Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Impact of Indian Total Sanitation Campaign on Latrine Coverage and Use: A Cross-Sectional Study in Orissa Three Years following Programme Implementation

Background

Faced with a massive shortfall in meeting sanitation targets, some governments have implemented campaigns that use subsidies focused on latrine construction to overcome income constraints and rapidly expand coverage. In settings like rural India where open defecation is common, this may result in sub-optimal compliance (use), thereby continuing to leave the population exposed to human excreta.

Methods

We conducted a cross-sectional study to investigate latrine coverage and use among 20 villages (447 households, 1933 individuals) in Orissa, India where the Government of India’s Total Sanitation Campaign had been implemented at least three years previously. We defined coverage as the proportion of households that had a latrine; for use we identified the proportion of households with at least one reported user and among those, the extent of reported use by each member of the household.

Results

Mean latrine coverage among the villages was 72% (compared to <10% in comparable villages in the same district where the Total Sanitation Campaign had not yet been implemented), though three of the villages had less than 50% coverage. Among these households with latrines, more than a third (39%) were not being used by any member of the household. Well over a third (37%) of the members of households with latrines reported never defecating in their latrines. Less than half (47%) of the members of such households reported using their latrines at all times for defecation. Combined with the 28% of households that did not have latrines, it appears that most defecation events in these communities are still practiced in the open.

Conclusion

A large-scale campaign to implement sanitation has achieved substantial gains in latrine coverage in this population. Nevertheless, gaps in coverage and widespread continuation of open defecation will result in continued exposure to human excreta, reducing the potential for health gains.     

In vitro biosynthesis, core glycosylation and membrane integration of opsin

A membrane-integrated , core-glycosylated form of bovine opsin was synthesized in vitro when bovine retina mRNA was translated in a wheat germ cell-free system supplemented with dog pancreas microsomal vesicles; glycosylation and integration of opsin into membranes were coupled to translation. Proteolysis with themolysin was used to probe the orientation of opsin within the dog pancreas microsomal membrane, and to compare it with that of opsin in rod cell disk membranes isolated from bovine retina. Intact microsomal or disk vesicles were required for production of discrete, membrane-associated thermolysin fragments of opsin; no discrete opsin fragments were detected when membranes were incubated with thermolysin in the presence of the nonionic detergent, Triton X-100. The major opsin fragments produced by themosylin treatment of intact microsomal vesicles resembled those from disk vesicles in their size, oligosaccharide content, and order of appearance. In each case, the first cleavage of opsin took place at the COOH-terminus, generating a glycosylated fragment, O’, which was only slightly smaller than intact opsin. Both the microsomal and disk membrane forms of O’ were next cleaved internally; glycosylated fragments of similar sizes in both cases were detected which were derived from the NH(2)-terminal portion of O’. Several smaller NH(2)-terminal fragments of opsin were detected only in thermolysin-treated microsomal membranes, and not in disk membranes. The data suggest that the topology of opsin integrated into dog pancreas microsomal vesicles is similar to that in rod cell disk vesicles, although not identical. In each case, the glycosylated NH(2)-terminal region of opsin is located within the lumen of the vesicle, while discrete COOH-terminal and internal segments of opsin apparently emerge at the outer, cytoplasmic face of the membrane. Thus, opsin in the heterologous microsomal membrane, like its counterpart in the native disk membrane, may cross the bilayer at least three times. The internal domain of the polypeptide that emerges at the outer membrane surface is apparently more highly exposed in the case of opsin in microsomal membranes, evidenced by the additional internal thermolysin cleavage sites detected.     

Assessing Water Filtration and Safe Storage in Households with Young Children of HIV-Positive Mothers: A Randomized,Controlled Trial in Zambia

Background

Unsafe drinking water presents a particular threat to people living with HIV/AIDS (PLHIV) due to the increased risk of opportunistic infections, diarrhea-associated malabsorption of essential nutrients, and increased exposure to untreated water for children of HIV-positive mothers who use replacement feeding to reduce the risk of HIV transmission. This population may particularly benefit from an intervention to improve water quality in the home.

Methods and Findings

We conducted a 12-month randomized, controlled field trial in Zambia among 120 households with children <2 years (100 with HIV-positive mothers and 20 with HIV-negative mothers to reduce stigma of participation) to assess a high-performance water filter and jerry cans for safe storage. Households were followed up monthly to assess use, drinking water quality (thermotolerant coliforms (TTC), an indicator of fecal contamination) and reported diarrhea (7-day recall) among children <2 years and all members of the household. Because previous attempts to blind the filter have been unsuccessful, we also assessed weight-for-age Z-scores (WAZ) as an objective measure of diarrhea impact. Filter use was high, with 96% (596/620) of household visits meeting the criteria for users. The quality of water stored in intervention households was significantly better than in control households (3 vs. 181 TTC/100 mL, respectively, p<0.001). The intervention was associated with reductions in the longitudinal prevalence of reported diarrhea of 53% among children <2 years (LPR = 0.47, 95% CI: 0.30–0.73, p = 0.001) and 54% among all household members (LPR = 0.46, 95% CI: 0.30–0.70, p<0.001). While reduced WAZ was associated with reported diarrhea (−0.26; 95% CI: −0.37 to −0.14, p<0.001), there was no difference in WAZ between intervention and control groups.

Conclusion

In this population living with HIV/AIDS, a water filter combined with safe storage was used correctly and consistently, was highly effective in improving drinking water quality, and was protective against diarrhea.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01116908","term_id":"NCT01116908"}}NCT01116908     

Effect of fruit maturity,seed weight and storage time on the viability and germination of the seed of candelilla (<i>Euphorbia antisiphylitica</i> Zucc.)

