Internal bulge and tetraloop of the catalytic domain 5 of a group II intron ribozyme are flexible: implications for catalysis

RNA molecules have an inherent flexibility that enables recognition of other interacting partners through potential disorder-order transitions, yet studies to quantify such motional dynamics remain few. With an increasing database of three-dimensional structures of biologically important RNA molecules, quantifying such motions becomes important to link structural deformations with function. One such system studied intensely is domain 5 (D5) from the self-splicing group II introns, which is at the heart of its catalytic machinery. We report the dynamics of a 36 nucleotide D5 from the Pylaiella littoralis group II intron in the presence and absence of magnesium ions, and at a range of temperatures (298K-318 K). Using high-resolution NMR experiments of heteronuclear nuclear Overhauser enhancement (NOE), spin-lattice (R(1)), and spin-spin (R(2)) (13)C relaxation rates, we determined the rotational diffusion tensor of D5 using the ROTDIF program modified for RNA dynamic analysis (ROTDIF_RNA). The D5 rotational diffusion tensor has an axial symmetric ratio (D(||)/D(perpendicular)) of 1.7+/-0.3, consistent with an estimated overall rotational correlation time of tau(m)=(2D(||)+4D(perpendicular))(-1) of 6.1(+/-0.3) ns at 298 K and 4.1(+/-0.2) ns at 318 K. The measured relaxation data were analyzed with the reduced spectral density mapping formalism using assumed values of the chemical shift anisotropy of the (13)C spins. Both the relaxation data and the values of the spectral density function reveal that the functional groups in D5 implicated in magnesium ion binding and catalysis (catalytic triad, internal bulge, and tetraloop regions) exhibit thermally induced motion on a wide variety of timescales. Because these motions parallel those observed in the intramolecular stem-loop of the U6 element within the spliceosome, we hypothesize that such extensive dynamic disorder likely facilitates D5 engaging both binding and catalytic regions of the ribozyme, and these may be a conserved feature of the catalytic machinery essential for catalysis.     

DNA sequence and haplotype variation in two candidate genes for dilated cardiomyopathy in the turkey Meleagris gallopavo

Determining variation in genes is fundamental to understanding their function in the disease state. Cardiac troponin T (cTnT) and phospholamban (PLN) genes have been implicated in dilated cardiomyopathy (DCM) in human and model species. To investigate the role of these 2 candidate genes in DCM in the turkey Meleagris gallopavo, understanding sequence variants and map position distribution is necessary. To this end, a total of 1854 and 1771 bp of cTnT and PLN gene sequences, respectively, were scanned for single nucleotide polymorphisms (SNPs) in a randomly bred population. A total of 15 SNPs was identified in the cTnT and PLN genomic sequences. Nine haplotypes, 5 in cTnT and 4 in PLN, were identified. Observed heterozygosities (0.02-0.39) in the turkey population were low for both genes. Within each gene, 1 SNP corresponding to a restriction enzyme site was identified and used to develop a PCR-restriction fragment length polymorphism (RFLP) genotyping assay. The PLN gene was genetically mapped to turkey chromosome 2, equivalent to Gallus gallus chromosome 3, and cTnT mapped to a turkey microchromosome. Although limited because of the relatively small sample size of 55 birds, the data from this SNP analysis of PLN and cTnT provide a foundation from which to evaluate the function of cTnT and PLN in the turkey. Information about the distribution of the SNPs and haplotypes will facilitate future association and linkage studies.     

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on (13)C-2-glycerol and (13)C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in (13)C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3' and C5' carbon positions. Consequently the C1', C2' and C4' positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with (13)C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a (13)C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4' carbon position, such that the C2' and C3' positions suffer from multiplet splitting but the C5' position remains singlet and the C1' position shows a small amount of residual C1'-C2' coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with (13)C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5' position (~90%) that makes it particularly attractive for NMR applications involving CH(2)-TROSY modules without the need for decoupling the C4' carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems.     

Default Mode,Dorsal Attention and Auditory Resting State Networks Exhibit Differential Functional Connectivity in Tinnitus and Hearing Loss

We investigated auditory, dorsal attention, and default mode networks in adults with tinnitus and hearing loss in a resting state functional connectivity study. Data were obtained using continuous functional magnetic resonance imaging (fMRI) while the participants were at “rest” and were not performing any task. Participants belonged to one of three groups: middle-aged adults with tinnitus and mild-to-moderate high frequency hearing loss (TIN), age-matched controls with normal hearing and no tinnitus (NH), and a second control group with mild-to-moderate high frequency hearing loss without tinnitus (HL). After standard preprocessing, (a) a group independent component analysis (ICA) using 30 components and (b) a seeding-based connectivity analysis were conducted. In the group ICA, the default mode network was the only network to display visual differences between subject groups. In the seeding analysis, we found increased connectivity between the left parahippocampus and the auditory resting state network in the TIN group when compared to NH controls. Similarly, there was also an increased correlation between the right parahippocampus and the dorsal attention network when compared to HL controls. Other group differences in this attention network included decreased correlations between the seed regions and the right supramarginal gyrus in TIN patients when compared to HL controls. In the default mode network, there was a strong decrease in correlation between the seed regions and the precuneus when compared to both control groups. The findings of this study identify specific alterations in the connectivity of the default mode, dorsal attention, and auditory resting state networks due to tinnitus. The results suggest that therapies for tinnitus that mitigate the increased connectivity of limbic regions with auditory and attention resting state networks and the decreased coherence of the default mode network could be effective at reducing tinnitus-related distress.     

