Longitudinal urinary neopterin is associated with hearing threshold change over time in independent older adults.

Low-grade chronic inflammation is associated with many age-related conditions. Non-invasive methods to monitor low-grade chronic inflammation may improve the management of older people at risk of poorer outcomes. This longitudinal cohort study has determined baseline inflammation using neopterin volatility in monthly urine samples of 45 independent older adults (aged 65-75 years). Measurement of neopterin, an inflammatory metabolite, enabled stratification of individuals into risk categories based on how often in a 12-month period their neopterin level was raised. Hearing was measured (pure-tone audiometry) at baseline, 1 year and 3 years of the study. Results show that those in the highest risk category (neopterin raised greater than 50% of the time) saw greater deterioration, particularly in high-frequency, hearing. A one-way Welch's ANOVA showed a significant difference between the risk categories for change in high-frequency hearing (W (3, 19.6) = 9.164, p = 0.0005). Despite the study size and duration individuals in the highest risk category were more than twice as likely to have an additional age-related morbidity than those in the lowest risk category. We conclude that volatility of neopterin in urine may enable stratification of those at greatest risk of progression of hearing loss.

Tau-mediated axonal degeneration is prevented by activation of the WldS pathway.

Tauopathy is characterized by neuronal dysfunction and degeneration occurring as a result of changes to the microtubule-associated protein tau. The neuronal changes evident in tauopathy bear striking morphological resemblance to those reported in models of Wallerian degeneration. The mechanisms underpinning Wallerian degeneration are not fully understood although it can be delayed by the expression of the slow Wallerian degeneration (WldS) protein, which has also been demonstrated to delay axonal degeneration in some models of neurodegenerative disease. Given the morphological similarities between tauopathy and Wallerian degeneration, this study investigated whether tau-mediated phenotypes can be modulated by co-expression of WldS. In a Drosophila model of tauopathy in which expression of human 0N3R tau protein leads to progressive age-dependent phenotypes, WldS was expressed with and without activation of the downstream pathway. The olfactory receptor neuron circuit OR47b was used for these studies in adults, and the larval motor neuron system was employed in larvae. Tau phenotypes studied included neurodegeneration, axonal transport, synaptic deficits and locomotor behaviour. Impact on total tau was ascertained by assessing total, phosphorylated and misfolded tau levels by immunohistochemistry. Activation of the pathway downstream of WldS completely suppressed tau-mediated degeneration. This protective effect was evident even if the pathway downstream of WldS was activated several weeks after tau-mediated degeneration had become established. Though total tau levels were not altered, the protected neurons displayed significantly reduced MC1 immunoreactivity suggestive of clearance of misfolded tau, as well as a trend for a decline in tau species phosphorylated at the AT8 and PHF1 epitopes. In contrast, WldS expression without activation of the downstream protective pathway did not rescue tau-mediated degeneration in adults or improve tau-mediated neuronal dysfunction including deficits in axonal transport, synaptic alterations and locomotor behaviour in tau-expressing larvae. This collectively implies that the pathway mediating the protective effect of WldS intersects with the mechanism(s) of degeneration initiated by tau and can effectively halt tau-mediated degeneration at both early and late stages. Understanding the mechanisms underpinning this protection could identify much-needed disease-modifying targets for tauopathies.

Leveraging real-world data to improve cochlear implant outcomes: Is the data available?

OBJECTIVES: A small but persistent proportion of individuals do not gain the expected benefit from cochlear implants(CI). A step-change in the understanding of factors affecting outcomes could come through data science. This study evaluates clinical data capture to assess the quality and utility of CI user's health records for data science, by assessing the recording of otitis media. Otitis media was selected as it is associated with the development of sensorineural hearing loss and may affect cochlear implant outcomes. METHODS: A retrospective service improvement project evaluating the medical records of 594 people with a CI under the care of the University of Southampton Auditory Implant Service between 2014 and 2020. RESULTS: The clinical records are suitable for data science research. Of the cohort studied 20% of Adults and more than 40% of the paediatric cases have a history of middle ear inflammation. DISCUSSION: Data science has potential to improve cochlear implant outcomes and improve understanding of the mechanisms underlying poor performance, through retrospective secondary analysis of real-world data. CONCLUSION: Implant centres and the British Cochlear Implant Group National Hearing Implant Registry are urged to consider the importance of consistently and accurate recording of patient data over time for each CI user. Data where links to hearing loss have been identified, such as middle ear inflammation, may be particularly valuable in future analyses and to inform clinical trials.

