The 35S U5 snRNP is generated from the activated spliceosome during In vitro splicing

Primary gene transcripts of eukaryotes contain introns, which are removed during processing by splicing machinery. Biochemical studies In vitro have identified a specific pathway in which introns are recognised and spliced out. This occurs by progressive formation of spliceosomal complexes designated as E, A, B, and C. The composition and structure of these spliceosomal conformations have been characterised in many detail. In contrast, transitions between the complexes and the intermediates of these reactions are currently less clear. We have previously isolated a novel 35S U5 snRNP from HeLa nuclear extracts. The protein composition of this particle differed from the canonical 20S U5 snRNPs but was remarkably similar to the activated B* spliceosomes. Based on this observation we have proposed a hypothesis that 35S U5 snRNPs represent a dissociation product of the spliceosome after both transesterification reactions are completed. Here we provide experimental evidence that 35S U5 snRNPs are generated from the activated B* spliceosomes during In vitro splicing

Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post- translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B’. Results also show that unstructured post- ranslationally modified C-terminal tails are responsible for the dynamics of Sm-B/B’ and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.This work was funded by: BBSRC (OVM), BBSRC and EPSRC (HH and NM), EU Prospects (HH), European Science Foundation (NM), the Royal Society (CVR), and fellowship from JSPS and HFSP (YM and DAPK respectively)

Bridging topological and functional information in protein interaction networks by short loops profiling

Protein-protein interaction networks (PPINs) have been employed to identify potential novel interconnections between proteins as well as crucial cellular functions. In this study we identify fundamental principles of PPIN topologies by analysing network motifs of short loops, which are small cyclic interactions of between 3 and 6 proteins. We compared 30 PPINs with corresponding randomised null models and examined the occurrence of common biological functions in loops extracted from a cross-validated high-confidence dataset of 622 human protein complexes. We demonstrate that loops are an intrinsic feature of PPINs and that specific cell functions are predominantly performed by loops of different lengths. Topologically, we find that loops are strongly related to the accuracy of PPINs and define a core of interactions with high resilience. The identification of this core and the analysis of loop composition are promising tools to assess PPIN quality and to uncover possible biases from experimental detection methods. More than 96% of loops share at least one biological function, with enrichment of cellular functions related to mRNA metabolic processing and the cell cycle. Our analyses suggest that these motifs can be used in the design of targeted experiments for functional phenotype detection.This research was supported by the Biotechnology and Biological Sciences Research Council (BB/H018409/1 to AP, ACCC and FF, and BB/J016284/1 to NSBT) and by the Leukaemia & Lymphoma Research (to NSBT and FF). SSC is funded by a Leukaemia & Lymphoma Research Gordon Piller PhD Studentship

Healing of surgical site after total hip and knee replacements show similar telethermographic patterns

BACKGROUND: Isolated reports indicate the efficacy of infrared thermography for monitoring wound healing and septic complications, but no long-term analysis has ever been performed on this, and there are no data on the telethermographic patterns of surgical site healing after uncomplicated total hip prosthesis and after knee prosthesis. MATERIALS AND METHODS: In this prospective, observational, nonrandomized cohort study, two groups with forty consecutive patients each, who were operated on respectively for total hip and for total knee replacements, underwent telethermographic examination of the operated and contralateral joints prior to and at fixed intervals for up to 1\ua0year after uncomplicated surgery. A digital, portable telethermocamera and dedicated software were used for data acquisition and processing. RESULTS: No thermographic difference was observed preoperatively between the affected side and the contralateral side in both groups. After the intervention, a steep increase in the temperature of the operated joint was recorded after total hip replacement and after knee replacement, with a peak mean differential temperature measured three days postoperatively between the operated and unoperated joint of 3.1\ua0\ub1\ua00.8\ub0C after total hip replacement, and 3.4\ua0\ub1\ua00.7\ub0C after total knee replacement. Thereafter, the mean differential temperature declined slowly to 0.7\ua0\ub1\ua01.1\ub0C and to 0.5\ua0\ub1\ua01.3\ub0C at 60\ua0days, and to 0.0\ua0\ub1\ua01.0\ub0C and -0.1\ua0\ub1\ua01.1\ub0C 90\ua0days post-operatively, respectively. No further changes were observed for up to 1\ua0year after surgery. Results were similar when comparing the average telethermographic values of an elliptical area where the main axis corresponded to the surgical wound. CONCLUSIONS: The surgical sites after uncomplicated total hip or total knee replacement show similar telethermographic patterns for up to 1\ua0year from surgery, and can easily be monitored using a portable, digital, telethermocamera

