Selective Replacement of Cholesterol with Cationic Amphiphilic Drugs Enables the Design of Lipid Nanoparticles with Improved RNA Delivery.

The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

Differential alternative splicing analysis links variation in ZRSR2 to a novel type of oral-facial-digital syndrome.

PURPOSE: Oral-facial-digital (OFD) syndromes are genetically heterogeneous developmental disorders, caused by pathogenic variants in genes involved in primary cilia formation and function. We identified a previously undescribed type of OFD with brain anomalies, ranging from alobar holoprosencephaly to pituitary anomalies, in 6 unrelated families. METHODS: Exome sequencing of affected probands was supplemented with alternative splicing analysis in patient and control lymphoblastoid and fibroblast cell lines, and primary cilia structure analysis in patient fibroblasts. RESULTS: In 1 family with 2 affected males, we identified a germline variant in the last exon of ZRSR2, NM_005089.4:c.1211_1212del NP_005080.1:p.(Gly404GlufsTer23), whereas 7 affected males from 5 unrelated families were hemizygous for the ZRSR2 variant NM_005089.4:c.1207_1208del NP_005080.1:p.(Arg403GlyfsTer24), either occurring de novo or inherited in an X-linked recessive pattern. ZRSR2, located on chromosome Xp22.2, encodes a splicing factor of the minor spliceosome complex, which recognizes minor introns, representing 0.35% of human introns. Patient samples showed significant enrichment of minor intron retention. Among differentially spliced targets are ciliopathy-related genes, such as TMEM107 and CIBAR1. Primary fibroblasts containing the NM_005089.4:c.1207_1208del ZRSR2 variant had abnormally elongated cilia, confirming an association between defective U12-type intron splicing, OFD and abnormal primary cilia formation. CONCLUSION: We introduce a novel type of OFD associated with elongated cilia and differential splicing of minor intron-containing genes due to germline variation in ZRSR2.

Proof of stability of an RSV Controlled Human Infection Model challenge agent.

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze-thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze-thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

The association of hypoglycemia with outcome of critically ill children in relation to nutritional and blood glucose control strategies.

BACKGROUND: Withholding parenteral nutrition (PN) until one week after PICU admission facilitated recovery from critical illness and protected against emotional and behavioral problems 4 years later. However, the intervention increased the risk of hypoglycemia, which may have counteracted part of the benefit. Previously, hypoglycemia occurring under tight glucose control in critically ill children receiving early PN did not associate with long-term harm. We investigated whether hypoglycemia in PICU differentially associates with outcome in the context of withholding early PN, and whether any potential association with outcome may depend on the applied glucose control protocol. METHODS: In this secondary analysis of the multicenter PEPaNIC RCT, we studied whether hypoglycemia in PICU associated with mortality (N = 1440) and 4-years neurodevelopmental outcome (N = 674) through univariable comparison and multivariable regression analyses adjusting for potential confounders. In patients with available blood samples (N = 556), multivariable models were additionally adjusted for baseline serum NSE and S100B concentrations as biomarkers of neuronal, respectively, astrocytic damage. To study whether an association of hypoglycemia with outcome may be affected by the nutritional strategy or center-specific glucose control protocol, we further adjusted the models for the interaction between hypoglycemia and the randomized nutritional strategy, respectively, treatment center. In sensitivity analyses, we studied whether any association with outcome was different in patients with iatrogenic or spontaneous/recurrent hypoglycemia. RESULTS: Hypoglycemia univariably associated with higher mortality in PICU, at 90 days and 4 years after randomization, but not when adjusted for risk factors. After 4 years, critically ill children with hypoglycemia scored significantly worse for certain parent/caregiver-reported executive functions (working memory, planning and organization, metacognition) than patients without hypoglycemia, also when adjusted for risk factors including baseline NSE and S100B. Further adjustment for the interaction of hypoglycemia with the randomized intervention or treatment center revealed a potential interaction, whereby tight glucose control and withholding early PN may be protective. Impaired executive functions were most pronounced in patients with spontaneous or recurrent hypoglycemia. CONCLUSION: Critically ill children exposed to hypoglycemia in PICU were at higher risk of impaired executive functions after 4 years, especially in cases of spontaneous/recurrent hypoglycemia.

