RNA binding properties of the AU-rich element-binding recombinant Nup475/TIS11/tristetraprolin protein

Regulation of messenger RNA stability by AU-rich elements is an important means of regulating genes induced by growth factors and cytokines. Nup475 (also known as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding Cys(3)His motif proteins required for proper regulation of tumor necrosis factor mRNA stability in macrophages. We developed an Escherichia coli expression system to produce soluble Nup475 protein in quantity to study its RNA binding properties. Nup475 protein bound a tumor necrosis factor AU-rich element over a broad range of pH and salt concentrations by RNA gel shift. This binding was inhibited by excess zinc metal, providing a potential mechanism for previous reports of zinc stabilization of AU-rich element (ARE) containing messenger RNAs. Immobilized Nup475 protein was used to select its optimal binding site by RNA SELEX and revealed a strong preference for the extended sequence UUAUUUAUU, rather than a simple AUUUA motif. These findings were confirmed by site-directed mutagenesis of the tumor necrosis factor ARE and RNA gel shifts on c-fos, interferon-gamma, and interferon-beta ARE fragments. A weaker binding activity toward adenine-rich sites, such as a poly(A) tail RNA fragment, can partially disrupt the Nup475-tumor necrosis factor AU-rich element complex.     

Muscle contraction: energy rate equations in relation to efficiency and step-size distance

We derive the energy rate equation for muscle contraction. Our equation has only two parameters m, the maintenance heat rate and 1/S, the shortening heat coefficient. The impulsive model (previously described in earlier papers) provides a physical basis for parameter 1/S as well as for constants a and b in Hill’s force–velocity equation. We develop new theory and relate the efficiency and the step-size distance to our energy rate equation. Correlation between the efficiency and the step-size distance is established. The various numbers are listed in Table 1: we use data from five different muscles in the literature. In summary, our analysis strongly supports the impulsive model as the correct model of contraction.     

Association of PADI4 and rheumatoid arthritis: a successful multidisciplinary approach

The identification of functionally relevant polymorphisms of peptidylarginine deiminase 4, an enzyme that catalyzes the post-translational citrullination of proteins, as a rheumatoid arthritis gene is one of the most convincing success stories of complex disease gene mapping to date. In addition to an extensive single nucleotide polymorphism-based association study in a Japanese cohort, a range of techniques have been used to validate this finding.     

Muscle contraction: viscous-like frictional forces and the impulsive model

Apart from a few experimental studies muscle viscosity has not received much recent analytical attention as a determinant of the contractile process. This is surprising, since any muscle cell is 80% water, and may undergo large shape changes during its working cycle. Intuitively one might expect the viscosity of the solvent to be an important determinant of the physiological activity of muscle tissue. This was apparent to pioneers of the study of muscle contraction such as Hill and his contemporaries, whose putative theoretical formulations contained terms related to muscle viscosity. More recently, though, a hydrodynamic calculation by Huxley, using a solvent viscosity close to that of water, has been held to demonstrate that viscous forces are negligible in muscle contraction. We have re-examined the role of viscosity in contraction, postulating impulsive acto-myosin forces that are opposed by a viscous resistance between the filaments. The viscous force required, 10⁴ times the hydrodynamic estimate, is close to recent experimental measurements, themselves 10²–10³ times the hydrodynamic estimate. This also agrees with contemporary measurements of cytoplasmic viscosity in other biological cells using magnetic bead micro-rheometry. These are several orders of magnitude greater than the viscosity of water. In the course of the analysis we have derived the force-velocity equation for an isolated half-sarcomere containing a single actin filament for the first time, and from first principles. We conclude that muscle viscosity is indeed important for the contractile process, and that it has been too readily discounted.     

PATTERNS OF FLORAL NECTAR-SUGAR COMPOSITION OF IPOMOPSIS LONGIFLORA (POLEMONIACEAE) NEAR THE CONTACT ZONE OF ITS SUBSPECIES LONGIFLORA AND AUSTRALIS

The nectar-sugar compositions of 338 individuals from 27 populations in two subspecies of Ipomopsis longiflora from Arizona, New Mexico, Texas, and Chihuahua were determined by high-performance liquid chromatography. Mean sucrose compositions of populations ranged from 73.2% to 91.9%, and the standard deviations ranged from 1.9% to 9.1%. Pairs and aggregates of populations were statistically compared. The nectar-sugar compositions of the two subspecies were significantly different as aggregates. Subspecies australis averaged 9.5% fructose, 7.9% glucose, and 82.6% sucrose while subspecies longiflora averaged 7.8% fructose, 5.7% glucose, and 86.5% sucrose. Each subspecies was found to have two groups of populations. Groups of higher sucrose populations were found along the contact zone of the subspecies; away from the contact zone both subspecies had groups of lower sucrose populations. The data show that significant variability exists in this biochemical character, and the pattern of variation is clearly related to the geographic distribution of the populations. The genetic and selective mechanisms involved are unknown.     

Discrepancies in Outcome Reporting Exist Between Protocols and Published Oral Health Cochrane Systematic Reviews

Objectives

To assess discrepancies in the analyzed outcomes between protocols and published reviews within Cochrane oral health systematic reviews (COHG) on the Cochrane Database of Systematic Reviews (CDSR).

Study Design and Setting

All COHG systematic reviews on the CDSR and the corresponding protocols were retrieved in November 2014 and information on the reported outcomes was recorded. Data was collected at the systematic review level by two reviewers independently.

Results

One hundred and fifty two reviews were included. In relation to primary outcomes, 11.2% were downgraded to secondary outcomes, 9.9% were omitted altogether in the final publication and new primary outcomes were identified in 18.4% of publications. For secondary outcomes, 2% were upgraded to primary, 12.5% were omitted and 30.9% were newly introduced in the publication. Overall, 45.4% of reviews had at least one discrepancy when compared to the protocol; these were reported in 14.5% reviews. The number of review updates appears to be associated with discrepancies between final review and protocol (OR: 3.18, 95% CI: 1.77, 5.74, p<0.001). The risk of reporting significant results was lower for both downgraded outcomes [RR: 0.52, 95% CI: 0.17, 1.58, p = 0.24] and upgraded or newly introduced outcomes [RR: 0.77, 95% CI: 0.36, 1.64, p = 0.50] compared to outcomes with no discrepancies. The risk of reporting significant results was higher for upgraded or newly introduced outcomes compared to downgraded outcomes (RR = 1.19, 95% CI: 0.65, 2.16, p = 0.57). None of the comparisons reached statistical significance.

Conclusion

While no evidence of selective outcome reporting was found in this study, based on the present analysis of SRs published within COHG systematic reviews, discrepancies between outcomes in pre-published protocols and final reviews continue to be common. Solutions such as the use of standardized outcomes to reduce the prevalence of this issue may need to be explored.     

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.