Fibroblast Growth Factor–21 Ameliorates Rheumatoid Arthritis by Maintaining Articular Integrity

International Journal of Peptide Research and Therapeutics - Rheumatoid arthritis, a chronic degenerative autoimmune disease is hallmarked by tenacious inflammation of synovial membranes causing...     

The erythrocyte receptor for Fusobacterium necrophorum hemolysin: phosphatidylcholine as a possible candidate

An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A₂ but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A₂ were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.     

Multiple conformations of SAM-II riboswitch detected with SAXS and NMR spectroscopy

Riboswitches are a newly discovered large family of structured functional RNA elements that specifically bind small molecule targets out of a myriad of cellular metabolites to modulate gene expression. Structural studies of ligand-bound riboswitches by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have provided insights into detailed RNA-ligand recognition and interactions. However, the structures of ligand-free riboswitches remain poorly characterized. In this study, we have used a variety of biochemical, biophysical and computational techniques including small-angle X-ray scattering and NMR spectroscopy to characterize the ligand-free and ligand-bound forms of SAM-II riboswitch. Our data demonstrate that the RNA adopts multiple conformations along its folding pathway and suggest that the RNA undergoes marked conformational changes upon Mg(2+) compaction and S-adenosylmethionine (SAM) metabolite binding. Further studies indicated that Mg(2+) ion is not essential for the ligand binding but can stabilize the complex by facilitating loop/stem interactions. In the presence of millimolar concentration of Mg(2+) ion, the RNA samples a more compact conformation. This conformation is near to, but distinct from, the native fold and competent to bind the metabolite. We conclude that the formation of various secondary and tertiary structural elements, including a pseudoknot, occur to sequester the putative Shine-Dalgarno sequence of the RNA only after metabolite binding.     

Factors Influencing Health Facility Delivery in Predominantly Rural Communities across the Three Ecological Zones in Ghana: A Cross-Sectional Study

Background

Maternal and neonatal mortality indicators remain high in Ghana and other sub-Saharan African countries. Both maternal and neonatal health outcomes improve when skilled personnel provide delivery services within health facilities. Determinants of delivery location are crucial to promoting health facility deliveries, but little research has been done on this issue in Ghana. This study explored factors influencing delivery location in predominantly rural communities in Ghana.

Methods

Data were collected from 1,500 women aged 15–49 years with live or stillbirths that occurred between January 2011 and April 2013. This was done within the three sites operating Health and Demographic Surveillance Systems, i.e., the Dodowa (Greater Accra Region), Kintampo (Brong Ahafo Region), and Navrongo (Upper-East Region) Health Research Centers in Ghana. Multivariable logistic regression was used to identify the determinants of delivery location, controlling for covariates that were statistically significant in univariable regression models.

Results

Of 1,497 women included in the analysis, 75.6% of them selected health facilities as their delivery location. After adjusting for confounders, the following factors were associated with health facility delivery across all three sites: healthcare provider’s influence on deciding health facility delivery, (AOR = 13.47; 95% CI 5.96–30.48), place of residence (AOR = 4.49; 95% CI 1.14–17.68), possession of a valid health insurance card (AOR = 1.90; 95% CI 1.29–2.81), and socio-economic status measured by wealth quintiles (AOR = 2.83; 95% CI 1.43–5.60).

Conclusion

In addition to known factors such as place of residence, socio-economic status, and possession of valid health insurance, this study identified one more factor associated with health facility delivery: healthcare provider’s influence. Ensuring care provider’s counseling of clients could improve the uptake of health facility delivery in rural communities in Ghana.     

Second-generation aryl isonitrile compounds targeting multidrug-resistant Staphylococcus aureus

Antibiotic resistance remains a major global public health threat that requires sustained discovery of novel antibacterial agents with unexploited scaffolds. Structure-activity relationship of the first-generation aryl isonitrile compounds we synthesized led to an initial lead molecule that informed the synthesis of a second-generation of aryl isonitriles. From this new series of 20 compounds, three analogues inhibited growth of methicillin-resistant Staphylococcus aureus (MRSA) (from 1 to 4?µM) and were safe to human keratinocytes. Compound 19, with an additional isonitrile group exhibited improved activity against MRSA compared to the first-generation lead compound. This compound emerged as a candidate worthy of further investigation and further reinforced the importance of the isonitrile functionality in the compounds’ anti-MRSA activity. In a murine skin wound model, 19 significantly reduced the burden of MRSA, similar to the antibiotic fusidic acid. In summary, 19 was identified as a new lead aryl isonitrile compound effective against MRSA.     

The influence of medication on salivary flow of the elderly: preliminary study

doi:10.1111/j.1741‐2358.2009.00326.x
The influence of medication on salivary flow of the elderly: preliminary study Objective: This study was to evaluate the influence of medications on unstimulated and stimulated salivary flow in elderly men and women. Background: Several diseases and conditions are associated with decreasing salivary flow. Medications can be risk factors for hyposalivation due to the effect particular drug categories. Methods: Seventy‐five elderly of both gender (sixty years old or over) from the Geriatric Dental Clinic at the Federal Fluminense University Dental School, were interviewed about their health status and chronic use of medication. After the interview, unstimulated and stimulated saliva were collected from each subject. The collection time was five minutes, and the flow rate was calculated as ml/min. Results: The mean unstimulated salivary flow was 0.25 ml/min for women and 0.30 ml/min for men, while the mean for stimulated salivary flow was 1.23 ml/min for women and 1.31 ml/min for men, without both differences being non‐significant (p > 0.05). The difference between the mean production of unstimulated and stimulated salivary flow was statistically significant, regardless of gender (p < 0.01). A significant reduction of stimulated salivary flow was observed with the usage of cardiovascular agents (p < 0.05). Conclusion: Drugs used in cardiovascular disease influence the flow of stimulated saliva.