The impact of a structured clinical training course on interns' self-reported confidence with core clinical urology skills.

BACKGROUND: Undergraduate training in core urology skills is lacking in many Irish training programmes. AIMS: Our aim was to assess newly qualified doctors' experience and confidence with core urological competencies. METHODS: A questionnaire survey covering exposure to urology and confidence with core clinical skills was circulated to all candidates. The group then attended a skills course covering male/female catheterisation, insertion of three-way catheters, bladder irrigation and management of long-term suprapubic catheters. The groups were re-surveyed following the course. RESULTS: Forty-five interns completed the pre-course questionnaire (group 1) and 27 interns completed the post-course questionnaire (group 2). 24/45 (53%) had no experience of catheter insertion on a patient during their undergraduate training. 26/45 (58%) were unsupervised during their first catheter insertion. 12/45 (27%) had inserted a female catheter. 18/45 (40%) had inserted a three-way catheter. 12/45 (27%) had changed a suprapubic catheter. 40/45 (89%) in group 1 reported 'good' or 'excellent' confidence with male urinary catheterisation, compared to 25/27 (92.5%) in group 2. 18/45 (40%) in group 1 reported 'none' or 'poor' confidence with female catheterisation, compared to 7/27 (26%) in group 2. 22/45 (49%) in group 1 reported 'none' or 'poor' confidence with insertion of three-way catheters, compared to 2/27 (7%) in group 2. 32/45 (71%) in group 1 reported 'none' or 'poor' confidence in changing long-term suprapubic catheters, falling to 3/27 (11%) in group 2. CONCLUSION: This study raises concerns about newly qualified doctors' practical experience in urology. We suggest that this course improves knowledge and confidence with practical urology skills and should be incorporated into intern induction.

Non-Dietary Correlates and Determinants of Plasma Lutein and Zeaxanthin Concentrations in the Irish Population.

OBJECTIVE: To investigate non-dietary correlates and determinants of plasma lutein (L) and zeaxanthin (Z) concentrations in The Irish Longitudinal Study on Ageing (TILDA) sample. DESIGN: Cross-sectional study. SETTING: Community dwelling adults in the Republic of Ireland (ROI). PARTICIPANTS: 3,681 participants aged 50 years and older. MEASUREMENTS: TILDA is a nationally representative prospective cohort study of community dwelling adults aged 50 years and over in the ROI. Demographic and health variables were collected during a face-to-face interview carried out in the home (n=8175), and a substantial proportion of these (n=5035; 62%) also attended a study visit in a health assessment centre. Blood samples collected at baseline (wave 1, the subject of the current study), were analysed for plasma concentrations of L and Z by reversed-phase high performance liquid chromatography, and macular pigment (MP) optical density was also measured (using customized heterochromatic flicker photometry). RESULTS: After excluding participants with eye disease, data from 3,681 participants were available for analysis. For this group of participants, plasma L and Z were inversely and significantly associated with body mass index (BMI), and were positively and significantly associated with MP, total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) (p<0.001, for all). Plasma L and Z were significantly lower in males, current smokers, participants reporting less physical exercise, and participants reporting lower levels of education (p<0.05, for all). Plasma L was significantly higher in participants reporting a family history of age-related macular degeneration (AMD) (p=0.001), and in the group of ≥75 years old (p<0.05). For each of these variables, the significant associations remained after controlling for other potential confounding variables. CONCLUSION: The findings of this large study indicate that plasma concentrations of L and Z were lower in association with indicators of a poor lifestyle (high BMI, tobacco use, and less physical exercise) and in association with lower education, indicating that modifying lifestyle in a positive way is likely to be reflected in higher concentrations of plasma carotenoids, with consequential and putative health benefits.

Phospholipid oxidation and carotenoid supplementation in Alzheimer's disease patients.