An Exploratory Study Provides Insights into MMP9 and Aß Levels in the Vitreous and Blood across Different Ages and in a Subset of AMD Patients.

Matrix metalloproteinase-9 (MMP9) and total amyloid-beta (Aß) are prospective biomarkers of ocular ageing and retinopathy. These were quantified by ELISA in the vitreous and blood from controls (n = 55) and in a subset of age-related macular degeneration (AMD) patients (n = 12) for insights and possible additional links between the ocular and systemic compartments. Vitreous MMP9 levels in control and AMD groups were 932.5 ± 240.9 pg/mL and 813.7 ± 157.6 pg/mL, whilst serum levels were 2228 ± 193 pg/mL and 2386.8 ± 449.4 pg/mL, respectively. Vitreous Aß in control and AMD groups were 1173.5 ± 117.1 pg/mL and 1275.6 ± 332.9 pg/mL, whilst plasma Aß were 574.3 ± 104.8 pg/mL and 542.2 ± 139.9 pg/mL, respectively. MMP9 and Aß showed variable levels across the lifecourse, indicating no correlation to each other or with age nor AMD status, though the smaller AMD cohort was a limiting factor. Aß and MMP9 levels in the vitreous and blood were unrelated to mean arterial pressure. Smoking, another modifiable risk, showed no association with vitreous Aß. However, smoking may be linked with vitreous (p = 0.004) and serum (p = 0.005) MMP9 levels in control and AMD groups, though this did not reach our elevated (p = 0.001) significance. A bioinformatics analysis revealed promising MMP9 and APP/Aß partners for further scrutiny, many of which are already linked with retinopathy.

Repeated short-term exposure to diesel exhaust reduces honey bee colony fitness.

Production of insect-pollinated crops is often reliant on honey bee (Apis mellifera) pollination services. Colonies can be managed and moved to meet the demands of modern intensified monoculture farming systems. Increased colony mortalities have been observed, which are thought be caused by interacting factors including exposure to pesticides, parasites, viruses, agricultural intensification, and changes in global and regional climate. However, whilst common tropospheric air pollutants (e.g. NOx, particulate matter etc) are known to cause a range of negative effects on human health, there is little evidence of their impact on the health of A. mellifera. This study investigates the effects of exposure to diesel exhaust on A. mellifera, both at the level of individual foragers and on the whole colony. We exposed a series of colonies to diesel exhaust fumes for 2 h a day over the course of three weeks and contrasted their performance to a series of paired control colonies located at the same field site. We investigated markers of neuronal health in the brains of individual foragers and measured the prevalence of common viruses. Electronic counters monitored daily colony activity patterns and pollen samples from returning foragers were analysed to investigate plant species richness and diversity. The amounts of honey, brood and pollen in each colony were measured regularly. We demonstrated an upregulation of the synapse protein Neurexin 1 in forager brains repeatedly exposed to diesel exhaust. Furthermore, we found that colonies exposed to diesel exhaust lost colony weight after the exposure period until the end of the summer season, whereas control colonies gained weight towards the end of the season. Further investigations are required, but we hypothesise that such effects on both individual foragers and whole colony fitness parameters could ultimately contribute to winter losses of honey bee colonies, particularly in the presence of additional stressors.

Macrophages in the cochlea; an immunological link between risk factors and progressive hearing loss.