Impact of critical illness and withholding of early parenteral nutrition in the pediatric intensive care unit on long-term physical performance of children: a 4-year follow-up of the PEPaNIC randomized controlled trial.

BACKGROUND: Many critically ill children face long-term developmental impairments. The PEPaNIC trial attributed part of the problems at the level of neurocognitive and emotional/behavioral development to early use of parenteral nutrition (early-PN) in the PICU, as compared with withholding it for 1 week (late-PN). Insight in long-term daily life physical functional capacity after critical illness is limited. Also, whether timing of initiating PN affects long-term physical function of these children remained unknown. METHODS: This preplanned follow-up study of the multicenter PEPaNIC randomized controlled trial subjected 521 former critically ill children (253 early-PN, 268 late-PN) to quantitative physical function tests 4 years after PICU admission in Leuven or Rotterdam, in comparison with 346 age- and sex-matched healthy children. Tests included handgrip strength measurement, timed up-and-go test, 6-min walk test, and evaluation of everyday overall physical activity with an accelerometer. We compared these functional measures for the former critically ill and healthy children and for former critically ill children randomized to late-PN versus early-PN, with multivariable linear or logistic regression analyses adjusting for risk factors. RESULTS: As compared with healthy children, former critically ill children showed less handgrip strength (p < 0.0001), completed the timed up-and-go test more slowly (p < 0.0001), walked a shorter distance in 6 min (p < 0.0001) during which they experienced a larger drop in peripheral oxygen saturation (p ≤ 0.026), showed a lower energy expenditure (p ≤ 0.024), performed more light and less moderate physical activity (p ≤ 0.047), and walked fewer steps per day (p = 0.0074). Late-PN as compared with early-PN did not significantly affect these outcomes. CONCLUSIONS: Four years after PICU admission, former critically ill children showed worse physical performance as compared with healthy children, without impact of timing of supplemental PN in the PICU. This study provides further support for de-implementing the early use of PN in the PICU. Trial registration ClinicalTrials.gov, NCT01536275 ; registered on February 22, 2012.

Postoperative Cerebral Oxygen Saturation in Children After Congenital Cardiac Surgery and Long-Term Total Intelligence Quotient: A Prospective Observational Study.

OBJECTIVES: During the early postoperative period, children with congenital heart disease can suffer from inadequate cerebral perfusion, with possible long-term neurocognitive consequences. Cerebral tissue oxygen saturation can be monitored noninvasively with near-infrared spectroscopy. In this prospective study, we hypothesized that reduced cerebral tissue oxygen saturation and increased intensity and duration of desaturation (defined as cerebral tissue oxygen saturation < 65%) during the early postoperative period, independently increase the probability of reduced total intelligence quotient, 2 years after admission to a PICU. DESIGN: Single-center, prospective study, performed between 2012 and 2015. SETTING: The PICU of the University Hospitals Leuven, Belgium. PATIENTS: The study included pediatric patients after surgery for congenital heart disease admitted to the PICU. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Postoperative cerebral perfusion was characterized with the mean cerebral tissue oxygen saturation and dose of desaturation of the first 12 and 24 hours of cerebral tissue oxygen saturation monitoring. The independent association of postoperative mean cerebral tissue oxygen saturation and dose of desaturation with total intelligence quotient at 2-year follow-up was evaluated with a Bayesian linear regression model adjusted for known confounders. According to a noninformative prior, reduced mean cerebral tissue oxygen saturation during the first 12 hours of monitoring results in a loss of intelligence quotient points at 2 years, with a 90% probability (posterior ß estimates [80% credible interval], 0.23 [0.04-0.41]). Similarly, increased dose of cerebral tissue oxygen saturation desaturation would result in a loss of intelligence quotient points at 2 years with a 90% probability (posterior ß estimates [80% credible interval], -0.009 [-0.016 to -0.001]). CONCLUSIONS: Increased dose of cerebral tissue oxygen saturation desaturation and reduced mean cerebral tissue oxygen saturation during the early postoperative period independently increase the probability of having a lower total intelligence quotient, 2 years after PICU admission.

