Insights into the mechanisms of eukaryotic translation gained with ribosome profiling

The development of Ribosome Profiling (RiboSeq) has revolutionized functional genomics. RiboSeq is based on capturing and sequencing of the mRNA fragments enclosed within the translating ribosome and it thereby provides a â snapshotâ of ribosome positions at the transcriptome wide level. Although the method is predominantly used for analysis of differential gene expression and discovery of novel translated ORFs, the RiboSeq data can also be a rich source of information about molecular mechanisms of polypeptide synthesis and translational control. This review will focus on how recent findings made with RiboSeq have revealed important details of the molecular mechanisms of translation in eukaryotes. These include mRNA translation sensitivity to drugs affecting translation initiation and elongation, the roles of upstream ORFs in response to stress, the dynamics of elongation and termination as well as details of intrinsic ribosome behavior on the mRNA after translation termination. As the RiboSeq method is still at a relatively early stage we will also discuss the implications of RiboSeq artifacts on data interpretation

Ischemic preconditioning in the liver is independent of regulatory T cell activity

Ischemic preconditioning (IPC) protects organs from ischemia reperfusion injury (IRI) through unknown mechanisms. Effector T cell populations have been implicated in the pathogenesis of IRI, and T regulatory cells (Treg) have become a putative therapeutic target, with suggested involvement in IPC. We explored the role of Treg in hepatic IRI and IPC in detail. IPC significantly reduced injury following ischemia reperfusion insults. Treg were mobilized rapidly to the circulation and liver after IRI, but IPC did not further increase Treg numbers, nor was it associated with modulation of circulating pro-inflammatory chemokine or cytokine profiles. We used two techniques to deplete Treg from mice prior to IRI. Neither Treg depleted FoxP3.LuciDTR mice, nor wildtyoe mice depleted of Tregs with PC61, were more susceptible to IRI compared with controls. Despite successful enrichment of Treg in the liver, by adoptive transfer of both iTreg and nTreg or by in vivo expansion of Treg with IL-2/anti-IL-2 complexes, no protection against IRI was observed.We have explored the role of Treg in IRI and IPC using a variety of techniques to deplete and enrich them within both the liver and systemically. This work represents an important negative finding that Treg are not implicated in IPC and are unlikely to have translational potential in hepatic IRI

Effects of Aerobic Exercise Training in Community-Based Subjects Aged 80 and Older: A Pilot Study

To assess the ability of sedentary, frail subjects aged 80 and older to train in a community-based exercise program and to evaluate clinical factors that predict improvements in peak oxygen consumption (VO 2 peak). DESIGN: Pretest, posttest. SETTING: Charlestown Retirement Community, Catonsville, Maryland PARTICIPANTS: Twenty-two (11 male, 11 female; mean age ± standard deviation = 84 ± 4.0, range 80–92) self-referred. INTERVENTION: Six months of moderate-intensity aerobic exercise training, two to three sessions/week, 20 to 30 minutes per session. Training modes included treadmill walking and/or stationary cycling. MEASUREMENTS: Baseline and follow-up maximal exercise treadmill tests (ETTs) with electrocardiogram monitoring and respiratory gas analysis. RESULTS: Six months of aerobic exercise training resulted in significant increases (mean ± standard deviation) in ETT duration (11.9 ± 3.3 vs 15.9 ± 4.3 minutes; P = .01), VO 2 peak (1.23 ± 0.37 vs 1.31 ± 0.36 L/min; P = .04), and oxygen pulse (9.3 ± 2.8 vs 10.1 ± 3.2; P = .03). Mean heart rate was significantly lower during submaximal ETT stages 1 through 4 ( P < .05), and resting systolic blood pressure decreased (146 ± 18 vs 133 ± 14 mmHg; P = .01) after training. Multiple regression analysis indicated that baseline VO 2 peak ( r = 0.75, P = .002) and the total amount of time spent in exercise training ( r = 0.55, P = .008) were independent predictors of the training-related improvements in VO 2 peak. CONCLUSION: Subjects aged 80 and older can increase aerobic capacity and reduce systolic blood pressure in a community-based exercise program of moderate intensity. The most important predictors of change in VO 2 peak were baseline VO 2 peak and the time spent in exercise training. Subjects with a lower baseline VO 2 peak had the greatest improvements in VO 2 peak after training.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65501/1/j.1532-5415.2002.50613.x.pd

AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation.