Macrophages are abundant in the cochlea; however, their role in hearing loss is not well understood. Insults to the cochlea, such as noise or insertion of a cochlear implant, cause an inflammatory response, which includes activation of tissue-resident macrophages. Activation is characterized by changes in macrophage morphology, mediator expression, and distribution. Evidence from other organs shows activated macrophages can become primed, whereby subsequent insults cause an elevated inflammatory response. Primed macrophages in brain pathologies respond to circulating inflammatory mediators by disproportionate synthesis of inflammatory mediators. This signaling occurs behind an intact blood-brain barrier, similar to the blood-labyrinth barrier in the cochlea. Local tissue damage can occur as the result of mediator release by activated macrophages. Damage is typically localized; however, if it is to structures with limited ability to repair, such as neurons or hair cells within the cochlea, it is feasible that this contributes to the progressive loss of function seen in hearing loss. We propose that macrophages in the cochlea link risk factors and hearing loss. Injury to the cochlea causes local macrophage activation that typically resolves. However, in susceptible individuals, some macrophages enter a primed state. Once primed, these macrophages can be further activated, as a consequence of circulating inflammatory molecules associated with common co-morbidities. Hypothetically, this would lead to further cochlear damage and loss of hearing. We review the evidence for the role of tissue-resident macrophages in the cochlea and propose that cochlear macrophages contribute to the trajectory of hearing loss and warrant further study.

Antibiotic-Loaded Polymersomes for Clearance of Intracellular Burkholderia thailandensis.

Melioidosis caused by the facultative intracellular pathogen Burkholderia pseudomallei is difficult to treat due to poor intracellular bioavailability of antibiotics and antibiotic resistance. In the absence of novel compounds, polymersome (PM) encapsulation may increase the efficacy of existing antibiotics and reduce antibiotic resistance by promoting targeted, infection-specific intracellular uptake. In this study, we developed PMs composed of widely available poly(ethylene oxide)-polycaprolactone block copolymers and demonstrated their delivery to intracellular B. thailandensis infection using multispectral imaging flow cytometry (IFC) and coherent anti-Stokes Raman scattering microscopy. Antibiotics were tightly sequestered in PMs and did not inhibit the growth of free-living B. thailandensis. However, on uptake of antibiotic-loaded PMs by infected macrophages, IFC demonstrated PM colocalization with intracellular B. thailandensis and a significant inhibition of their growth. We conclude that PMs are a viable approach for the targeted antibiotic treatment of persistent intracellular Burkholderia infection.

Oligomeric Aß1-42 Induces an AMD-Like Phenotype and Accumulates in Lysosomes to Impair RPE Function.

Alzheimer's disease-associated amyloid beta (Aß) proteins accumulate in the outer retina with increasing age and in eyes of age-related macular degeneration (AMD) patients. To study Aß-induced retinopathy, wild-type mice were injected with nanomolar human oligomeric Aß1-42, which recapitulate the Aß burden reported in human donor eyes. In vitro studies investigated the cellular effects of Aß in endothelial and retinal pigment epithelial (RPE) cells. Results show subretinal Aß-induced focal AMD-like pathology within 2 weeks. Aß exposure caused endothelial cell migration, and morphological and barrier alterations to the RPE. Aß co-localized to late-endocytic compartments of RPE cells, which persisted despite attempts to clear it through upregulation of lysosomal cathepsin B, revealing a novel mechanism of lysosomal impairment in retinal degeneration. The rapid upregulation of cathepsin B was out of step with the prolonged accumulation of Aß within lysosomes, and contrasted with enzymatic responses to internalized photoreceptor outer segments (POS). Furthermore, RPE cells exposed to Aß were identified as deficient in cargo-carrying lysosomes at time points that are critical to POS degradation. These findings imply that Aß accumulation within late-endocytic compartments, as well as lysosomal deficiency, impairs RPE function over time, contributing to visual defects seen in aging and AMD eyes.

Inflammation at the Tissue-Electrode Interface in a Case of Rapid Deterioration in Hearing Performance Leading to Explant After Cochlear Implantation.