Alzheimer's disease (AD) is a progressive, neurodegenerative disease, characterised by decline of memory, cognitive function and changes in behaviour. Generic markers of lipid peroxidation are increased in AD and reactive oxygen species have been suggested to be involved in the aetiology of cognitive decline. Carotenoids are depleted in AD serum, therefore we have compared serum lipid oxidation between AD and age-matched control subjects before and after carotenoid supplementation. The novel oxidised phospholipid biomarker 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) was analysed using electrospray ionisation tandem mass spectrometry (MS) with multiple reaction monitoring (MRM), 8-isoprostane (IsoP) was measured by ELISA and ferric reducing antioxidant potential (FRAP) was measured by a colorimetric assay. AD patients (n=21) and healthy age-matched control subjects (n=16) were supplemented with either Macushield™ (10mg meso-zeaxanthin, 10mg lutein, 2mg zeaxanthin) or placebo (sunflower oil) for six months. The MRM-MS method determined serum POVPC sensitively (from 10µl serum) and reproducibly (CV=7.9%). At baseline, AD subjects had higher serum POVPC compared to age-matched controls, (p=0.017) and cognitive function was correlated inversely with POVPC (r=-0.37; p=0.04). After six months of carotenoid intervention, serum POVPC was not different in AD patients compared to healthy controls. However, POVPC was significantly higher in control subjects after six months of carotenoid intervention compared to their baseline (p=0.03). Serum IsoP concentration was unrelated to disease or supplementation. Serum FRAP was significantly lower in AD than healthy controls but was unchanged by carotenoid intervention (p=0.003). In conclusion, serum POVPC is higher in AD patients compared to control subjects, is not reduced by carotenoid supplementation and correlates with cognitive function.

The evidence informing the surgeon's selection of intraocular lens on the basis of light transmittance properties.

In recent years, manufacturers and distributors have promoted commercially available intraocular lenses (IOLs) with transmittance properties that filter visible short-wavelength (blue) light on the basis of a putative photoprotective effect. Systematic literature review. Out of 21 studies reporting on outcomes following implantation of blue-light-filtering IOLs (involving 8914 patients and 12 919 study eyes undergoing cataract surgery), the primary outcome was vision, sleep pattern, and photoprotection in 9 (42.9%), 9 (42.9%), and 3 (14.2%) respectively, and, of these, only 7 (33.3%) can be classed as high as level 2b (individual cohort study/low-quality randomized controlled trials), all other studies being classed as level 3b or lower. Of the level 2b studies, only one (14.3%) found in favor of blue-light-filtering IOLs vs ultraviolet (UV)-only filtering IOLs on the basis of an association between better post-operative contrast sensitivity (CS) at select frequencies with the former; however, that study did not measure or report CS preoperatively in either group, and the finding may simply reflect better preoperative CS in the eyes scheduled to be implanted with the blue-light-filtering IOL; moreover, that study failed to measure macular pigment, a natural preceptoral filter of blue-light, augmentation of which is now known to improve CS. In terms of photoprotection, there is no level 2b (or higher) evidence in support of blue filtering IOLs vs UV-only filtering IOLs. On the basis of currently available evidence, one cannot advocate for the use of blue-light-filtering IOLs over UV-only filtering IOLs.

Sustained supplementation and monitored response with differing carotenoid formulations in early age-related macular degeneration.

PURPOSE: To compare the impact of sustained supplementation using different macular carotenoid formulations on macular pigment (MP) and visual function in early age-related macular degeneration (AMD). PATIENTS AND METHODS: Sixty-seven subjects with early AMD were randomly assigned to: Group 1 (20 mg per day lutein (L), 0.86 mg per day zeaxanthin (Z); Ultra Lutein), Group 2 (10 mg per day meso-zeaxanthin (MZ), 10 mg per day L, 2 mg per day Z; Macushield; Macuhealth), Group 3 (17 mg per day MZ, 3 mg per day L, 2 mg per day Z). MP was measured using customised heterochromatic flicker photometry and visual function was assessed by measuring contrast sensitivity (CS) and best-corrected visual acuity (BCVA). AMD was graded using the Wisconsin Age-Related Maculopathy Grading System (AREDS 11-step severity scale). RESULTS: At 3 years, a significant increase in MP from baseline was observed in all groups at each eccentricity (P<0.05), except at 1.75° in Group 1 (P=0.160). Between 24 and 36 months, significant increases in MP at each eccentricity were seen in Group 3 (P<0.05 for all), and at 0.50° in Group 2 (P<0.05), whereas no significant increases were seen in Group 1 (P>0.05 for all). At 36 months, compared with baseline, the following significant improvements (P<0.05) in CS were observed: Group 2-1.2, 6, and 9.6 cycles per degree (c.p.d.); Group 1-15.15 c.p.d.; and Group 3-6, 9.6, and 15.15 c.p.d. No significant changes in BCVA, or progression to advanced AMD, were observed. CONCLUSION: In early AMD, MP can be augmented with a variety of supplements, although the inclusion of MZ may confer benefits in terms of panprofile augmentation and in terms of CS enhancement.