Augmented Reality-Assisted Neurosurgical Drain Placement (ARANED): Technical Note.

BACKGROUND: Many surgical procedures, such as placement of intracranial drains, are currently being performed blindly, relying on anatomical landmarks. As a result, accuracy results still have room for improvement. Neuronavigation could address this issue, but its application in an urgent setting is often impractical. Augmented reality (AR) provided through a head-worn device has the potential to tackle this problem, but its implementation should meet physicians' needs. METHODS: The Surgical Augmented Reality Assistance (SARA) project aims to develop an AR solution that is suitable for preoperative planning, intraoperative visualisation and navigational support in an everyday clinical setting, using a Microsoft HoloLens. RESULTS: Proprietary hardware and software adaptations and dedicated navigation algorithms are applied to the Microsoft HoloLens to optimise it specifically for neurosurgical navigation. This includes a pipeline with an additional set of advanced, semi-automated algorithms responsible for image processing, hologram-to-patient registration and intraoperative tracking using infrared depth-sensing. A smooth and efficient workflow while maintaining high accuracy is prioritised. The AR solution provides a fully integrated and completely mobile navigation setup. Initial preclinical and clinical validation tests applying the solution to intracranial drain placement are described. CONCLUSION: AR has the potential to vastly increase accuracy of everyday procedures that are frequently performed without image guidance, but could still benefit from navigational support, such as intracranial drain placements. Technical development should go hand in hand with preclinical and clinical validation in order to demonstrate improvements in accuracy and clinical outcomes.

The effect of augmented reality on the accuracy and learning curve of external ventricular drain placement.

OBJECTIVE: The traditional freehand technique for external ventricular drain (EVD) placement is most frequently used, but remains the primary risk factor for inaccurate drain placement. As this procedure could benefit from image guidance, the authors set forth to demonstrate the impact of augmented-reality (AR) assistance on the accuracy and learning curve of EVD placement compared with the freehand technique. METHODS: Sixteen medical students performed a total of 128 EVD placements on a custom-made phantom head, both before and after receiving a standardized training session. They were guided by either the freehand technique or by AR, which provided an anatomical overlay and tailored guidance for EVD placement through inside-out infrared tracking. The outcome was quantified by the metric accuracy of EVD placement as well as by its clinical quality. RESULTS: The mean target error was significantly impacted by either AR (p = 0.003) or training (p = 0.02) in a direct comparison with the untrained freehand performance. Both untrained (11.9 ± 4.5 mm) and trained (12.2 ± 4.7 mm) AR performances were significantly better than the untrained freehand performance (19.9 ± 4.2 mm), which improved after training (13.5 ± 4.7 mm). The quality of EVD placement as assessed by the modified Kakarla scale (mKS) was significantly impacted by AR guidance (p = 0.005) but not by training (p = 0.07). Both untrained and trained AR performances (59.4% mKS grade 1 for both) were significantly better than the untrained freehand performance (25.0% mKS grade 1). Spatial aptitude testing revealed a correlation between perceptual ability and untrained AR-guided performance (r = 0.63). CONCLUSIONS: Compared with the freehand technique, AR guidance for EVD placement yielded a higher outcome accuracy and quality for procedure novices. With AR, untrained individuals performed as well as trained individuals, which indicates that AR guidance not only improved performance but also positively impacted the learning curve. Future efforts will focus on the translation and evaluation of AR for EVD placement in the clinical setting.

A Digital Platform to Support Self-management of Multiple Chronic Conditions (ProACT): Findings in Relation to Engagement During a One-Year Proof-of-Concept Trial.

