics and long-range interactions that cannot be predicted. We demonstrate a pair of aerobic, high-throughput growth selection platforms inEscherichia colifor oxygenase evolution, based on NADPH or NADH redox balance. We utilize the NADPH-dependent selection in the directed evolution of thermostable CHMO and discover the variant CHMO GV (A245G-A288V) with a 2.7-fold improvement in residual activity compared to the wild type after 40 °C incubation. Addition of a previously reported mutation resulted in A245G-A288V-T415C which has further improved thermostability at 45 °C. We apply the NADH-dependent selection to alter the cofactor specificity of CHMO to accept NADH, a less expensive cofactor than NADPH. We identified the variant CHMO DTNP (S208D-K326T-K349N-L143P) with a 21-fold cofactor specificity switch from NADPH to NADH compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and backbone torsions, and CHMO DTNP activity is driven by cooperative fine-tuning of cofactor contacts. Our introduced tools for oxygenase evolution enable the rapid engineering of properties critical to industrial scalability.
AmpliconDesign – An interactive web server for the design of high-throughput targeted DNA methylation assays
Authors: Schönung Maximilian , Hess Jana , Bawidamann Pascal , Stäble Sina , Hey Joschka , Langstein Jens , Assenov Yassen , Weichenhan Dieter , Lutsik Pavlo , Lipka Daniel B. 
Published: May 2020

ABSTRACTTargeted analysis of DNA methylation patterns based on bisulfite-treated genomic DNA (BT-DNA) is considered as a gold-standard for epigenetic biomarker development. Existing software tools facilitate primer design, primer quality control or visualization of primer localization. However, high-throughput design of primers for BT-DNA amplification is hampered by limits in throughput and functionality of existing tools, requiring users to repeatedly perform specific tasks manually. Consequently, the design of PCR primers for BT-DNA remains a tedious and time-consuming process. To bridge this gap, we developedAmpliconDesign, a webserver providing a scalable and user-friendly platform for the design and analysis of targeted DNA methylation studies based on BT-DNA, e.g. deep amplicon bisulfite sequencing (ampBS-seq), EpiTYPER MassArray, or pyrosequencing. Core functionality of the web server includes high-throughput primer design and binding site validation based onin silicobisulfite-converted DNA sequences, prediction of fragmentation patterns for EpiTYPER MassArray, an interactive quality control as well as a streamlined analysis workflow for ampBS-seq.Availability and ImplementationTheAmpliconDesignwebserver is freely available online at:https://amplicondesign.dkfz.de/.AmpliconDesignhas been implemented using the RShinyframework (Changet al., 2018). The source code is publicly available under the GNU General Public License v3.0 (https://github.com/MaxSchoenung/AmpliconDesign).ContactDaniel B. Lipka (d.lipka@dkfz.de) & Maximilian Schönung (m.schoenung@dkfz.de)
Dramatic differences in male and female mortality trends for selected European cohorts over ∼20 years
Authors: Clark Jeremy S.C. , Rydzewska Kamila , Podsiadło Konrad , van de Wetering Thierry , Ciechanowicz Andrzej 
Published: May 2020

ABSTRACTLongevity is of considerable interest. Collation of recent data after World War II by the Human Mortality Database allowed analyses, previously unattainable, of modal death-ages for sufficient numbers of selected European cohorts. The aim was to track modes and medians/means (≥60 years old (y)) of all-cause mortality for both sexes. The only highest-quality, large-number Lexis data available were analyzed: from nine countries: Denmark, Finland, France, Iceland, Italy, Netherlands, Norway, Sweden and Switzerland; raw-data modes (and medians/means ≥60y, plus thin-plate-spline averages), were analyzed, plus pooled data. Here we show that for cohorts 1880-∼1900 dramatic sex differences existed between death-age changes with all countries except Iceland showing male modal negative trends lasting ∼10-20 years and medians in all countries near-constant or negative lasting ∼10-20 years; whereas females from most countries showed fairly constant positive trends (except Finnish modes and Norwegian medians). For cohorts ∼1900-1919 male and female modal trends were positive (except Dutch and Icelandic cohorts and Finnish females). The net results were that male mortality modes for Danish, Icelandic, Italian, Dutch, Swedish and Norwegian 1919 cohorts were roughly the same as for 1880 cohorts, whereas female death-age modes increased. Results clarify previously knowledge concerning sex differences during this period. Despite improved environment in late adulthood over this period, this did not translate into increased male longevity and earlier events might have sealed their fate, especially in Denmark, Italy, Netherlands, Norway, aand Sweden (and, later, Iceland).
Modeling the transmission and vaccination strategy for porcine reproductive and respiratory syndrome virus
Authors: Galvis Jason A. , Prada Joaquin M. , Corzo Cesar A. , Machado Gustavo 
Published: May 2020

