N-Way FRET Microscopy of Multiple Protein-Protein Interactions in Live Cells

Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.     

Anti-Inflammatory Effects of Adult Stem Cells in Sustained Lung Injury: A Comparative Study

Lung diseases are a major cause of global morbidity and mortality that are treated with limited efficacy. Recently stem cell therapies have been shown to effectively treat animal models of lung disease. However, there are limitations to the translation of these cell therapies to clinical disease. Studies have shown that delayed treatment of animal models does not improve outcomes and that the models do not reflect the repeated injury that is present in most lung diseases. We tested the efficacy of amnion mesenchymal stem cells (AM-MSC), bone marrow MSC (BM-MSC) and human amniotic epithelial cells (hAEC) in C57BL/6 mice using a repeat dose bleomycin-induced model of lung injury that better reflects the repeat injury seen in lung diseases. The dual bleomycin dose led to significantly higher levels of inflammation and fibrosis in the mouse lung compared to a single bleomycin dose. Intravenously infused stem cells were present in the lung in similar numbers at days 7 and 21 post cell injection. In addition, stem cell injection resulted in a significant decrease in inflammatory cell infiltrate and a reduction in IL-1 (AM-MSC), IL-6 (AM-MSC, BM-MSC, hAEC) and TNF-α (AM-MSC). The only trophic factor tested that increased following stem cell injection was IL-1RA (AM-MSC). IL-1RA levels may be modulated by GM-CSF produced by AM-MSC. Furthermore, only AM-MSC reduced collagen deposition and increased MMP-9 activity in the lung although there was a reduction of the pro-fibrogenic cytokine TGF-β following BM-MSC, AM-MSC and hAEC treatment. Therefore, AM-MSC may be more effective in reducing injury following delayed injection in the setting of repeated lung injury.     

Production of cyclodextrin glycosyltransferase by immobilized <Emphasis Type="Italic">Bacillus</Emphasis> sp. on chitosan matrix

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol–sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml^?1 (36 h), 47.50 U ml^?1 (36 h) and 68.36 U ml^?1 (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml^?1 (18 h) on cassava, 79.17 U ml^?1 (12 h) on potato and 55.37 U ml^?1 (in 6 h and max 77.75 U ml^?1 in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.     

Reliability and Validity of an Interviewer-Administered Adaptation of the Youth Self-Report for Mental Health Screening of Vulnerable Young People in Ethiopia

Objective

Evaluate the reliability and validity of the Youth Self-Report (YSR) as a screening tool for mental health problems among young people vulnerable to HIV in Ethiopia.

Design

A cross-sectional assessment of young people currently receiving social services.

Methods

Young people age 15–18 participated in a study where a translated and adapted version of the YSR was administered by trained nurses, followed by an assessment by Ethiopian psychiatrists. Internal reliability of YSR syndrome scales were assessed using Chronbach''s alpha. Test-retest reliability was assessed through repeating the YSR one month later. To assess validity, analysis of the sensitivity and specificity of the YSR compared to the psychiatrist assessment was conducted.

Results

Across the eight syndrome scales, the YSR best measured the diagnosis of anxiety/depression and social problems among young women, and attention problems among young men. Among individual YSR syndrome scales, internal reliability ranged from unacceptable (Chronback’s alpha = 0.11, rule-breaking behavior among young women) to good (α≥0.71, anxiety/depression among young women). Anxiety/depression scores of ≥8.5 among young women also had good sensitivity (0.833) and specificity (0.754) to predict a true diagnosis. The YSR syndrome scales for social problems among young women and attention problems among young men also had fair consistency and validity measurements. Most YSR scores had significant positive correlations between baseline and post-one month administration. Measures of reliability and validity for most other YSR syndrome scales were fair to poor.

Conclusions

The adapted, personally administered, Amharic version of the YSR has sufficient reliability and validity in identifying young vulnerable women with anxiety/depression and/or social problems, and young men with attention problems; which were the most common mental health disorders observed by psychiatrists among the migrant populations in this study. Further assessment of the applicability of the YSR among vulnerable young people for less common disorders in Ethiopia is needed.     