BACKGROUND: Populations globally are ageing, resulting in higher incidence rates of chronic diseases. Digital health platforms, designed to support those with chronic conditions to self-manage at home, offer a promising solution to help people monitor their conditions and lifestyle, maintain good health, and reduce unscheduled clinical visits. However, despite high prevalence rates of multimorbidity or multiple chronic conditions, most platforms tend to focus on a single disease. A further challenge is that despite the importance of users actively engaging with such systems, little research has explored engagement. OBJECTIVE: The objectives of this study are to design and develop a digital health platform, ProACT, for facilitating older adults self-managing multimorbidity, with support from their care network, and evaluate end user engagement and experiences with this platform through a 12-month trial. METHODS: The ProACT digital health platform is presented in this paper. The platform was evaluated in a year-long proof-of-concept action research trial with 120 older persons with multimorbidity in Ireland and Belgium. Alongside the technology, participants had access to a clinical triage service responding to symptom alerts and a technical helpdesk. Interactions with the platform during the trial were logged to determine engagement. Semistructured interviews were conducted with participants and analyzed using inductive thematic analysis, whereas usability and user burden were examined using validated questionnaires. RESULTS: This paper presents the ProACT platform and its components, along with findings on engagement with the platform and its usability. Of the 120 participants who participated, 24 (20%) withdrew before the end of the study, whereas 3 (2.5%) died. The remaining 93 participants actively used the platform until the end of the trial, on average, taking 2 or 3 health readings daily over the course of the trial in Ireland and Belgium, respectively. The participants reported ProACT to be usable and of low burden. Findings from interviews revealed that participants experienced multiple benefits as a result of using ProACT, including improved self-management, health, and well-being and support from the triage service. For those who withdrew, barriers to engagement were poor health and frustration when technology, in particular sensing devices, did not work as expected. CONCLUSIONS: This is the first study to present findings from a longitudinal study of older adults using digital health technology to self-manage multimorbidity. Our findings show that older adults sustained engagement with the technology and found it usable. Potential reasons for these results include a strong focus on user-centered design and engagement throughout the project lifecycle, resulting in a platform that meets user needs, as well as the integration of behavior change techniques and personal analytics into the platform. The provision of triage and technical support services alongside the platform during the trial were also important facilitators of engagement. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/22125.

Social Processes: What Determines Industrial Workers' Intention to Use Exoskeletons?

OBJECTIVE: The aim of this study is to test the unified theory of acceptance and use of technology (UTAUT) model for explaining the intention to use exoskeletons among industrial workers. BACKGROUND: Exoskeletons could help reduce physical workload and risk for injuries among industrial workers. Therefore, it is crucial to understand which factors play a role in workers' intention to use such exoskeletons. METHOD: Industrial workers (N = 124) completed a survey on their attitudes regarding the use of exoskeletons at their workplace. Using partial least squares (PLS) path modeling, the UTAUT model and a revised version of the UTAUT model were fitted to these data. RESULTS: The adapted UTAUT model of Dwivedi et al. (2017) was able to explain up to 75.6% of the variance in intention to use exoskeletons, suggesting a reasonable model fit. CONCLUSION: The model fit suggests that effort expectancy (how easy it seems to use an exoskeleton) plays an important role in predicting the intention to use exoskeletons. Social influence (whether others think workers should use exoskeletons) and performance expectancy (how useful exoskeletons seem to be for work) play a smaller role in predicting the intention to use. APPLICATIONS: This research informs companies about the optimal implementation of exoskeletons by improving the determinants of acceptance among their workers.

Role of nanoparticle size and sialic acids in the distinct time-evolution profiles of nanoparticle uptake in hematopoietic progenitor cells and monocytes.

