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

Highly purified extracellular vesicles from human cardiomyocytes demonstrate preferential uptake by human endothelial cells

Extracellular vesicles (EVs) represent a promising cell-free alternative for treatment of cardiovascular diseases. Nevertheless, the lack of standardised and reproducible isolation methods capable of recovering pure, intact EVs presents a significant obstacle. Additionally, there is significant interest in investigating the interactions of EVs with different cardiac cell types. Here we established a robust technique for the production and isolation of EVs harvested from an enriched (>97% purity) population of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) with size exclusion chromatography. Utilizing an advanced fluorescence labelling strategy, we then investigated the interplay of the CM-EVs with the three major cellular components of the myocardium (fibroblasts, cardiomyocytes and endothelial cells) and identified that cardiac endothelial cells show preferential uptake of these EVs. Overall, our findings provide a great opportunity to overcome the translational hurdles associated with the isolation of intact, non-aggregated human iPSC-CM EVs at high purity. Furthermore, understanding in detail the interaction of the secreted EVs with their surrounding cells in the heart may open promising new avenues in the field of EV engineering for targeted delivery in cardiac regeneration

Highly purified extracellular vesicles from human cardiomyocytes demonstrate preferential uptake by human endothelial cells

Extracellular vesicles (EVs) represent a promising cell-free alternative for treatment of cardiovascular diseases. Nevertheless, the lack of standardised and reproducible isolation methods capable of recovering pure, intact EVs presents a significant obstacle. Additionally, there is significant interest in investigating the interactions of EVs with different cardiac cell types. Here we established a robust technique for the production and isolation of EVs harvested from an enriched (>97% purity) population of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) with size exclusion chromatography. Utilizing an advanced fluorescence labelling strategy, we then investigated the interplay of the CM-EVs with the three major cellular components of the myocardium (fibroblasts, cardiomyocytes and endothelial cells) and identified that cardiac endothelial cells show preferential uptake of these EVs. Overall, our findings provide a great opportunity to overcome the translational hurdles associated with the isolation of intact, non-aggregated human iPSC-CM EVs at high purity. Furthermore, understanding in detail the interaction of the secreted EVs with their surrounding cells in the heart may open promising new avenues in the field of EV engineering for targeted delivery in cardiac regeneration

Application of the speed-duration relationship to normalize the intensity of high-intensity interval training

The tolerable duration of continuous high-intensity exercise is determined by the hyperbolic Speed-tolerable duration (S-tLIM) relationship. However, application of the S-tLIM relationship to normalize the intensity of High-Intensity Interval Training (HIIT) has yet to be considered, with this the aim of present study. Subjects completed a ramp-incremental test, and series of 4 constant-speed tests to determine the S-tLIM relationship. A sub-group of subjects (n = 8) then repeated 4 min bouts of exercise at the speeds predicted to induce intolerance at 4 min (WR4), 6 min (WR6) and 8 min (WR8), interspersed with bouts of 4 min recovery, to the point of exercise intolerance (fixed WR HIIT) on different days, with the aim of establishing the work rate that could be sustained for 960 s (i.e. 4×4 min). A sub-group of subjects (n = 6) also completed 4 bouts of exercise interspersed with 4 min recovery, with each bout continued to the point of exercise intolerance (maximal HIIT) to determine the appropriate protocol for maximizing the amount of high-intensity work that can be completed during 4×4 min HIIT. For fixed WR HIIT tLIM of HIIT sessions was 399±81 s for WR4, 892±181 s for WR6 and 1517±346 s for WR8, with total exercise durations all significantly different from each other (P<0.050). For maximal HIIT, there was no difference in tLIM of each of the 4 bouts (Bout 1: 229±27 s; Bout 2: 262±37 s; Bout 3: 235±49 s; Bout 4: 235±53 s; P>0.050). However, there was significantly less high-intensity work completed during bouts 2 (153.5±40. 9 m), 3 (136.9±38.9 m), and 4 (136.7±39.3 m), compared with bout 1 (264.9±58.7 m; P>0.050). These data establish that WR6 provides the appropriate work rate to normalize the intensity of HIIT between subjects. Maximal HIIT provides a protocol which allows the relative contribution of the work rate profile to physiological adaptations to be considered during alternative intensity-matched HIIT protocols

Reproducibility of the mfERG between instruments

Purpose First, to examine both the reproducibility of the multifocal electroretinogram (mfERG) recorded on different versions of the same instrument, and the repeatability of the mfERG recorded on a single instrument using two different amplifiers. Second, to demonstrate a means by which multicenter and longitudinal studies that use more than one recording instrument can compare and combine data effectively. Methods Three different amplifiers and two mfERG setups, one using VERIS™ 4.3 software (mfERG1) and another using VERIS™ Pro 5.2 software (mfERG2), were evaluated. A total of 73 subjects with normal vision were tested in three groups. Group 1 (n = 42) was recorded using two amplifiers in parallel on mfERG1. Group 2 (n = 52) was recorded on mfERG2 using a single amplifier. Group 3 was a subgroup of 21 subjects from groups 1 and 2 that were tested sequentially on both instruments. A fourth group of 26 subjects with diabetes were also recorded using the two parallel amplifiers on mfERG1. P1 implicit times and N1-P1 amplitudes of the 103 local first order mfERGs were measured, and the differences between the instruments and amplifiers were evaluated as raw scores and Z-scores based on normative data. Measurements of individual responses and measurements averaged over the 103 responses were analyzed. Results Simultaneous recordings made on mfERG1 with the two different amplifiers showed differences in implicit times but similar amplitudes. There was a mean implicit time difference of 2.5 ms between the amplifiers but conversion to Z-scores improved their agreement. Recordings made on different days with the two instruments produced similar but more variable results, with amplitudes differing between them more than implicit times. For local response implicit times, the 95% confidence interval of the difference between instruments was approximately ±1 Z-score (±0.9 ms) in either direction. For local response amplitude, it was approximately ±1.6 Z-scores (±0.3 μV). Conclusions Different amplifiers can yield quite different mfERG P1 implicit times, even with identical band-pass settings. However, the reproducibility of mfERG Z-scores across recording instrumentation is relatively high. Comparison of data across systems and laboratories, necessary for multicenter or longitudinal investigations, is facilitated if raw data are converted into Z-scores based on normative data