What is meso-zeaxanthin, and where does it come from?

The carotenoids lutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ) accumulate in the central retina, where they are collectively known as macular pigment (MP). Each of these three compounds exhibit a regional dominance, with MZ, Z, and L being the dominant carotenoids at the epicentre, mid-periphery, and periphery of the macula, respectively. There is a growing and evidence-based consensus that MP is important for optimal visual performance, because of its blue light-filtering properties and consequential attenuation of chromatic aberration, veiling luminance, and blue haze. It has also been hypothesised that MP may protect against age-related macular degeneration because of the same optical properties and also because of the antioxidant capacity of the three macular carotenoids. Challenges inherent in the separation and quantification of MZ have resulted in a paucity of data on the content of this carotenoid in foodstuffs, and have rendered the study of tissue concentrations of this compound problematic. As a consequence, the few studies that have investigated MZ have, perhaps, been disproportionately influential in the ongoing debate about the origins of this macular carotenoid. Certainly, the narrative that retinal MZ is derived wholly and solely from retinal L needs to be revisited.

The utility of using customized heterochromatic flicker photometry (cHFP) to measure macular pigment in patients with age-related macular degeneration.

The purpose of this study was to assess the utility and validity of using customized heterochromatic flicker photometry (cHFP) to measure macular pigment optical density (MPOD) in patients with intermediate stages of age-related macular degeneration (AMD). The measurement procedure was optimized to accommodate individual differences in temporal vision related to age, disease, or other factors. The validity criteria were based on the similarity of the spectral absorption curves to ex vivo curves of lutein and zeaxanthin and the similarity of spatial density profiles to those measured in subjects without retinal disease. Macular pigment optical density (MPOD) spatial profiles were measured with an LED-based macular densitometer; spectral absorption curves were measured with a 3-channel Maxwellian view system including a monochromator. All patients were characterized via clinical exams and all but 2 subjects from whom data were obtained had masked grading of color fundus photographs using the Wisconsin Age-Related Maculopathy Grading System. Most of the patients were in AREDS category 2 (27%) or 3 (57%). Patients with visual acuity as poor as 20/80 were included, and could perform the task as long as they could see the stimulus. Eighty-one percent of the patients screened were able to perform the cHFP task, and data were obtained from 30 AMD patients. Spatial profiles of MPOD were measured in 19 subjects who could see the stimulus at all tested loci. These profiles were highly similar to those that have been measured with HFP in subjects without retinal disease. The average shape of the spectral absorption curves for the AMD subjects corresponded well to an ex vivo template. These data support both the utility and validity of the cHFP method for measuring MPOD in subjects with intermediate stages of AMD. The ability to measure the retinal response to nutritional intervention is of practical importance for monitoring patients being supplemented with lutein and zeaxanthin in hopes of retarding visual loss and/or disease progression.

The rationale and evidence base for a protective role of macular pigment in age-related maculopathy.

Age-related maculopathy (ARM) remains the most common cause of blind registration in people aged 50 years or over in the developed world, and its prevalence continues to rise. Although effective new treatments have become available in the recent past, these are expensive and cumbersome to the healthcare provider and to the patient, and many cases remain resistant to such therapy. There is a biologically plausible rationale whereby macular pigment, which is entirely of dietary origin, may prevent or delay the onset, or ameliorate the clinical course, of ARM. In this article, we review this rationale, and critically appraise the current evidence base germane to the use of supplements containing the macular carotenoids in patients with, or at risk of developing, ARM.

Bacterial ribonuclease P holoenzyme crosslinking analysis reveals protein interaction sites on the RNA subunit.

