Molecular imaging of extracellular vesicles in vitro via Raman metabolic labelling

Extracellular vesicles (EVs) are biologically-derived nanovectors important for intercellular communication and trafficking. As such, EVs show great promise as disease biomarkers and therapeutic drug delivery vehicles. However, despite the rapidly growing interest in EVs, understanding of the biological mechanisms that govern their biogenesis, secretion, and uptake remains poor. Advances in this field have been hampered by both the complex biological origins of EVs, which make them difficult to isolate and identify, and a lack of suitable imaging techniques to properly study their diverse biological roles. Here, we present a new strategy for simultaneous quantitative in vitro imaging and molecular characterisation of EVs in 2D and 3D based on Raman spectroscopy and metabolic labelling. Deuterium, in the form of deuterium oxide (D2O), deuterated choline chloride (d-Chol), or deuterated D-glucose (d-Gluc), is metabolically incorporated into EVs through the growth of parent cells on medium containing one of these compounds. Isolated EVs are thus labelled with deuterium, which acts as a bio-orthogonal Raman-active tag for direct Raman identification of EVs when introduced to unlabelled cell cultures. Metabolic deuterium incorporation demonstrates no apparent adverse effects on EV secretion, marker expression, morphology, or global composition, indicating its capacity for minimally obstructive EV labelling. As such, our metabolic labelling strategy could provide integral insights into EV biocomposition and trafficking. This approach has the potential to enable a deeper understanding of many of the biological mechanisms underpinning EVs, with profound implications for the design of EVs as therapeutic delivery vectors and applications as disease biomarkers

Natural orifice surgery: initial clinical experience

Natural orifice translumenal endoscopic surgery (NOTES) has moved quickly from preclinical investigation to clinical implementation. However, several major technical problems limit clinical NOTES including safe access, retraction and dissection of the gallbladder, and clipping of key structures. This study aimed to identify challenges and develop solutions for NOTES during the initial clinical experience. Under an Institutional Review Board (IRB)-approved protocol, patients consented to a natural orifice operation for removal of either the gallbladder or the appendix via either the vagina or the stomach using a single umbilical trocar for safety and assistance. Nine transvaginal cholecystectomies, one transgastric appendectomy, and one transvaginal appendectomy have been completed to date. All but one patient were discharged on postoperative day 1 as per protocol. No complications occurred. The limited initial evidence from this study demonstrates that NOTES is feasible and safe. The addition of an umbilical trocar is a bridge allowing safe performance of NOTES procedures until better instruments become available. The addition of a flexible long grasper through the vagina and a flexible operating platform through the stomach has enabled the performance of NOTES in a safe and easily reproducible manner. The use of a uterine manipulator has facilitated visualization of the cul de sac in women with a uterus to allow for safe transvaginal access

QCD and strongly coupled gauge theories : challenges and perspectives

We highlight the progress, current status, and open challenges of QCD-driven physics, in theory and in experiment. We discuss how the strong interaction is intimately connected to a broad sweep of physical problems, in settings ranging from astrophysics and cosmology to strongly coupled, complex systems in particle and condensed-matter physics, as well as to searches for physics beyond the Standard Model. We also discuss how success in describing the strong interaction impacts other fields, and, in turn, how such subjects can impact studies of the strong interaction. In the course of the work we offer a perspective on the many research streams which flow into and out of QCD, as well as a vision for future developments.Peer reviewe

Pathogen prevalence in commercially reared bumble bees and evidence of spillover in conspecific populations

Worldwide, wild bumble bees (Bombus spp.) are experiencing marked declines, with potentially up to 11% of species currently under threat. Recent studies from North America suggest that disease transmission from commercially reared bumble bees to wild populations has led to marked range contractions in some species. In Europe, data on the prevalence of pathogen spillover from commercial to wild bumble bee populations is lacking, despite the widespread production and transport of hives within the EU since the early 1980s. We determined the permeability of cropping systems to commercial bumble bees, and quantified the prevalence of four pathogens in commercial Bombus terrestris hives and adjacent conspecific populations at increasing distances from greenhouses in Ireland. Commercial bumble bees collected from 31% to 97% of non-crop pollen, depending on the cropping system, and hives had markedly higher frequencies of two gut parasites, Crithidia spp. and Nosema bombi, compared to adjacent populations, but were free of tracheal mites. The highest prevalence of Crithida was observed within 2. km of greenhouses and the probability of infection declined in a host sex- and pathogen-specific manner up to 10. km. We suggest implementing measures that prevent the interaction of commercially reared and wild bumble bees by integrating the enforcement of national best management practices for users of commercial pollinators with international legislation that regulates the sanitation of commercial hives in production facilities. © 2012 Elsevier Ltd