The Sir Jimmy Savile Scandal: Child Sexual Abuse and Institutional Denial at the BBC

This study advances research on scandal through an empirical examination of one of the most extraordinary UK institutional child sexual abuse (CSA) scandals in the post-war period. Sir Jimmy Savile (1926–2011) was a BBC celebrity, showbiz friend of the establishment and philanthropist. In October 2012, one year after his death, an ITV documentary alleged that Savile was also a prolific sexual predator who for decades had exploited his BBC status to abuse teenage girls. As we demonstrate, this incendiary documentary triggered a news media feeding frenzy that in less than one week destroyed Savile’s reputation and thrust the BBC – the institution that made him a star – into a multi-faceted, globally reported CSA scandal. This study has four purposes. First, we propose a model of institutional CSA scandals that can account for critical transitions between key phases in the scandal process. Second, we apply this model to analyse the transition between the ‘latent’ and ‘activated’ phases of the Savile scandal. This transition corresponded with a dramatic transformation in the inferential structuring of Savile from ‘national treasure’, who had devoted decades to working with children, to ‘prolific sexual predator’, who spent decades abusing them. Third, we demonstrate how the BBC’s denial of responsibility for Savile’s sexual offending and its subsequent institutional cover-up triggered a ‘trial by media’ which in turn initiated the next phase in the scandal’s development – ‘amplification’. Finally, we consider the significance of our analysis of the Sir Jimmy Savile scandal for understanding the activation and development of scandals more generally

Spectroscopic and biochemical correlations during the course of human lens aging

BACKGROUND: With age, the human lens accumulates variety of substances that absorbs and fluorescence, which explains the color of yellow, brunescent and nigrescent cataract in terms of aging. The aim of this study was to assess lens fluorophores with properties comparable to those of advanced glycated end products (AGEs) in relation to age in human lenses. These fluorescent compounds are believed to be involved in the development of cataract. METHODS: Spectroscopic (UV-Vis-NIR) and fluorescence photography (CCD-Digital based image analysis) studies were carried out in randomly selected intact human lenses (2–85 years). AGE-like fluorophores were also measured in water soluble and insoluble (alkali soluble) fractions of human lenses (20–80 years). RESULTS: Our experimental findings suggest that there was a progressive shift in the absorbance characteristic of intact lens in the range of λ(210 nm)-λ(470 nm). A relative increase in the absorptivity at λ((511–520 nm)), with age, was also observed. In addition, the ratio of absorptivity at λ((511–520 nm)) versus the maximum absorbance recorded at blue-end cut-off (210–470 nm) was also found to increase, with age. The fluorescent intensity in the intact lens at both UV-B (λ(Ex312 nm)) and UV-A (λ(Ex365 nm)) were found to be positively correlated (r(2 )= 0.91 & 0.94, respectively; Confidence interval 95%) upto 50 years of age. In addition, a concomitant changes in AGE- like fluorophores were also observed in the processed lens samples (soluble and insoluble fractions) along the age. A significant increase in the concentration of AGE- like fluorophores, both in intact and processed lens was observed during the period of 40 – 50 years. CONCLUSION: Based on the present investigation, it was concluded that significant changes do occur in the AGE-like fluorophores of human lenses during the period of 40–50 years

The effect of nutritional supplementation on the multifocal electroretinogram in healthy eyes

BACKGROUND: Previous studies have demonstrated an increase in macular pigment optical density (MPOD) with lutein (L)-based supplementation in healthy eyes. However, not all studies have assessed whether this increase in MPOD is associated with changes to other measures of retinal function such as the multifocal ERG (mfERG). Some studies also fail to report dietary levels of L and zeaxanthin (Z). Because of the associations between increased levels of L and Z, and reduced risk of AMD, this study was designed to assess the effects of L-based supplementation on mfERG amplitudes and latencies in healthy eyes. METHODS: Multifocal ERG amplitudes, visual acuity, contrast sensitivity, MPOD and dietary levels of L and Z were assessed in this longitudinal, randomized clinical trial. Fifty-two healthy eyes from 52 participants were randomly allocated to receive a L-based supplement (treated group), or no supplement (non-treated group). RESULTS: There were 25 subjects aged 18-77 (mean age ± SD; 48 ± 17) in the treated group and 27 subjects aged 21-69 (mean age ± SD; 43 ± 16) in the non-treated group. All participants attended for three visits: visit one at baseline, visit two at 20 weeks and visit three at 40 weeks. A statistically significant increase in MPOD (F = 17.0, p ≤ 0.001) and shortening of mfERG ring 2 P1 latency (F = 3.69, p = 0.04) was seen in the treated group. CONCLUSIONS: Although the results were not clinically significant, the reported trend for improvement in MPOD and mfERG outcomes warrants further investigation