SummaryMany aspects of the porcine reproductive and respiratory syndrome virus (PRRSV) between-farm transmission dynamics have been investigated, but uncertainty remains about the significance of farm type and different transmission routes on PRRSV spread. We developed a stochastic epidemiological model calibrated on weekly PRRSV outbreaks accounting for the population dynamics in different pig production phases, breeding herds, gilt development units, nurseries, and finisher farms, of three hog producer companies. Our model accounted for indirect contacts by the close distance between farms (local transmission), between-farm animal movements (pig flow), and reinfection of sow farms (re-break). The fitted model was used to examine the effectiveness of vaccination strategies and complementary interventions such as enhanced PRRSV detection and vaccination delays and forecast the spatial distribution of PRRSV outbreak. The results of our analysis indicated that for sow farms, 59% of the simulated infections were related to local transmission (e.g. airborne, feed deliveries, shared equipment) whereas 36% and 5% were related to animal movements and re-break, respectively. For nursery farms, 80% of infections were related to animal movements and 20% to local transmission; while at finisher farms it was split between local transmission and animal movements. Assuming that the current vaccines are 1% effective in mitigating between-farm PRRSV transmission, weaned pigs vaccination would reduce the incidence of PRRSV outbreaks by 2%, indeed under any scenario vaccination alone was insufficient for completely controlling PRRSV spread. Our results also showed that intensifying PRRSV detection and/or vaccination pigs at placement increased the effectiveness of all simulated vaccination strategies. Our model reproduced the incidence and PRRSV spatial distribution; therefore, this model could also be used to map current and future farms at-risk. Finally, this model could be a useful tool for veterinarians, allowing them to identify the effect of transmission routes and different vaccination interventions to control PRRSV spread.
A screen of FDA-approved drugs identifies inhibitors of Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3)
Authors: Rivas Dylan R. , Dela Cerna Mark Vincent C. , Smith Caroline N. , Patty Blaine G. , Lee Donghan , Blackburn Jessica S. 
Published: May 2020

ABSTRACTProtein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) is highly expressed in a variety of cancers, where it promotes tumor cell migration and metastasis leading to poor prognosis. Despite its clinical significance, there are currently no viable options to target PRL-3in vivo. Here, we screened 1,443 FDA-approved drugs for their ability to inhibit the activity of the PRL phosphatase family. We identified five specific inhibitors for PRL-3 as well as one selective inhibitor of PRL-2. Additionally, we found nine drugs that broadly and significantly suppressed PRL activity. Two of these broad PRL inhibitors, Salirasib and Candesartan blocked PRL-3-induced migration in human embryonic kidney (HEK) cells with no negative impact on cell viability. Both drugs prevented migration of PRL-3 expressing human colorectal cancer cells to a similar degree as the research-grade PRL inhibitor, Thienopyridone, and are selective towards PRLs over other phosphatases.In silicomodeling revealed that Salirasib binds a putative allosteric site near the WPD loop of PRL-3, while Candesartan binds a potentially novel targetable site adjacent to the CX5R motif. Inhibitor binding at either of these sites is predicted to trap PRL-3 in a closed conformation, preventing substrate binding and inhibiting function.
Mapping the architecture of regulatory variation provides insights into the evolution of complex traits
Authors: Lupo Offir , Krieger Gat , Jonas Felix , Barkai Naama 
Published: May 2020

AbstractBackgroundOrganisms evolve complex traits by recruiting existing programs to new contexts, referred as co-option. Within a species, single upstream regulators can trigger full differentiation programs. Distinguishing whether co-option of differentiation programs results from variation in single regulator, or in multiple genes, is key for understanding how complex traits evolve. As an experimentally accessible model for studying this question we turned to budding yeast, where a differentiation program (filamentous) is activated inS. cerevisiaeonly upon starvation, but used by the related speciesS. paradoxusalso in rich conditions.ResultsTo define expression variations associated with species-specific activation of the filamentous program, we profiled the transcriptome ofS. cerevisiae, S.paradoxusand their hybrid along two cell cycles at 5-minutes resolution. As expected in cases of co-option, expression of oscillating genes varies between the species in synchrony with their growth phenotypes and was dominated by upstreamtrans-variations. Focusing on regulators of filamentous growth, we identified gene-linked variations (cis) in multiple genes across regulatory layers, which propagated to affect expression of target genes, as well as binding specificities of downstream transcription factor. Unexpectedly, variations in regulators essential forS. cerevisiaefilamentation were individually too weak to explain activation of this program inS. paradoxus.ConclusionsOur study reveals the complex architecture of regulatory variation associated with species-specific use of a differentiation program. Based on these results, we suggest a new model in which evolutionary co-option of complex traits is stabilized in a distributed manner through multiple weak-effect variations accumulating throughout the regulatory network.
Hypoxia-induced SETX links replication stress with the unfolded protein response
Authors: Ramachandran Shaliny , Ma Tiffany , Ng Natalie , Foskolou Iosifina P. , Hwang Ming-Shih , Victori Pedro , Cheng Wei-Chen , Buffa Francesca M. , Leszczynska Katarzyna B. , Gromak Natalia , Hammond Ester M. 
Published: May 2020

