Resolution of Two Sub-Populations of Conformers and Their Individual Dynamics by Time Resolved Ensemble Level FRET Measurements

Most active biopolymers are dynamic structures; thus, ensembles of such molecules should be characterized by distributions of intra- or intermolecular distances and their fast fluctuations. A method of choice to determine intramolecular distances is based on Förster resonance energy transfer (FRET) measurements. Major advances in such measurements were achieved by single molecule FRET measurements. Here, we show that by global analysis of the decay of the emission of both the donor and the acceptor it is also possible to resolve two sub-populations in a mixture of two ensembles of biopolymers by time resolved FRET (trFRET) measurements at the ensemble level. We show that two individual intramolecular distance distributions can be determined and characterized in terms of their individual means, full width at half maximum (FWHM), and two corresponding diffusion coefficients which reflect the rates of fast ns fluctuations within each sub-population. An important advantage of the ensemble level trFRET measurements is the ability to use low molecular weight small-sized probes and to determine nanosecond fluctuations of the distance between the probes. The limits of the possible resolution were first tested by simulation and then by preparation of mixtures of two model peptides. The first labeled polypeptide was a relatively rigid Pro₇ and the second polypeptide was a flexible molecule consisting of (Gly-Ser)₇ repeats. The end to end distance distributions and the diffusion coefficients of each peptide were determined. Global analysis of trFRET measurements of a series of mixtures of polypeptides recovered two end-to-end distance distributions and associated intramolecular diffusion coefficients, which were very close to those determined from each of the pure samples. This study is a proof of concept study demonstrating the power of ensemble level trFRET based methods in resolution of subpopulations in ensembles of flexible macromolecules.     

Video Capsule Endoscopy in Patients with Chronic Abdominal Pain with or without Associated Symptoms: A Retrospective Study

Background

Chronic abdominal pain (CAP) is a common indication for gastroenterology referrals. More insidious causes of CAP isolated to the small bowel, such as malignancies and Crohn’s disease, are rising in incidence and causing more gastroenterologists to evaluate their patients with video capsule endoscopy (VCE). However, the role of VCE in patients with CAP is still unclear.

Aims

We assessed the efficacy of VCE in patients with CAP and whether it led to findings that contributed to disease management and meaningful interventions.

Methods

This retrospective study evaluated 607 capsule endoscopy studies at an open referral endoscopy unit. Ninety of the studies were for CAP. These studies were compared to those performed for other indications to compare diagnostic yield. In addition, we investigated whether VCE led to an intervention that improved clinical outcomes.

Results

Overall, the number of abnormal findings in CAP patients was significantly lower than VCE performed for other indications (24.4% vs 39.0%, respectively p = 0.009). When patients with CAP presented with other pertinent clinical findings (e.g. nausea, weight loss, anemia, history of in inflammatory bowel disease, etc.), the likelihood of an abnormal finding increased to a level that was not different from those who received VCE for other indications (27.1%, p = 0.10). The findings from VCE lead to changed management and improved outcomes in 16.2% of CAP patients with associated symptoms. However, the subgroup that benefited the most were those who had a prior history of Crohn’s disease. Patients with CAP who did not have any associated symptoms continued to have a significantly lower abnormal finding rate compared to those who received VCE for other indications (19.4%, p = 0.03) and VCE rarely led to a change in management that would improve outcomes (5.6%).

Conclusions

VCE for CAP has a lower rate of abnormal findings than other indications. However, VCE is a useful diagnostic tool that can help provide a possible etiology of CAP in patients with associated symptoms. However, a change in management from VCE is likely to be limited to those with a history of Crohn’s disease.     

Psychometric Properties of the Problem Areas in Diabetes (PAID) Instrument in Singapore

Background

Emotional distress is an important dimension in diabetes, and several instruments have been developed to measure this aspect. The Problem Areas in Diabetes (PAID) scale is one such instrument which has demonstrated validity and reliability in Western populations, but its psychometric properties in Asian populations have not been examined.

Methods

This was a secondary analysis of data from patients with Type 2 diabetes mellitus recruited through convenience sampling from a diabetes specialist outpatient clinic in Singapore. The following psychometric properties were assessed: Construct validity through confirmatory factor analysis (CFA) and Rasch analysis, concurrent validity through correlation with related scales (Kessler Psychological Distress Scale, Diabetes Health Profile—psychological distress, Audit of Diabetes Dependent Quality of Life), reliability through assessment of internal consistency and floor and ceiling effects, and sensitivity by estimating effect sizes for known clinical and social functioning groups.