BACKGROUND: Human hematopoietic progenitor cells (HPCs) are important for cell therapy in cancer and tissue regeneration. In vitro studies have shown a transient association of 40 nm polystyrene nanoparticles (PS NPs) with these cells, which is of interest for intelligent design and application of NPs in HPC-based regenerative protocols. In this study, we aimed to investigate the involvement of nanoparticles' size and membrane-attached glycan molecules in the interaction of HPCs with PS NPs, and compared it with monocytes. Human cord blood-derived HPCs and THP-1 cells were exposed to fluorescently labelled, carboxylated PS NPs of 40, 100 and 200 nm. Time-dependent nanoparticle membrane association and/or uptake was observed by measuring fluorescence intensity of exposed cells at short time intervals using flow cytometry. By pretreating the cells with neuraminidase, we studied the possible effect of membrane-associated sialic acids in the interaction with NPs. Confocal microscopy was used to visualize the cell-specific character of the NP association. RESULTS: Confocal images revealed that the majority of PS NPs was initially observed to be retained at the outer membrane of HPCs, while the same NPs showed immediate internalization by THP-1 monocytic cells. After prolonged exposure up to 4 h, PS NPs were also observed to enter the HPCs' intracellular compartment. Cell-specific time courses of NP association with HPCs and THP-1 cells remained persistent after cells were enzymatically treated with neuraminidase, but significantly increased levels of NP association could be observed, suggesting a role for membrane-associated sialic acids in this process. CONCLUSIONS: We conclude that the terminal membrane-associated sialic acids contribute to the NP retention at the outer cell membrane of HPCs. This retention behavior is a unique characteristic of the HPCs and is independent of NP size.

Performance of Pediatric Mortality Prediction Scores for PICU Mortality and 90-Day Mortality.

OBJECTIVES: The use of mortality prediction scores in clinical trials in the PICU is essential for comparing patient groups. Because of the decline in PICU mortality over the last decades, leading to a shift toward later deaths, recent trials use 90-day mortality as primary outcome for estimating mortality and survival more accurately. This study assessed and compared the performance of two frequently used PICU mortality prediction scores for prediction of PICU and 90-day mortality. DESIGN: This secondary analysis of the randomized controlled Early versus Late Parenteral Nutrition in the Pediatric Intensive Care Unit trial compared the discrimination (area under the receiver operating characteristic curve) and calibration of the Pediatric Index of Mortality 3 and the Pediatric Risk of Mortality III scores for prediction of PICU and 90-day mortality. SETTING: Three participating PICUs within academic hospitals in Belgium, the Netherlands, and Canada. PATIENTS: One-thousand four-hundred twenty-eight critically ill patients 0-17 years old. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Although Pediatric Index of Mortality 3 only includes information available at the time of PICU admission, thus before any intervention in the PICU, it showed good discrimination (area under the receiver operating characteristic curve, 0.894; 95% CI, 0.892-0.896) and good calibration (no deviation from the diagonal, p = 0.58) for PICU mortality. Pediatric Risk of Mortality III, which involves the worst values for the evaluated variables during the first 24 hours of PICU stay, was statistically more discriminant (area under the receiver operating characteristic curve, 0.920; 95% CI, 0.918-0.921; p = 0.04) but poor in calibration (significant deviation from the diagonal; p = 0.04). Pediatric Index of Mortality 3 and Pediatric Risk of Mortality III discriminated equally well between 90-day mortality and survival (area under the receiver operating characteristic curve, 0.867; 95% CI, 0.866-0.869 and area under the receiver operating characteristic curve, 0.882; 95% CI, 0.880-0.884, respectively, p = 0.77), but Pediatric Risk of Mortality III was not well calibrated (p = 0.04), unlike Pediatric Index of Mortality 3 (p = 0.34). CONCLUSIONS: Pediatric Index of Mortality 3 performed better in calibration for predicting PICU and 90-day mortality than Pediatric Risk of Mortality III and is not influenced by intervention or PICU quality of care. Therefore, Pediatric Index of Mortality 3 seems a better choice for use in clinical trials with 90-day mortality as primary outcome.

International survey of De-implementation of initiating parenteral nutrition early in Paediatric intensive care units.

