Miracle-dz/UniFAR-FTUs huggingface dataset:
Differential binding of ppGpp and pppGpp to E. coli RNA polymerase: photo‐labeling and mass spectral studies

(p)ppGpp, a secondary messenger, is induced under stress and shows pleiotropic response. It binds to RNA polymerase and regulates transcription in Escherichia coli. More than 25 years have passed since the first discovery was made on the direct interaction of ppGpp with E. coli RNA polymerase. Several lines of evidence suggest different modes of ppGpp binding to the enzyme. Earlier cross‐linking experiments suggested that the β‐subunit of RNA polymerase is the preferred site for ppGpp, whereas recent crystallographic studies pinpoint the interface of β’/ω‐subunits as the site of action. With an aim to validate the binding domain and to follow whether tetra‐ and pentaphosphate guanosines have different location on RNA polymerase, this work was initiated. RNA polymerase was photo‐labeled with 8‐azido‐ppGpp/8‐azido‐pppGpp, and the product was digested with trypsin and subjected to mass spectrometry analysis. We observed three new peptides in the trypsin digest of the RNA polymerase labeled with 8‐azido‐ppGpp, of which two peptides correspond to the same pocket on β’‐subunit as predicted by X‐ray structural analysis, whereas the third peptide was mapped on the β‐subunit. In the case of 8‐azido‐pppGpp‐labeled RNA polymerase, we have found only one cross‐linked peptide from the β’‐subunit. However, we were unable to identify any binding site of pppGpp on the β‐subunit. Interestingly, we observed that pppGpp at high concentration competes out ppGpp bound to RNA polymerase more efficiently, whereas ppGpp cannot titrate out pppGpp. The competition between tetraphosphate guanosine and pentaphosphate guanosine for E. coli RNA polymerase was followed by gel‐based assay as well as by a new method known as DRaCALA assay.
Ética de la investigación en la era MySpace

A total population of 29,395 neonates cared for in the six-year period from 1971 to 1976 was reviewed for evidence of autopsy-proven kernicterus. A total of 327 neonates died and 232 were autopsied. The only cases of kernicterus occurred in four near-term infants with antemortem proven sepsis. All four of these infants weighed more than 2,200 gm and were delivered after gestations of either 36 or 37 weeks. These cases of kernicterus occurred during a period when more aggressive management of hyperbilirubinemia in low-birth-weight infants had apparently eliminated immaturity as a predisposing factor in the development of kernicterus, uncovering bacterial infection as the major remaining etiologic co-factor.
Whole Mouse Brain Image Reconstruction from Serial Coronal Sections Using FIJI (ImageJ)

Whole‐brain reconstruction of the mouse enables comprehensive analysis of the distribution of neurochemical markers, the distribution of anterogradely labeled axonal projections or retrogradely labeled neurons projecting to a specific brain site, or the distribution of neurons displaying activity‐related markers in behavioral paradigms. This unit describes a method to produce whole‐brain reconstruction image sets from coronal brain sections with up to four fluorescent markers using the freely available image‐processing program FIJI (ImageJ). © 2015 by John Wiley & Sons, Inc.
Basic Confocal Microscopy

Confocal microscopy produces sharp images of structures within relatively thick specimens (up to several hundred microns). It is particularly useful for examining fluorescent specimens. This overview intended to provide background and practical tips needed to get started with confocal microscopy. It begins with a description of the basis of optical sectioning, then discusses various types of confocal microscopes, and concludes with practical guidelines for sample preparation and optimizing image acquisition parameters.
Ballistic Labeling with Fluorescent Dyes and Indicators