ABSTRACTThe levels of hypoxia associated with resistance to radiotherapy significantly impact cancer patient prognosis. These levels of hypoxia initiate a unique transcriptional response with the rapid activation of numerous transcription factors in a background of global repression of transcription. Here, we show that the biological response to radiobiological hypoxia includes the induction of the DNA/RNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. SETX plays a key role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we show that the mechanism of SETX induction is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to radiobiological hypoxia, which includes both a replication stress dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.
DNA Binding Induces acistotransSwitch in Cre Recombinase to Enable Intasome Assembly
Authors: Unnikrishnan Aparna , Amero Carlos D. , Yadav Deepak Kumar , Stachowski Kye , Potter Devante , Foster Mark P. 
Published: May 2020

ABSTRACTMechanistic understanding of DNA recombination in the Cre-loxPsystem has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution NMR to discover the link between intrinsic flexibility and function in Cre recombinase. TROSY-NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD, CreCat) to be structurally independent. Amide15N relaxation measurements of the CreCatdomain reveal fast time scale dynamics in most regions that exhibit conformational differences inactiveandinactiveCre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity viatransprotein-protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and inter-molecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative auto-inhibitory conformation for the αN region of free Cre, wherein it packsin cisover the protein DNA binding surface and active site. Moreover, binding toloxPDNA induces a conformational change that dislodge the C-terminus, resulting in acistotransswitch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a re-examination of the mechanisms by which this widely-utilized gene editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.SIGNIFICANCE STATEMENTThe Cre-loxPsystem is a widely used gene editing tool that has enabled transformative advances in immunology, neuroscience and cardiovascular research. Still, off-target activities confound research results and present obstacles to biomedical applications. Overcoming those limitations requires understanding the steps leading to assembly of recombination complexes,intasomes. We measured the magnetic properties of nitrogen nuclei in the backbone of the enzyme to correlate its intrinsic dynamics with its function in DNA recognition and cleavage. Remarkably, we found that in the absence of DNA the C-terminus of Cre appears to block the DNA binding surface and active site of the enzyme. Binding toloxPDNA induces a conformational switch that would enable the intermolecular protein-protein interactions required for assembly of recombinogenic Cre intasomes.
Evidence for the agricultural origin of antimicrobial resistance in a fungal pathogen of humans
Authors: Kang S. Earl , Sumabat Leilani G. , Melie Tina , Mangum Brandon , Momany Michelle , Brewer Marin T. 
Published: May 2020

AbstractResistance to clinical antimicrobials is an urgent problem, reducing our ability to combat deadly pathogens of humans. Azole antimicrobials target ergosterol synthesis and are highly effective against fungal pathogens of both humans and plants leading to their widespread use in clinical and agricultural settings1,2. The fungusAspergillus fumigatuscauses 300,000 life-threatening infections in susceptible human hosts annually and azoles are the most effective treatment3. Resistance to clinical azole antifungals has become a major problem in Europe and India over the last decade, where identical mutations incyp51A, an ergosterol biosynthetic gene, have been found in strains from both clinical and agricultural settings4. Sharedcyp51Agenotypes suggest that clinical azole resistance might have had an agricultural origin; however, until now, independent origins of clinical and agricultural mutations could not be ruled out. Here we show that azole-resistant isolates ofA. fumigatusfrom clinical and agricultural settings also carry mutations conferring resistance to quinone outside inhibitor (QoI) fungicides, which are used exclusively in agricultural settings. This is the first report of a clear marker for the agricultural origin of resistance to a clinical antifungal. We anticipate that our work will increase the understanding of interactions between pathogens of plants and pathogens of humans.