Bumblebee (Hymenoptera: Apidae) sample storage for a posteriori molecular studies: Interactions between sample storage and DNA-extraction techniques

A global decline in pollinator abundance and diversity has demanded increased research attention to the ecology and genetics of bumblebees. however, as progressively more restrictions are placed on sampling for insects, researchers are increasingly obliged to use archival specimens collected for purposes other than genetic analyses. in this study we assessed the suitability, for population genetic studies, of popular, low-cost methods for preservation and storage of bumblebee specimens. specimens of bombus terrestris l. were held under six storage regimes for up to two years. DNA was extracted from the samples using three extraction protocols and the quality of the DNA was examined using PCR amplification of a mitochondrial and a nuclear gene. all extraction and storage methods provided sufficient dna for successful PCR amplification. however, samples preserved in acetone or at freezing temperatures yielded the highest DNA concentrations. DNA yields from pinned specimens at room temperature declined over time, particularly when using standard extraction techniques. DNA concentrations were significantly lower from specimens preserved in 70% ethanol compared to all other extraction techniques and declined linearly over the two years of storage. these results indicate that two of the most popular insect storage methods (pinning and storage in ethanol) should be avoided for the long-term preservation of genetic material for future studies. we suggest that optimal insect preservation methods should be incorporated into research protocols in order to best capitalise on limited collection opportunities

Energy-dispersive X-ray microanalysis of air-dried microdroplets containing a macromolecular solute.

In the preparation of microdroplets of biological fluids for X-ray microanalysis, we have found that incorporation of a macromolecular solute, dextran, to a final concentration of 1.5-2.5% retards crystal formation and produces sufficiently uniform deposits on thin films to allow droplets to be analysed without prior freeze-drying. Analyses have been carried out at 20 kV in a scanning electron microscope, using energy-dispersive spectrometry. Absorption of Na X-rays by the added solute can be significant but its effect is minimized by preparing droplets as thin as possible, and by using standards of similar composition. The minimum detectable concentrations are increased because of the extra background contribution, and for a single determination are about 6 mM for Na and 2 mM for Cl and K. These concentrations can be further reduced by measuring replicates. The reproducibility of analysis is significantly improved (to less than 5% for Na and K) over the use of calibration curves by calculating the element concentrations from a known element in the sample, chlorine. Under our analytical conditions loss of Cl did not occur. This method requires that the Cl is measured separately by microcoulometry, but eliminates the need for a range of standard droplets on the grid, and determination of the unknowns is then independent of droplet volume, beam current, counting time and magnification. We have compared, with biological samples, the results from using Cl as an internal standard with those obtained using an added standard element, cobalt. The reproducibility using Cl was approximately two-times better than that obtained with Co, probably because of unavoidable volumetric errors when the Co is pipetted separately

Health information-seeking behaviors about attention deficit hyperactivity disorder on the internet and the analysis of top ranking websites

Health information-seeking behaviors about attention deficit hyperactivity disorder on the internet and the analysis of top ranking websites Objective: This study analyses the volume of searches conducted using Turkish keywords related to Attention Deficit Hyperactivity Disorder (ADHD) on internet search engines and the content of high-ranking websites in order to identify the interest of the public on ADHD. Method: The Google AdWords Keyword Planner Tool was used to find out the related Turkish keywords and web sites listed on the first default results pages were analyzed. Results/Conclusion: Although ADHD is a very common psychiatric disorder, our research reveals that public interest is not parallel with the prevalence of it, particularly with adult ADHD. The websites listed on the first results pages do not contain sufficient information on adult ADHD and only little content on these web pages is written by academicians or supported by scientific references. For a wider and more accurate public recognition of ADHD, academicians and professional practitioners should use the internet more proactively and effectively. © 2015 Cukurova Univ Tip Fakultesi Psikiyatri Anabilim Dali