Neuronal cell labeling is fundamental to investigations of the nervous system. Labeling of cells in live or fixed tissue with dyes or ion indicators using ballistic approaches has recently been developed for the study of neuronal architecture and function. In this approach, dye‐coated particles are propelled into cells by a pulse of pressurized helium. This unit provides step‐by‐step protocols for coating tungsten particles with fluorescent or indicator dyes and for delivering these particles into cells and tissue. The major advantage of the ballistic method of dye delivery is that large populations of neurons can be rapidly labeled within a piece of live or fixed tissue. Advantages and limitations of the approach are discussed and technical advice is provided. Curr. Protoc. Neurosci. 43:2.11. 1‐2.11. 10. © 2008 by John Wiley & Sons, Inc.
Confocal Laser Scanning Microscopic Photoconversion: A New Method to Stabilize Fluorescently Labeled Cellular Elements for Electron Microscopic Analysis

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low‐resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region‐of‐interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. Curr. Protoc. Neurosci. 58:2.15. 1‐2.15. 12. © 2012 by John Wiley & Sons, Inc.
Gene Expression Analysis Using cDNA Microarrays

This unit focuses on cDNA microarrays, which are being increasingly applied to large‐scale gene expression studies in the nervous system, concentrating on the steps of probe preparation and microarray hybridization. Four protocols for the attachment of fluorophores to cDNA (labeling) and cDNA hybridization onto microarrays are presented: direct labeling using either Klenow fragment or reverse transcriptase, and two signal amplification techniques represented by the tyramide signal amplification method (TSA), and detection and labeling of cDNA using PCR amplification. Basic issues in experimental design, sources of variability and potential pitfalls in microarray experiments are described. Anticipated results and follow up of microarray experiments, including confirmation of the results and troubleshooting are also discussed.
Zombie‐Science and Beyond

According to Haitian Creole lore, a zombie is a dead person brought back to life using magic. By analogy, zombie-science is research that should be dead, but magically lives on. So what is the origin of zombie-science in groundwater, why does the magic still work to keep research alive, and finally, do we really still have the capacity for innovation? Not every paper written can—nor should be—an innovative blockbuster. Thomas Kuhn’s view on progress in science was that more than just revolutions were needed and “something different must necessarily go on in between” (Kuhn, 2000). That something is what Kuhn called normal science—the incremental work of embellishing ideas and filling in details. Zombie-science is essentially normal science run amok, the land where embellishments and detail-filling never die. In some areas of groundwater, normal science, apparently uninterrupted by revolutionary spurts, has been in progress for more than 50 years. The cost is represented by an inefficient use of human capital in research, stagnation of the field, and journals full of information that only few care about. The cost of research-not-done on key problems is unknowable. I attributed stagnation in the past to comfort with research paradigms; unwillingness to part with methods, facilities, and field sites; or the maturing of the field. Undoubtedly, these factors contribute, but my view has shifted to simply linking zombie-science to a lack of funding. Some fields, most notably, medical science, (e.g., neurobiology, molecular genetics) are awash in full-time researchers taking their studies in unique directions. It is possible to do good science without money. But broadly speaking, even zombie magic cannot put life back into research founded on collections of ordinary data, routine project work, and too little time. Given the present economic conditions and the depressed government support for water sciences, perhaps the best we can hope for future funding is something like the status quo.
Rodent Models of Global Cerebral Ischemia

Brain damage after stroke and head injury remains a huge clinical problem. In stroke, the initial cause of the damage is a blockage in a blood vessel (often the middle cerebral artery) and this sets off several pathways that ultimately lead to cell death. Recent studies have demonstrated that several new mechanisms are involved in neuronal death and this has led to an increase in research into novel molecules that might prevent brain damage or improve recuperation post‐stroke. There are several models of global cerebral ischemia. Two of the most widely‐used models are discussed in detail in UNIT , the gerbil bilateral carotid artery occlusion (BCAO) model and rat 4‐vessel occlusion (4‐VO) model. Additionally, several models of focal cerebral ischemia have been developed to mimic the effects of human stroke. The rationale behind the use of animal models, the various types of models and advantage and disadvantages of each model are presented.
Overview of Rodent Models for Obesity Research