In addition to acting as template for protein synthesis, messenger RNA (mRNA) often contains sensory sequence elements that regulate this process1,2. Here we report a new mechanism that limits the number of complete protein molecules that can be synthesized from a single mRNA molecule of the human AMD1 gene encoding adenosylmethionine decarboxylase 1 (AdoMetDC). A small proportion of ribosomes translating AMD1 mRNA stochastically read through the stop codon of the main coding region. These readthrough ribosomes then stall close to the next in-frame stop codon, eventually forming a ribosome queue, the length of which is proportional to the number of AdoMetDC molecules that were synthesized from the same AMD1 mRNA. Once the entire spacer region between the two stop codons is filled with queueing ribosomes, the queue impinges upon the main AMD1 coding region halting its translation. Phylogenetic analysis suggests that this mechanism is highly conserved in vertebrates and existed in their common ancestor. We propose that this mechanism is used to count and limit the number of protein molecules that can be synthesized from a single mRNA template. It could serve to safeguard from dysregulated translation that may occur owing to errors in transcription or mRNA damage

Definitions and sharpness of the extratropical tropopause : a trace gas perspective

Author Posting. © American Geophysical Union, 2004. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 109 (2004): D23103, doi:10.1029/2004JD004982.Definitions of the extratropical tropopause are examined from the perspective of chemical composition. Fine-scale measurements of temperature, ozone, carbon monoxide, and water vapor from approximately 70 aircraft flights, with ascending and descending tropopause crossings near 40°N and 65°N, are used in this analysis. Using the relationship of the stratospheric tracer O3 and the tropospheric tracer CO, we address the issues of tropopause sharpness and where the transitions from troposphere to stratosphere occur in terms of the chemical composition. Tracer relationships indicate that mixing of stratospheric and tropospheric air masses occurs in the vicinity of the tropopause to form a transition layer. Statistically, this transition layer is centered on the thermal tropopause. Furthermore, we show that the transition is much sharper near 65°N (a region away from the subtropical jet) but spans a larger altitude range near 40°N (in the vicinity of the subtropical jet). This latter feature is consistent with enhanced stratosphere-troposphere exchange and mixing activity near the tropopause break.This work is supported in part by the National Science Foundation through its support to the University Corporation for Atmospheric Research, by the NASA Upper Atmosphere Research Satellite guest investigator program, and by the NASA Atmospheric Chemistry Modeling and Analysis Program. Work performed at the Jet Propulsion Laboratory, California Institute of Technology, was carried out under a contract with the National Aeronautics and Space Administration

Neisseria gonorrhoeae Activates the Proteinase Cathepsin B to Mediate the Signaling Activities of the NLRP3 and ASC-Containing Inflammasome

Neisseria gonorrhoeae is a common sexually transmitted pathogen that significantly impacts female fertility, neonatal health, and transmission of HIV worldwide. N. gonorrhoeae usually causes localized inflammation of the urethra and cervix by inducing production of IL-1β and other inflammatory cytokines. Several NLR (Nucleotide binding domain, Leucine Rich Repeat) proteins are implicated in the formation of pro-IL-1β-processing complexes called inflammasomes in response to pathogens. We demonstrate that NLRP3 (cryopyrin,NALP3) is the primary NLR required for IL-1β/IL-18 secretion in response to N. gonorrhoeae in monocytes. We also show that N. gonorrhoeae infection promotes NLRP3-dependent monocytic cell death via pyronecrosis, a recently described pathway with morphological features of necrosis, including release of the strong inflammatory mediator HMBG1. Additionally, N. gonorrhoeae activates the cysteine protease Cathepsin-B as measured by the breakdown of a Cathepsin B substrate. Inhibition of Cathepsin B shows that this protease is an apical controlling step in the downstream activities of NLRP3 including IL-1β production, pyronecrosis, and HMGB1 release. Non-pathogenic Neisseria strains (N. cinerea and N. flavescens) do not activate NLRP3 as robustly as N. gonorrhoeae. Conditioned media from N. gonorrhoeae contains factors capable of initiating the NLRP3 mediated signaling events. Isolated N. gonorrhoeae lipooligosaccharide, a known virulence factor from this bacterium that is elaborated from the bacterium in the form of outer membrane blebs, activates both NLRP3-induced IL-1β secretion and pyronecrosis. Our findings indicate that activation of NLRP3-mediated inflammatory response pathways is an important venue associated with host response and pathogenesis of N. gonorrhoeae

Care management for Type 2 diabetes in the United States: a systematic review and meta-analysis