BACKGROUND: Initiating parenteral nutrition (PN) within 24 h in critically ill children is inferior to withholding PN during the first week, as was found in the PEPaNIC study. The aims of this study were to investigate de-implementation of early initiation of PN at PICUs worldwide, and to identify factors influencing de-implementation. METHODS: A cross-sectional online survey was conducted (May - October 2017), consisting of 41 questions addressing current PN practices, the degree of de-implementation, and factors affecting de-implementation. RESULTS: We analysed 81 responses from 39 countries. Of these 81 respondents, 53 (65%) were aware of the findings of the PEPaNIC study, and 43 (53%) have read the article. In these 43 PICUs, PN was completely withheld during the first week in 10 PICUs, of which 5 already withheld PN (12%), and 5 de-implemented early initiation of PN (12%). Partial de-implementation was reported by 17 (40%) and no de-implementation by 16 (37%). Higher de-implementation rates were observed when the interpreted level of evidence and grade of recommendation of PEPaNIC was high. Predominant reasons for retaining early initiation of PN were concerns on withholding amino acids, the safety in undernourished children and neonates, and the long-term consequences. Furthermore, the respondents were waiting for updated guidelines. CONCLUSIONS: One year after the publication of the PEPaNIC trial, only two-thirds of the respondents was aware of the study results. Within this group, early initiation of PN was de-implemented completely in 12% of the PICUs, while 40% asserted partial de-implementation. Increasing the awareness, addressing the intervention-specific questions and more frequently revising international guidelines might help to accelerate de-implementation of ineffective, unproven or harmful healthcare.

Xenopus laevis as a Bioindicator of Endocrine Disruptors in the Region of Central Chile.

One of the direct causes of biodiversity loss is environmental pollution resulting from the use of chemicals. Different kinds of chemicals, such as persistent organic pollutants and some heavy metals, can be endocrine disruptors, which act at low doses over a long period of time and have a negative effect on the reproductive and thyroid system in vertebrates worldwide. Research on the effects of endocrine disruptors and the use of bioindicators in neotropical ecosystems where pressure on biodiversity is high is scarce. In Chile, although endocrine disruptors have been detected at different concentrations in the environments of some ecosystems, few studies have been performed on their biological effects in the field. In this work, Xenopus laevis (African clawed frog), an introduced species, is used as a bioindicator for the presence of endocrine disruptors in aquatic systems with different degrees of contamination in a Mediterranean zone in central Chile. For the first time for Chile, alterations are described that can be linked to exposure to endocrine disruptors, such as vitellogenin induction, decreased testosterone in male frogs, and histological changes in gonads. Dioxin-like and oestrogenic activity was detected in sediments at locations where it seem to be related to alterations found in the frogs. In addition, an analysis of land use/cover use revealed that urban soil was the best model to explain the variations in frog health indicators. This study points to the usefulness of an invasive species as a bioindicator for the presence of endocrine-disruptive chemicals.

The comet assay in human biomonitoring: cryopreservation of whole blood and comparison with isolated mononuclear cells.

The comet assay is often applied in human biomonitoring. Most of the time the assay is performed with isolated peripheral blood mononuclear cells (PBMC). However, using whole blood instead of isolated cells reduces processing time, and only 20 µl is sufficient for analysis. In this study, a cryopreservation protocol for human whole blood for application in the comet assay was optimised by removing excess plasma before adding freezing medium. Cryopreservation of whole blood samples (n = 30) did not increase the detected level of strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites. Although there was no significant correlation with breaks measured in fresh whole blood, strand breaks detected in frozen whole blood were significantly correlated with breaks measured in frozen PBMC (Pearson correlation r = 0.54, P < 0.01). This correlation was however not observed for FPG-sensitive sites. Since we do not yet know the full extent to which cryopreservation might influence the blood cell population, care should be taken to ensure a similar cell type and storage conditions for all samples in one study.

A Novel Exposure System Termed NAVETTA for In Vitro Laminar Flow Electrodeposition of Nanoaerosol and Evaluation of Immune Effects in Human Lung Reporter Cells.