Animal obesity models differ widely in type and extent of obesity. They are either based on environmental factors (e.g., high‐fat diet‐induced obesity), spontaneous mutants (i.e., ob/ob mice), genetically engineered animals (e.g., mice with melanocortin receptor subtype‐4 gene disruption (knock‐out), or mechanical intervention (e.g., chemical lesion of the ventromedial hypothalamus). This unit reviews available rodent models to study obesity and attempts to highlight the greatest utility for each model.
Diet‐Induced Models of Obesity (DIO) in Rodents

Obesity results from a complex interplay between a susceptible genotype and an environment that both promotes increased caloric intake and enables sustained decreases in energy expenditure. One commonly employed approach to modeling obesity in preclinical species is the diet‐induced obese (DIO) rodent. Here, theoretical and practical considerations for producing obese rodents via dietary manipulation, and for assessing drug‐induced changes in food intake and body weight are described. Based on these considerations, a standardized protocol for diet‐induced obesity in both mouse and rat is provided and sample data from these models are also described. Curr. Protoc. Neurosci. 59:9.38. 1‐9.38. 13. © 2012 by John Wiley & Sons, Inc.
A New Zonation Algorithm with Parameter Estimation Using Hydraulic Head and Subsidence Observations

Parameter estimation codes such as UCODE_2005 are becoming well‐known tools in groundwater modeling investigations. These programs estimate important parameter values such as transmissivity (T) and aquifer storage values (Sa) from known observations of hydraulic head, flow, or other physical quantities. One drawback inherent in these codes is that the parameter zones must be specified by the user. However, such knowledge is often unknown even if a detailed hydrogeological description is available. To overcome this deficiency, we present a discrete adjoint algorithm for identifying suitable zonations from hydraulic head and subsidence measurements, which are highly sensitive to both elastic (Sske) and inelastic (Sskv) skeletal specific storage coefficients. With the advent of interferometric synthetic aperture radar (InSAR), distributed spatial and temporal subsidence measurements can be obtained. A synthetic conceptual model containing seven transmissivity zones, one aquifer storage zone and three interbed zones for elastic and inelastic storage coefficients were developed to simulate drawdown and subsidence in an aquifer interbedded with clay that exhibits delayed drainage. Simulated delayed land subsidence and groundwater head data are assumed to be the observed measurements, to which the discrete adjoint algorithm is called to create approximate spatial zonations of T, Sske, and Sskv. UCODE‐2005 is then used to obtain the final optimal parameter values. Calibration results indicate that the estimated zonations calculated from the discrete adjoint algorithm closely approximate the true parameter zonations. This automation algorithm reduces the bias established by the initial distribution of zones and provides a robust parameter zonation distribution.
Assessment of Parkinson Disease Manifestations

Parkinson disease (PD) is a progressive neurologic condition that causes motor and nonmotor manifestations. Treatment provides symptomatic benefit but no current treatment has been proven to slow disease progression. Research studies of PD require a means of rating the severity of disease by measurement of motor manifestations, assessment of ability to perform daily functional activities, and symptomatic response to medication. The most common rating scales are the Unified Parkinson Disease Rating Scale (UPDRS), Hoehn and Yahr staging, and the Schwab and England rating of activities of daily living. Each of these rating scales are described, including detailed instructions on how to implement these ratings. Although these are the most widely applied rating scales of PD, there are still substantial limitations to these scales that must be considered when using them for research. Finally, some common applications of these scales are described. Curr. Protoc. Neurosci. 49:10.1. 1‐10.1. 14. © 2009 by John Wiley & Sons, Inc.
Development of a Watershed‐Based Geospatial Groundwater Specific Vulnerability Assessment Tool