The structure of the Escherichia coli ribonuclease P (RNase P) holoenzyme was investigated by site-directed attachment of an aryl azide crosslink reagent to specific sites in the protein subunit of the enzyme. The sites of crosslinking to the RNase P RNA subunit were mapped by primer extension to several conserved residues and structural features throughout the RNA. The results suggest rearrangement of current tertiary models of the RNA subunit, particularly in regions poorly constrained by earlier data. Crosslinks to the substrate precursor-tRNA were also detected, consistent with previous crosslinking results in the Bacillus subtilis RNase P holoenzyme.

A practical six-step synthesis of (S)-camptothecin.

An asymmetric synthesis of (S)-camptothecin (1) has been accomplished in six steps starting from two commercially available heterocycles. [reaction: see text]

Guanosine 2-NH2 groups of Escherichia coli RNase P RNA involved in intramolecular tertiary contacts and direct interactions with tRNA.

We have identified by nucleotide analog interference mapping (NAIM) exocyclic NH2 groups of guanosines in RNase P RNA from Escherichia coli that are important for tRNA binding. The majority of affected guanosines represent phylogenetically conserved nucleotides. Several sites of interference could be assigned to direct contacts with the tRNA moiety, whereas others were interpreted as reflecting indirect effects on tRNA binding due to the disruption of tertiary contacts within the catalytic RNA. Our results support the involvement of the 2-NH2 groups of G292/G293 in pairing with C74 and C75 of tRNA CCA-termini, as well as formation of two consecutive base triples involving C75 and A76 of CCA-ends interacting with G292/A258 and G291/G259, respectively. Moreover, we present first biochemical evidence for two tertiary contacts (L18/P8 and L8/P4) within the catalytic RNA, whose formation has been postulated previously on the basis of phylogenetic comparative analyses. The tRNA binding interference data obtained in this and our previous studies are consistent with the formation of a consecutive nucleotide triple and quadruple between the tetraloop L18 and helix P8. Formation of the nucleotide triple (G316 and A94:U104 in wild-type E. coli RNase P RNA) is also supported by mutational analysis. For the mutant RNase P RNA carrying a G94:C104 double mutation, an additional G316-to-A mutation resulted in a restoration of binding affinity for mature and precursor tRNA.

Analysis of the conformation of the 3' major domain of Escherichia coli16S ribosomal RNA using site-directed photoaffinity crosslinking.

The 3' major domain of Escherichia coli 16S rRNA, which occupies the head of the small ribosomal subunit, is involved in several functions of the ribosome. We have used a site-specific crosslinking procedure to gain further insights into the higher-order structure of this domain. Circularly permuted RNAs were used to introduce an azidophenacyl group at specific positions within the 3' major domain. Crosslinks were generated in a high-ionic strength buffer that has been used for ribosome reconstitution studies and so enables the RNA to adopt a structure recognized by ribosomal proteins. The crosslinking sites were identified by primer extension and confirmed by assessing the mobility of the crosslinked RNA lariats in denaturing polyacrylamide gels. Eight crosslinks were characterized. Among them, one crosslink demonstrates that helix 28 is proximal to the top of helix 34, and two others show that the 1337 region, located in an internal loop at the junction of helices 29, 30, 41, and 42, is proximal to the center of helix 30 and to a segment connecting helix 28 to helix 29. These relationships of vicinity have previously been observed in native 30S subunits, which suggests that the free domain adopts a conformation similar to that within the 30S subunit. Furthermore, crosslinks were obtained in helix 34, which suggest that the upper and lower portions of this helix are in close proximity.

Comparative photocross-linking analysis of the tertiary structures of Escherichia coli and Bacillus subtilis RNase P RNAs.

Bacterial ribonuclease P contains a catalytic RNA subunit that cleaves precursor sequences from the 5' ends of pre-tRNAs. The RNase P RNAs from Bacillus subtilis and Escherichia coli each contain several unique secondary structural elements not present in the other. To understand better how these phylogenetically variable elements affect the global architecture of the ribozyme, photoaffinity cross-linking studies were carried out. Photolysis of photoagents attached at homologous sites in the two RNAs results in nearly identical cross-linking patterns, consistent with the homology of the RNAs and indicating that these RNAs contain a common, core tertiary structure. Distance constraints were used to derive tertiary structure models using a molecular mechanics-based modeling protocol. The resulting superimposition of large sets of equivalent models provides a low resolution (5-10 A) structure for each RNA. Comparison of these structure models shows that the conserved core helices occupy similar positions in space. Variably present helical elements that may play a role in global structural stability are found at the periphery of the core structure. The P5.1 and P15.1 helical elements, unique to the B.subtilis RNase P RNA, and the P6/16/17 helices, unique to the E.coli RNA, occupy similar positions in the structure models and, therefore, may have analogous structural function.