<p>Abstract</p> <p>Background</p> <p>This systematic review and meta-analysis aims at assessing the composition and performance of care management models evaluated in the last decade and their impact on patient important outcomes.</p> <p>Methods</p> <p>A comprehensive literature search of electronic bibliographic databases was performed to identify care management trials in type 2 diabetes. Random effects meta-analysis was used when feasible to pool outcome measures.</p> <p>Results</p> <p>Fifty-two studies were eligible. Most commonly reported were surrogate outcomes (such as HbA1c and LDL), followed by process measures (clinic visit or testing frequency). Less frequently reported were quality of life, patient satisfaction, self-care, and healthcare utilization. Most care management modalities were carved out from primary care. Meta-analysis demonstrated a statistically significant but trivial reduction of HbA1c (weighted difference in means -0.21%, 95% confidence interval -0.40 to -0.03, p < .03) and LDL-cholesterol (weighted difference in means -3.38 mg/dL, 95% confidence interval -6.27 to -0.49, p < .02).</p> <p>Conclusions</p> <p>Most care management programs for patients with type 2 diabetes are 'carved-out', accomplish limited effects on metabolic outcomes, and have unknown effects on patient important outcomes. Comparative effectiveness research of different models of care management is needed to inform the design of medical homes for patients with chronic conditions.</p

Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use.

Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is frameshifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression.This work was supported by grants from Science Foundation Ireland [12/IP/1492 and 13/1A/1853 to J.F.A; 12/IA/1335 to P.V.B.], US. National Institutes of Health [RO3 MH098688 to J.F.A.], the Wellcome Trust [106207 to A.E.F and 094423 to P.V.B.] and the European Research Council (ERC) grant No. 646891 to A.E.F.]This is the final version of the article. It first appeared from Oxford University Press via https://doi.org/10.1093/nar/gkw53

LSST: from Science Drivers to Reference Design and Anticipated Data Products

(Abridged) We describe here the most ambitious survey currently planned in the optical, the Large Synoptic Survey Telescope (LSST). A vast array of science will be enabled by a single wide-deep-fast sky survey, and LSST will have unique survey capability in the faint time domain. The LSST design is driven by four main science themes: probing dark energy and dark matter, taking an inventory of the Solar System, exploring the transient optical sky, and mapping the Milky Way. LSST will be a wide-field ground-based system sited at Cerro Pach\'{o}n in northern Chile. The telescope will have an 8.4 m (6.5 m effective) primary mirror, a 9.6 deg$^2$ field of view, and a 3.2 Gigapixel camera. The standard observing sequence will consist of pairs of 15-second exposures in a given field, with two such visits in each pointing in a given night. With these repeats, the LSST system is capable of imaging about 10,000 square degrees of sky in a single filter in three nights. The typical 5$\sigma$ point-source depth in a single visit in $r$ will be $\sim 24.5$ (AB). The project is in the construction phase and will begin regular survey operations by 2022. The survey area will be contained within 30,000 deg$^2$ with $\delta<+34.5^\circ$, and will be imaged multiple times in six bands, $ugrizy$, covering the wavelength range 320--1050 nm. About 90\% of the observing time will be devoted to a deep-wide-fast survey mode which will uniformly observe a 18,000 deg$^2$ region about 800 times (summed over all six bands) during the anticipated 10 years of operations, and yield a coadded map to $r\sim27.5$. The remaining 10\% of the observing time will be allocated to projects such as a Very Deep and Fast time domain survey. The goal is to make LSST data products, including a relational database of about 32 trillion observations of 40 billion objects, available to the public and scientists around the world.Comment: 57 pages, 32 color figures, version with high-resolution figures available from https://www.lsst.org/overvie

Patients' and Observers' Perceptions of Involvement Differ. Validation Study on Inter-Relating Measures for Shared Decision Making

OBJECTIVE: Patient involvement into medical decisions as conceived in the shared decision making method (SDM) is essential in evidence based medicine. However, it is not conclusively evident how best to define, realize and evaluate involvement to enable patients making informed choices. We aimed at investigating the ability of four measures to indicate patient involvement. While use and reporting of these instruments might imply wide overlap regarding the addressed constructs this assumption seems questionable with respect to the diversity of the perspectives from which the assessments are administered. METHODS: The study investigated a nested cohort (N = 79) of a randomized trial evaluating a patient decision aid on immunotherapy for multiple sclerosis. Convergent validities were calculated between observer ratings of videotaped physician-patient consultations (OPTION) and patients' perceptions of the communication (Shared Decision Making Questionnaire, Control Preference Scale & Decisional Conflict Scale). RESULTS: OPTION reliability was high to excellent. Communication performance was low according to OPTION and high according to the three patient administered measures. No correlations were found between observer and patient judges, neither for means nor for single items. Patient report measures showed some moderate correlations. CONCLUSION: Existing SDM measures do not refer to a single construct. A gold standard is missing to decide whether any of these measures has the potential to indicate patient involvement. PRACTICE IMPLICATIONS: Pronounced heterogeneity of the underpinning constructs implies difficulties regarding the interpretation of existing evidence on the efficacy of SDM. Consideration of communication theory and basic definitions of SDM would recommend an inter-subjective focus of measurement. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN25267500