This study assesses and characterizes the vulnerability of unregulated groundwater systems to microbial contamination in 18 counties in the state of Georgia using a contamination risk screening strategy based on watershed characteristics and elements of the Safe Drinking Water Act's Wellhead Protection program. Environmental data sources analyzed include septic systems, elevation, land use and land cover data, soil, vegetation coverage, demographics, and livestock. A geospatial overlay/index modeling approach was developed to identify areas of higher vulnerability for groundwater pollution by taking into consideration watershed land use, hydrology, and topography (LHT). Sensitivity analysis was used to evaluate the effectiveness of model variables. The results of the model were validated by using field data and output from U.S. EPA's DRASTIC model, a widely used intrinsic vulnerability assessment tool. The validation showed a higher risk of microbial contamination for wells located in a high to medium LHT vulnerability zones. LHT provided a clear distribution of satisfactory and unsatisfactory wells in the three vulnerability zones; however, the majority of wells (>75%), with both satisfactory and unsatisfactory test results, are located in medium DRASTIC vulnerability zone. This difference between LHT and DRASTIC can be attributed to the microbial contamination specific factors incorporated into LHT index. It is concluded that although inclusion of potential contamination sources on adjacent land uses in the vulnerability assessment framework adds to the complexity of the processes involved in a vulnerability assessment, such inclusion provides a meaningful perspective to groundwater protection efforts as an effective screening tool.
Isolation , endocrine regulation and transcript distribution of a putative primary JH-responsive gene from the pine engraver , Ips pini ( Coleoptera : Scolytidae )

We isolated a cDNA of unknown function from a juvenile hormone III (JH III)-treated male midgut cDNA library prepared from the pine engraver beetle, Ips pini, and examined its genomic structure. The gene, tentatively named ‘‘Ipi10G08’’, encoded a 410 amino acid translation product that shared 26–37% identity with unannotated matches from several insects. Semi-quantitative RT-PCR analysis of Ipi10G08 following application of a 10 mg dose of JH III demonstrated an early induction for both male and female beetles, with transcripts being detectable after 45min. An expression profile of male midgut tissue indicated Ipi10G08 transcript levels reach a maximum induction of 22.5-fold control levels at 4 h post-treatment. Tissue distribution studies displayed a large induction of Ipi10G08 mRNA in the alimentary canal of JH III-treated beetles, especially in males. A dose curve from both sexes suggested there may be a difference in the ability to respond to lower levels of JH III and immunoblot analysis indicated that although JH III highly induces transcript levels in females, protein levels are not similarly induced, while protein levels are induced in males. Ipi10G08 is likely a primary JH response gene and may provide insight into how this hormone exerts its actions. Published by Elsevier Ltd.
Effects of Changing Meteoric Precipitation Patterns on Groundwater Temperature in Karst Environments

Climate predictions indicate that precipitation patterns will change and average air temperatures will increase across much of the planet. These changes will alter surface water and groundwater temperatures which can significantly affect the local and regional environment. Here, we examine the role of precipitation timing in changes to groundwater temperature in carbonate‐karst aquifers using measured groundwater level and temperature data from the Konza Prairie Long‐Term Ecological Research Site, Kansas. We demonstrate that shifts to increased cool‐season precipitation may mitigate the increases in groundwater temperature produced by increases in average annual air temperature. In karst, the solution‐enlarged conduits allow faster and focused recharge, and the recharge‐event temperature can strongly influence the groundwater temperature in the aquifer. Our field data and analysis show that predictions of future groundwater conditions in karst aquifers need to consider changes in precipitation patterns, in addition to changes to average annual air temperature.
Solithromycin, a novel macrolide, does not prolong cardiac repolarization: a randomized, three-way crossover study in healthy subjects