Knowledge and Perceptions about Clinical Trials and the Use of Biomedical Samples: Findings from a Qualitative Study in Rural Northern Ghana

IntroductionClinical trials conducted in sub-Saharan Africa have helped to address the prevalent health challenges. The knowledge about how communities perceive clinical trials is however only now evolving. This study was conducted among parents whose children participated in past clinical trials in northern Ghana to assess their knowledge and perceptions of clinical trials and the use of biomedical samples.MethodThis was a qualitative study based on eighty in-depth interviews with parents. The participants were randomly selected from among parents whose children were enrolled in a clinical trial conducted in the Kassena-Nankana districts between 2000 and 2003. The interviews were transcribed and coded into emergent themes using Nvivo 9 software. The thematic analysis framework was used to analyze the data.ResultsStudy participants reported that clinical trials were carried out to determine the efficacy of drugs and to make sure that these drugs were suitable for human beings to use. The conduct of clinical trials was perceived to have helped to reduce the occurrence of diseases such as malaria, cerebrospinal meningitis and diarrhea. Quality of care was reported to be better in clinical trials than in the routine care. Parents indicated that participation in clinical trials positively influenced their health-seeking behavior. Apprehensions about blood draw and the use to which samples were put were expressed, with suspicion by a few participants that researchers sold blood samples. The issue of blood draw was most contentious.ConclusionParents perception about the conduct of clinical trials in the study districts is generally positive. However, misconceptions made about the use of blood samples in this study must be taken seriously and strategies found to improve transparency and greater community acceptability.     

Dealing with paralogy in RADseq data: in silico detection and single nucleotide polymorphism validation in Robinia pseudoacacia L.

The RADseq technology allows researchers to efficiently develop thousands of polymorphic loci across multiple individuals with little or no prior information on the genome. However, many questions remain about the biases inherent to this technology. Notably, sequence misalignments arising from paralogy may affect the development of single nucleotide polymorphism (SNP) markers and the estimation of genetic diversity. We evaluated the impact of putative paralog loci on genetic diversity estimation during the development of SNPs from a RADseq dataset for the nonmodel tree species Robinia pseudoacacia L. We sequenced nine genotypes and analyzed the frequency of putative paralogous RAD loci as a function of both the depth of coverage and the mismatch threshold allowed between loci. Putative paralogy was detected in a very variable number of loci, from 1% to more than 20%, with the depth of coverage having a major influence on the result. Putative paralogy artificially increased the observed degree of polymorphism and resulting estimates of diversity. The choice of the depth of coverage also affected diversity estimation and SNP validation: A low threshold decreased the chances of detecting minor alleles while a high threshold increased allelic dropout. SNP validation was better for the low threshold (4×) than for the high threshold (18×) we tested. Using the strategy developed here, we were able to validate more than 80% of the SNPs tested by means of individual genotyping, resulting in a readily usable set of 330 SNPs, suitable for use in population genetics applications.     

A recipe for postfledging survival in great tits Parus major: be large and be early (but not too much)

Survival of juveniles during the postfledging period can be markedly low, which may have major consequences on avian population dynamics. Knowing which factors operating during the nesting phase affect postfledging survival is crucial to understand avian breeding strategies. We aimed to obtain a robust set of predictors of postfledging local survival using the great tit (Parus major) as a model species. We used mark–recapture models to analyze the effect of hatching date, temperatures experienced during the nestling period, fledging size and body mass on first‐year postfledging survival probability of great tit juveniles. We used data from 5192 nestlings of first clutches ringed between 1993 and 2010. Mean first‐year postfledging survival probability was 15.2%, and it was lower for smaller individuals, as well as for those born in either very early or late broods. Our results stress the importance of choosing an optimum hatching period, and raising large chicks to increase first‐year local survival probability in the studied population.     