Comparative analysis of ribonuclease P RNA using gene sequences from natural microbial populations reveals tertiary structural elements.

PCR amplification of template DNAs extracted from mixed, naturally occurring microbial populations, using oligonucleotide primers complementary to highly conserved sequences, was used to obtain a large collection of diverse RNase P RNA-encoding genes. An alignment of these sequences was used in a comparative analysis of RNase P RNA secondary and tertiary structure. The new sequences confirm the secondary structure model based on sequences from cultivated organisms (with minor alterations in helices P12 and P18), providing additional support for nearly every base pair. Analysis of sequence covariation using the entire RNase P RNA data set reveals elements of tertiary structure in the RNA; the third nucleotides (underlined) of the GNRA tetraloops L14 and L18 are seen to interact with adjacent Watson-Crick base pairs in helix P8, forming A:G/C or G:A/U base triples. These experiments demonstrate one way in which the enormous diversity of natural microbial populations can be used to elucidate molecular structure through comparative analysis.

Analysis of conserved positions in nuclear RNase P RNA.

Secondary structure models of eubacterial and eukaryotic nuclear RNase P RNA subunits show extensive structural similarities, allowing the identification of highly conserved nucleotide positions and molecular modeling of the enzyme-substrate complex in three dimensions. Based on this information, we present a preliminary tertiary structure model of the yeast nuclear RNase P RNA. In addition, the most conserved positions in the structure have been subjected to sequence randomization, with viable sequence variations identified by selection in vivo and characterized for phenotypic consequences.

Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3-D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. A molecular mechanics-based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre-tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.

Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure.

Regions of Escherichia coli ribonuclease P (RNase P) RNA in proximity to a bound transfer RNA (tRNA) substrate were mapped by photoaffinity. A photoaffinity cross-linking reagent was introduced at specific sites in the interior of the native tRNA structure by modification of the 5' ends of circularly permuted tRNAs (cptRNAs). The polymerase chain reaction was used for the production of cptRNA templates. After the amplification of a segment of a tandemly duplicated tRNA gene, the cptRNA gene was transcribed in vitro to produce cptRNA. Modified cptRNAs were cross-linked to RNase P RNA, and the conjugation sites in RNase P RNA were determined by primer extension. These sites occur in phylogenetically conserved structures and sequences and identify regions of the ribozyme that form part of the tRNA binding site. The use of circularly permuted molecules to position specific modifications is applicable to the study of many inter- and intramolecular interactions.

Effects of cigarette smoking on solid and liquid intragastric distribution and gastric emptying.

BACKGROUND: The acute effects of cigarette smoking on gastric emptying are controversial, whereas its effects on the intragastric distribution of solids and liquids are not established. METHODS: Dual isotope gastric scintigraphy was performed in 15 habitual smokers (studied twice, either sham smoking or actively smoking) and in 15 age- and sex-matched nonsmokers. RESULTS: Acute smoking was associated with an increased prevalence of episodes of retrograde intragastric movement of solids (3 of 15 sham subjects vs. 12 of 15 actively smoking subjects; P < 0.01) and of liquids (0 of 15 vs. 7 of 15; P < 0.01) from distal to proximal stomach. Fundal half-emptying time (T1/2) for liquids was also prolonged by smoking (43 +/- 19 minutes sham vs. 125 +/- 216 minutes active; P < 0.05). Acute smoking delayed solid lag time (13 +/- 6 minutes sham vs. 32 +/- 18 active; P < 0.05) and liquid T1/2 (46 +/- 21 vs. 90 +/- 50 minutes; P < 0.05). In the nonsmokers, such episodes of proximal intragastric redistribution did not occur, and intragastric and overall emptying parameters did not differ significantly from those of habitual sham smokers. CONCLUSIONS: Acute cigarette smoking produces excessive antrofundal redistribution of both solid and liquid contents and delays solid and liquid gastric emptying.