Background Macrolide antibiotics may cause QT prolongation. Objectives To study the QT effect of a novel macrolide, solithromycin. Methods This was a thorough QT study with a three-way crossover design performed in healthy male and female subjects to evaluate the ECG effects of a novel macrolide, solithromycin. Forty-eight subjects were randomized to receive 800 mg of intravenous (iv) solithromycin, 400 mg of oral moxifloxacin and placebo in three separate treatment periods. Continuous 12 lead ECGs were recorded at a pre-dose baseline and serially after drug administration for 24 h. Results After the 40 min infusion of 800 mg of solithromycin, the geometric mean solithromycin peak plasma concentration (Cmax) reached 5.9 (SD: 1.30) &mgr;g/mL. Solithromycin infusion caused a heart rate increase with a peak effect of 15 bpm immediately after the end of the infusion. The change-from-baseline QTcF (&Dgr;QTcF) was similar after dosing with solithromycin and placebo and the resulting placebo-corrected &Dgr;QTcF (&Dgr;&Dgr;QTcF) for solithromycin was therefore small at all timepoints with a peak effect at 4 h of only 2.8 ms (upper bound of the 90% CI: 4.9 ms). Using a linear exposure–response model, a statistically significant, slightly negative slope of −0.86 ms per ng/mL (90% CI: −1.19 to −0.53; P = 0.0001) was observed with solithromycin. The study's ability to detect small QT changes was confirmed by the moxifloxacin response. Solithromycin did not have a clinically meaningful effect on the PR or QRS interval. Conclusions The study demonstrated that solithromycin, unlike other macrolide antibiotics, does not cause QT prolongation.
Impaired Intrinsic Immunity to HSV-1 in Human iPSC-Derived TLR3-Deficient CNS Cells

FG Lafaille, IM Pessach, SY Zhang. Nature. 2012;491(7426):769–773 Toll-like receptors (TLR) are innate immune receptors that recognize pathogen-associated molecules and are critical for the development of proper antimicrobial responses. Children with defects in the TLR3-signaling pathway are specifically susceptible to developing herpes simplex virus (HSV) encephalitis but not disseminated infections with HSV or other viruses. Therefore, the authors investigated whether TLR3 activity in neuronal cells was necessary for controlling HSV infection in the central nervous system (CNS). Studies were performed by using cells from patients with …
The impact of social influences in the context of attitude, self-efficacy, intention, and previous behavior as predictors of smoking onset

Three different constructs for measuring social influence were utilized in the present study to explain adolescents' present and future smoking behavior at 6 (T2), 12 (T3), and 18 months (T4) after the first test. Social influence was assessed by measuring the social norms, perceived smoking behavior, and direct pressure. The impact of the social influence constructs was also assessed in the context of broader models, including attitudes and self-efficacy expectations, intention, and previous behavior. The three social influence measures correlated significantly with intention and behavior. Stepwise regression analyses showed that perceived behavior and pressure made significant contributions, after entering social norms, in explaining actual and future adolescent smoking behavior. Adding attitudes and self-efficacy increased the predictive power of the model significantly. In agreement with the theory of Fishbein & Ajzen (1975), intention was the most powerful predictor in explaining present and future smoking behavior. Attitudes, self-efficacy, and the social influences also made small unique contributions improving the explanatory power by approximately 5%. Previous behavior, however, had a substantial unique contribution in predicting future behavior after attitudes, social influences, self-efficacy, and intention were entered in the equations. Since social influences may exert their impact via different routes, it is recommended that smoking prevention programs discuss not only overt pressures such as direct pressure from peers, parents, and media, but also address the more covert social pressures such as modeling and the adolescents' ability to cope with these covert influences. Furthermore, norms on nonsmoking should be made explicit.
Mapping 3′ mRNA Isoforms on a Genomic Scale

Most eukaryotic genes are transcribed into mRNAs with alternative poly(A) sites. Emerging evidence suggests that mRNA isoforms with alternative poly(A) sites can perform critical regulatory functions in numerous biological processes. In recent years, a number of strategies utilizing high‐throughput sequencing technologies have been developed to aid in the identification of genome‐wide poly(A) sites. This unit describes a modified protocol for a recently published 3′READS (3′ region extraction and deep sequencing) method that accurately identifies genome‐wide poly(A) sites and that can be used to quantify the relative abundance of the resulting 3′ mRNA isoforms. This approach minimizes nonspecific sequence reads due to internal priming and typically yields a high percentage of sequence reads that are ideally suited for accurate poly(A) identification. © 2015 by John Wiley & Sons, Inc.