Morphological Diversity of the Rod Spherule: A Study of Serially Reconstructed Electron Micrographs

Purpose

Rod spherules are the site of the first synaptic contact in the retina’s rod pathway, linking rods to horizontal and bipolar cells. Rod spherules have been described and characterized through electron micrograph (EM) and other studies, but their morphological diversity related to retinal circuitry and their intracellular structures have not been quantified. Most rod spherules are connected to their soma by an axon, but spherules of rods on the surface of the Mus musculus outer plexiform layer often lack an axon and have a spherule structure that is morphologically distinct from rod spherules connected to their soma by an axon. Retraction of the rod axon and spherule is often observed in disease processes and aging, and the retracted rod spherule superficially resembles rod spherules lacking an axon. We hypothesized that retracted spherules take on an axonless spherule morphology, which may be easier to maintain in a diseased state. To test our hypothesis, we quantified the spatial organization and subcellular structures of rod spherules with and without axons. We then compared them to the retracted spherules in a disease model, mice that overexpress Dscam (Down syndrome cell adhesion molecule), to gain a better understanding of the rod synapse in health and disease.

Methods

We reconstructed serial EM images of wild type and Dscam^GoF (gain of function) rod spherules at a resolution of 7 nm in the X-Y axis and 60 nm in the Z axis. Rod spherules with and without axons, and retracted spherules in the Dscam^GoF retina, were reconstructed. The rod spherule intracellular organelles, the invaginating dendrites of rod bipolar cells and horizontal cell axon tips were also reconstructed for statistical analysis.

Results

Stereotypical rod (R1) spherules occupy the outer two-thirds of the outer plexiform layer (OPL), where they present as spherical terminals with large mitochondria. This spherule group is highly uniform and composed more than 90% of the rod spherule population. Rod spherules lacking an axon (R2) were also described and characterized. This rod spherule group consists of a specific spatial organization that is strictly located at the apical OPL-facing layer of the Outer Nuclear Layer (ONL). The R2 spherule displays a large bowl-shaped synaptic terminal that hugs the rod soma. Retracted spherules in the Dscam^GoF retina were also reconstructed to test if they are structurally similar to R2 spherules. The misplaced rod spherules in Dscam^GoF have a gross morphology that is similar to R2 spherules but have significant disruption in internal synapse organization.

Conclusion

We described a morphological diversity within Mus musculus rod spherules. This diversity is correlated with rod location in the ONL and contributes to the intracellular differences within spherules. Analysis of the Dscam^GoF retina indicated that their R2 spherules are not significantly different than wild type R2 spherules, but that their retracted rod spherules have abnormal synaptic organization.     

Feasibility and Acceptability of Global Positioning System (GPS) Methods to Study the Spatial Contexts of Substance Use and Sexual Risk Behaviors among Young Men Who Have Sex with Men in New York City: A P18 Cohort Sub-Study

Background

No global positioning system (GPS) technology study has been conducted among a sample of young gay, bisexual, and other men who have sex with men (YMSM). As such, the purpose of this study was to evaluate the feasibility and acceptability of using GPS methods to understand the spatial context of substance use and sexual risk behaviors among a sample of YMSM in New York City, a high-risk population.

Methods

Data came from a subsample of the ongoing P18 Cohort Study (n = 75). GPS feasibility and acceptability among participants was measured with: 1) a pre- and post-survey and 2) adherence to the GPS protocol which included returning the GPS device, self-report of charging and carrying the GPS device as well as objective data analyzed from the GPS devices. Analyses of the feasibility surveys were treated as repeated measures as each participant had a pre- and post-feasibility survey. When comparing the similar GPS survey items asked at baseline and at follow-up, we present percentages and associated p-values based on chi-square statistics.

Results

Participants reported high ratings of pre-GPS acceptability, ease of use, and low levels of wear-related concerns in addition to few concerns related to safety, loss, or appearance, which were maintained after baseline GPS feasibility data collection. The GPS return rate was 100%. Most participants charged and carried the GPS device on most days. Of the total of 75 participants with GPS data, 75 (100%) have at least one hour of GPS data for one day and 63 (84%) had at least one hour on all 7 days.

Conclusions

Results from this pilot study demonstrate that utilizing GPS methods among YMSM is feasible and acceptable. GPS devices may be used in spatial epidemiology research in YMSM populations to understand place-based determinants of health such as substance use and sexual risk behaviors.