The Pioneer Anomaly

Radio-metric Doppler tracking data received from the Pioneer 10 and 11 spacecraft from heliocentric distances of 20-70 AU has consistently indicated the presence of a small, anomalous, blue-shifted frequency drift uniformly changing with a rate of ~6 x 10^{-9} Hz/s. Ultimately, the drift was interpreted as a constant sunward deceleration of each particular spacecraft at the level of a_P = (8.74 +/- 1.33) x 10^{-10} m/s^2. This apparent violation of the Newton's gravitational inverse-square law has become known as the Pioneer anomaly; the nature of this anomaly remains unexplained. In this review, we summarize the current knowledge of the physical properties of the anomaly and the conditions that led to its detection and characterization. We review various mechanisms proposed to explain the anomaly and discuss the current state of efforts to determine its nature. A comprehensive new investigation of the anomalous behavior of the two Pioneers has begun recently. The new efforts rely on the much-extended set of radio-metric Doppler data for both spacecraft in conjunction with the newly available complete record of their telemetry files and a large archive of original project documentation. As the new study is yet to report its findings, this review provides the necessary background for the new results to appear in the near future. In particular, we provide a significant amount of information on the design, operations and behavior of the two Pioneers during their entire missions, including descriptions of various data formats and techniques used for their navigation and radio-science data analysis. As most of this information was recovered relatively recently, it was not used in the previous studies of the Pioneer anomaly, but it is critical for the new investigation.Comment: 165 pages, 40 figures, 16 tables; accepted for publication in Living Reviews in Relativit

A prospective study of rural drinking water quality and acute gastrointestinal illness

BACKGROUND: This study examined the relationship between the bacteriological contamination of drinking water from private wells and acute gastrointestinal illness (AGII), using current government standards for safe drinking water. METHODS: A prospective cohort study was conducted using 235 households (647 individuals) randomly selected from four rural hamlets. Data were collected by means of a self-administered questionnaire, a self-report diary of symptoms and two drinking water samples. RESULTS: Twenty percent of households sampled, had indicator bacteria (total coliform or Escherichia coli (E. coli)) above the current Canadian and United States standards for safe drinking water. No statistically significant associations between indicator bacteria and AGII were observed. The odds ratio (OR) for individuals exposed to E. coli above the current standards was 1.52 (95% confidence interval (CI), 0.33–6.92), compared to individuals with levels below current standards. The odds ratio estimate for individuals exposed to total coliforms above the current standards was 0.39 (95% CI, 0.10–1.50). CONCLUSIONS: This study observed a high prevalence of bacteriological contamination of private wells in the rural hamlets studied. Individual exposure to contaminated water defined by current standards may be associated with an increased risk of AGII

Predicting the demand of physician workforce: an international model based on "crowd behaviors"

<p>Abstract</p> <p>Background</p> <p>Appropriateness of physician workforce greatly influences the quality of healthcare. When facing the crisis of physician shortages, the correction of manpower always takes an extended time period, and both the public and health personnel suffer. To calculate an appropriate number of Physician Density (PD) for a specific country, this study was designed to create a PD prediction model, based on health-related data from many countries.</p> <p>Methods</p> <p>Twelve factors that could possibly impact physicians' demand were chosen, and data of these factors from 130 countries (by reviewing 195) were extracted. Multiple stepwise-linear regression was used to derive the PD prediction model, and a split-sample cross-validation procedure was performed to evaluate the generalizability of the results.</p> <p>Results</p> <p>Using data from 130 countries, with the consideration of the correlation between variables, and preventing multi-collinearity, seven out of the 12 predictor variables were selected for entry into the stepwise regression procedure. The final model was: PD = (5.014 - 0.128 × proportion under age 15 years + 0.034 × life expectancy)², with R²of 80.4%. Using the prediction equation, 70 countries had PDs with "negative discrepancy", while 58 had PDs with "positive discrepancy".</p> <p>Conclusion</p> <p>This study provided a regression-based PD model to calculate a "norm" number of PD for a specific country. A large PD discrepancy in a country indicates the needs to examine physician's workloads and their well-being, the effectiveness/efficiency of medical care, the promotion of population health and the team resource management.</p

The Confrontation between General Relativity and Experiment

The status of experimental tests of general relativity and of theoretical frameworks for analysing them is reviewed. Einstein's equivalence principle (EEP) is well supported by experiments such as the Eotvos experiment, tests of special relativity, and the gravitational redshift experiment. Future tests of EEP and of the inverse square law are searching for new interactions arising from unification or quantum gravity. Tests of general relativity at the post-Newtonian level have reached high precision, including the light deflection, the Shapiro time delay, the perihelion advance of Mercury, and the Nordtvedt effect in lunar motion. Gravitational-wave damping has been detected in an amount that agrees with general relativity to better than half a percent using the Hulse-Taylor binary pulsar, and other binary pulsar systems have yielded other tests, especially of strong-field effects. When direct observation of gravitational radiation from astrophysical sources begins, new tests of general relativity will be possible.Comment: 89 pages, 8 figures; an update of the Living Review article originally published in 2001; final published version incorporating referees' suggestion