Results

203 patients with mean age of 45±12 years were analysed. None of the previously published model structures achieved a good fit on CFA. On Rasch analysis, four items showed poor fit and were removed. The abridged 16-item PAID mapped to a single latent trait, with a high degree of internal consistency (Cronbach ɑ 0.95), but significant floor effect (24.6% scoring at floor). Both 20-item and 16-item PAID scores were moderately correlated with scores of related scales, and sensitive to differences in clinical and social functioning groups, with large effect sizes for glycemic control and diabetes related complications, nephropathy and neuropathy.

Conclusion

The abridged 16-item PAID measures a single latent trait of emotional distress due to diabetes whereas the 20-item PAID appears to measures more than one latent trait. However, both the 16-item and 20-item PAID versions are valid, reliable and sensitive for use among Singaporean patients with diabetes.     

Is ‘Self-Medication’ a Useful Term to Retrieve Related Publications in the Literature? A Systematic Exploration of Related Terms

BackgroundSelf-Medication (SM), i.e. using medications to treat oneself, is a major concern for health researchers and policy makers. The terms “self medication” or “self-medication” (SM terms) have been used to explain various concepts while several terms have also been employed to define this practice. Hence, retrieving relevant publications would require exhaustive literature screening. So, we assessed the current situation of SM terms in the literature to improve the relevancy of search outcomes.MethodsIn this Systematic exploration, SM terms were searched in the 6 following databases and publisher’s portals till April 2012: Web of Science, Scopus, PubMed, Google scholar, ScienceDirect, and Wiley. A simple search query was used to include only publications with SM terms. We used Relative-Risk (RR) to estimate the probability of SM terms use in related compared to unrelated publications. Sensitivity and specificity of SM terms as keywords in search query were also calculated. Relevant terms to SM practice were extracted and their Likelihood Ratio positive and negative (LR+/-) were calculated to assess their effect on the probability of search outcomes relevancy in addition to previous search queries. We also evaluated the content of unrelated publications. All mentioned steps were performed in title (TI) and title or abstract (TIAB) of publications.Results1999 related and 1917 unrelated publications were found. SM terms RR was 4.5 in TI and 2.1 in TIAB. SM terms sensitivity and specificity respectively were 55.4% and 87.7% in TI and 84.0% and 59.5% in TIAB. “OTC” and “Over-The-Counter Medication”, with LR+ 16.78 and 16.30 respectively, provided the most conclusive increase in the probability of the relevancy of publications. The most common unrelated SM themes were self-medication hypothesis, drug abuse and Zoopharmacognosy.ConclusionsDue to relatively low specificity or sensitivity of SM terms, relevant terms should be employed in search queries and clear definitions of SM applications should be applied to improve the relevancy of publications.     

Complete Genome Sequence and Genome Analysis of Eggplant mottled dwarf virus‐Iranian Isolate

The full‐length nucleotide sequence of the Iranian isolate of Eggplant mottled dwarf virus (EMDV), a phytorhabdovirus, was determined using the random polymerase chain reaction method (rPCR) followed by PCR with specific primers to fill in the gaps. The negative‐sense RNA genome of the Iranian isolate of EMDV contains 13154 nucleotides and seven open‐reading frames (ORFs) in the order 3′‐leader‐N‐X‐P‐Y‐M‐G‐L‐trailer‐5′. These ORFs encode the nucleocapsid, X protein (of unknown function), phosphoprotein, Y protein (putative movement protein), matrix protein, glycoprotein and RNA‐dependent RNA polymerase, respectively. EMDV has a 199 nt 3′ leader RNA and a 151 nt 5′ trailer, and the ORFs are separated by conserved intergenic sequences. Phylogenetic analyses indicate that EMDV is most closely related to Potato yellow dwarf virus, which has a distinctly different geographical distribution.     

Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP)

Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct subcellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11 family of interacting proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain at their C termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic, and cellular analyses of the interactions between Rab14 and one of the class I FIPs, the Rab-coupling protein (RCP), to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11-FIP and Rab25-FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a noncanonical 1:2 stoichiometry between Rab14-RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14-RCP reveals that Rab14 interacts with the canonical Rab-binding domain and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab coupling protein and Rab14 function in neuritogenesis.