Candelilla (Euphorbia antisiphylitica Zucc.) is a very important plant resource in the arid lands of Northern Mexico. This is because the wax content coating the stem has unique properties which have been useful for multiple applications in the food industry, electronics, cosmetics, etc. However, the intensive exploitation of this resource has caused a great decrease in the populations of this species making necessary to consider strategies for their conservation and sustainable use. One of the primary needs with regeneration purposes is to know their reproductive processes, particularly the biotic and/or abiotic factors that determine the viability and germination of seeds. The present study evaluated the (1) germination and seed viability in relation to the ripeness degree of the fruit at the time of collection, (2) weight of the seed (low, medium and high), and (3) storage time (1, 3, and 5 months). Fruits from four locations, two in the State of Coahuila (Las Coloradas and Candela) and two in the State of Nuevo Leon (Icamole 1 and Icamole 2), were collected. Three germination assays were carried out corresponding to each month of storage. Seed viability was determined by the tetrazolium test. The average weight of the candelilla seeds was 0.0029 ± 0.0010 g, with extreme average values of 0.0018 ± 0.0006 g at Las Coloradas and 0.0036 ± 0.0010 g in Icamole 2. Those seeds with heavier weight obtained from red fruits and with 1 month of storage showed the highest average percentage of viability (66.87 ± 24.19%). At the same time, seeds with around average weight, obtained from red fruits and five months of storage, showed the highest average germination percentage (50.00 ± 9.42%).     

Attentional modulation of central odor processing

Two studies were conducted to investigate the influence of attention on the components of the chemosensory event-related potential (CSERP). In the first study the odors linalool and eugenol were delivered to six male subjects, in the second study three male and two female subjects were presented with their own body odor (axillary hair) and the body odor of a same sex donor. In both studies the odors were presented in an oddball paradigm under ignore and attend conditions via a constant- flow olfactometer. In the ignore condition attention was diverted from the odors with a distractor task, while in the attend condition the subjects were asked to respond to the infrequently occurring odor. In both studies the allocation of attention led to a decrease in the latency of the early components (N1, P2, N2) and to an increase in the amplitude of the late positivities. The modulation of the early components suggests that attentional gating in olfaction might already be effective at an early processing level.      

Efficacy assessed in follow-ups of clinical trials: methodological conundrum

Increasingly, we see papers describing the long-term follow-up results of randomised clinical trials. Sometimes, like the article by Rantalaiho and colleagues in the previous issue of Arthritis Research & Therapy, the follow-up extends to more than 10 years. It is not uncommon that authors of such articles describe their results as a comparison of the original treatment groups in the original randomised clinical trial. Methodologically, such a comparison is fallible for several reasons. In this editorial, two important sources of bias that may jeopardise the results of such follow-up studies are discussed: confounding by indication and confounding by trial completion.     

Comparison of computational methods for identifying translation initiation sites in EST data

Background

Expressed Sequence Tag (EST) sequences are generally single-strand, single-pass sequences, only 200–600 nucleotides long, contain errors resulting in frame shifts, and represent different parts of their parent cDNA. If the cDNAs contain translation initiation sites, they may be suitable for functional genomics studies. We have compared five methods to predict translation initiation sites in EST data: first-ATG, ESTScan, Diogenes, Netstart, and ATGpr.

Results

A dataset of 100 EST sequences, 50 with and 50 without, translation initiation sites, was created. Based on analysis of this dataset, ATGpr is found to be the most accurate for predicting the presence versus absence of translation initiation sites. With a maximum accuracy of 76%, ATGpr more accurately predicts the position or absence of translation initiation sites than NetStart (57%) or Diogenes (50%). ATGpr similarly excels when start sites are known to be present (90%), whereas NetStart achieves only 60% overall accuracy. As a baseline for comparison, choosing the first ATG correctly identifies the translation initiation site in 74% of the sequences. ESTScan and Diogenes, consistent with their intended use, are able to identify open reading frames, but are unable to determine the precise position of translation initiation sites.

Conclusions

ATGpr demonstrates high sensitivity, specificity, and overall accuracy in identifying start sites while also rejecting incomplete sequences. A database of EST sequences suitable for validating programs for translation initiation site prediction is now available. These tools and materials may open an avenue for future improvements in start site prediction and EST analysis.

     

Identifying and quantifying metabolites by scoring peaks of GC-MS data

Background

Metabolomics is one of most recent omics technologies. It has been applied on fields such as food science, nutrition, drug discovery and systems biology. For this, gas chromatography-mass spectrometry (GC-MS) has been largely applied and many computational tools have been developed to support the analysis of metabolomics data. Among them, AMDIS is perhaps the most used tool for identifying and quantifying metabolites. However, AMDIS generates a high number of false-positives and does not have an interface amenable for high-throughput data analysis. Although additional computational tools have been developed for processing AMDIS results and to perform normalisations and statistical analysis of metabolomics data, there is not yet a single free software or package able to reliably identify and quantify metabolites analysed by GC-MS.

Results

Here we introduce a new algorithm, PScore, able to score peaks according to their likelihood of representing metabolites defined in a mass spectral library. We implemented PScore in a R package called MetaBox and evaluated the applicability and potential of MetaBox by comparing its performance against AMDIS results when analysing volatile organic compounds (VOC) from standard mixtures of metabolites and from female and male mice faecal samples. MetaBox reported lower percentages of false positives and false negatives, and was able to report a higher number of potential biomarkers associated to the metabolism of female and male mice.

Conclusions

Identification and quantification of metabolites is among the most critical and time-consuming steps in GC-MS metabolome analysis. Here we present an algorithm implemented in a R package, which allows users to construct flexible pipelines and analyse metabolomics data in a high-throughput manner.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0374-2) contains supplementary material, which is available to authorized users.