A new prototype air-liquid interface (ALI) exposure system, a flatbed aerosol exposure chamber termed NAVETTA, was developed to investigate deposition of engineered nanoparticles (NPs) on cultured human lung A549 cells directly from the gas phase. This device mimics human lung cell exposure to NPs due to a low horizontal gas flow combined with cells exposed at the ALI. Electrostatic field assistance is applied to improve NP deposition efficiency. As proof-of-principle, cell viability and immune responses after short-term exposure to nanocopper oxide (CuO)-aerosol were determined. We found that, due to the laminar aerosol flow and a specific orientation of inverted transwells, much higher deposition rates were obtained compared to the normal ALI setup. Cellular responses were monitored with postexposure incubation in submerged conditions, revealing CuO dissolution in a concentration-dependent manner. Cytotoxicity was the result of ionic and nonionic Cu fractions. Using the optimized inverted ALI/postincubation procedure, pro-inflammatory immune responses, in terms of interleukin (IL)-8 promoter and nuclear factor kappa B (NFκB) activity, were observed within short time, i.e. One hour exposure to ALI-deposited CuO-NPs and 2.5 h postincubation. NAVETTA is a novel option for mimicking human lung cell exposure to NPs, complementing existing ALI systems.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.

Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot-Marie-Tooth neuropathy.

Charcot-Marie-Tooth (CMT) neuropathies are common disorders of the peripheral nervous system caused by demyelination or axonal degeneration, or a combination of both features. We previously assigned the locus for autosomal dominant intermediate CMT neuropathy type C (DI-CMTC) to chromosome 1p34-p35. Here we identify two heterozygous missense mutations (G41R and E196K) and one de novo deletion (153-156delVKQV) in tyrosyl-tRNA synthetase (YARS) in three unrelated families affected with DI-CMTC. Biochemical experiments and genetic complementation in yeast show partial loss of aminoacylation activity of the mutant proteins, and mutations in YARS, or in its yeast ortholog TYS1, reduce yeast growth. YARS localizes to axonal termini in differentiating primary motor neuron and neuroblastoma cultures. This specific distribution is significantly reduced in cells expressing mutant YARS proteins. YARS is the second aminoacyl-tRNA synthetase found to be involved in CMT, thereby linking protein-synthesizing complexes with neurodegeneration.

Human speckle perception threshold for still images from a laser projection system.

We study the perception of speckle by human observers in a laser projector based on a 40 persons survey. The speckle contrast is first objectively measured making use of a well-defined speckle measurement method. We statistically analyse the results of the user quality scores, revealing that the speckle perception is not only influenced by the speckle contrast settings of the projector, but it is also strongly influenced by the type of image shown. Based on the survey, we derive a speckle contrast threshold for which speckle can be seen, and separately we investigate a speckle disturbance limit that is tolerated by the majority of test persons.

Pro-opiomelanocortin and ACTH-cortisol dissociation during pediatric cardiac surgery.

In critically ill adults, high plasma cortisol in face of low ACTH coincides with high pro-opiomelanocortin (POMC) levels. Glucocorticoids further lower ACTH without affecting POMC. We hypothesized that in pediatric cardiac surgery-induced critical illness, plasma POMC is elevated, plasma ACTH transiently rises intraoperatively but becomes suppressed post-operatively, and glucocorticoid administration amplifies this phenotype. From 53 patients (0-36 months), plasma was obtained pre-operatively, intraoperatively and on post-operative day 1 and 2. Plasma was also collected from 24 healthy children. In patients, POMC was supra-normal pre-operatively (p<0.0001) but no longer thereafter (p<0.05). ACTH was never high in patients. While in glucocorticoid-naive patients ACTH became suppressed by post-operative day 1 (p<0.0001), glucocorticoid-treated patients had suppressed ACTH already intraoperatively (p≤0.0001). Pre-operatively high POMC, not accompanied by increased plasma ACTH, suggests a centrally-activated HPA-axis with reduced pituitary processing of POMC into ACTH. Increasing systemic glucocorticoid availability with glucocorticoid treatment accelerated the suppression of plasma ACTH.