OBJECTIVE: The reasons for soft failure after cochlear implantation require investigation. This study proposes a method to study and characterize the tissue response to the array in a case of soft failure in a person undergoing reimplantation. CASE: The woman in her 50s, with an underlying autoimmune condition, received a cochlear implant using hearing preservation technique after developing profound hearing loss more than 2 kHz with a moderate loss of less than 500 Hz over a 10-year period. The case was identified as a soft failure due to deteriorating performance, discomfort, and migration over the 10 months after implantation. Impedance telemetry, speech perception measures, and audiometric thresholds are described. At explantation there was evidence of fibrosis. INTERVENTIONS: To use histology and immunohistochemistry to determine the cellular response of the tissue associated with the electrode array at time of explantation. MAIN OUTCOME MEASURES: Identification of the cell types, regional variations, and inflammatory marker expression in the fibrotic tissue associated with the array. RESULTS: Neutrophils and eosinophils were identified, along with a variable pattern of collagen deposition. CD68 and CD163-positive macrophages and T cells were variably distributed through the tissue and interleukin-1 beta and vascular endothelial growth factor receptor-2 expression was identified. CONCLUSIONS: The expression profile is evidence of active inflammation in the tissue despite the time since implantation. This study is the first to characterize the tissue response to the array in a person undergoing reimplantation, and who can be followed to determine the individual response to arrays. It establishes that the investigation of explanted devices after soft-failure is feasible.

Genetics of age-related hearing loss.

Age-related hearing loss (ARHL) has recently been confirmed as a common complex trait, that is, it is heritable with many genetic variants each contributing a small amount of risk, as well as environmental determinants. Historically, attempts to identify the genetic variants underlying the ARHL have been of limited success, relying on the selection of candidate genes based on the limited knowledge of the pathophysiology of the condition, and linkage studies in samples comprising related individuals. More recently genome-wide association studies have been performed, but these require very large samples having consistent and reliable phenotyping for hearing loss (HL), and early attempts suffered from lack of reliable replication of their findings. Replicated variants shown associated with ARHL include those lying in genes GRM7, ISG20, TRIOBP, ILDR1, and EYA4. The availability of large biobanks and the development of collaborative consortia have led to a breakthrough over the last couple of years, and many new genetic variants associated with ARHL are becoming available, through the analysis publicly available bioresources and electronic health records. These findings along with immunohistochemistry and mouse models of HL look set to help disentangle the genetic architecture of ARHL, and highlight the need for standardization of phenotyping methods to facilitate data sharing and collaboration across research networks.

High-Throughput Urinary Neopterin-to-Creatinine Ratio Monitoring of Systemic Inflammation.

BACKGROUND: Systemic inflammation is a marker of ill health and has prognostic implications in multiple health settings. Urinary neopterin is an excellent candidate as a nonspecific marker of systemic inflammation. Expression as urinary neopterin-to-creatinine ratio (UNCR) normalizes for urinary hydration status. Major attractions include (a) urine vs blood sampling, (b) integration of inflammation over a longer period compared with serum sampling, and (c) high stability of neopterin and creatinine. METHODS: A high-throughput ultraperformance LC-MS method was developed to measure neopterin and creatinine together from the same urine sample. The assay was applied in several clinical scenarios: healthy controls, symptomatic infections, and multiple sclerosis. Area under the curve was compared between weekly and monthly sampling scenarios. Analysis of a single pooled sample was compared with averaging results from analysis of individual samples. RESULTS: The assay has excellent intraassay and interassay precision, linearity of dilution, and spike and recovery. Higher UNCR was demonstrated in female vs male individuals, older age, inflammatory disease (multiple sclerosis), and symptomatic infections. In healthy controls, fluctuations in inflammatory state also occurred in the absence of symptomatic infection or other inflammatory triggers. Analysis of a single pooled sample, made up from weekly urine samples, integrates inflammatory activity over time. CONCLUSIONS: UNCR is a useful biomarker of systemic inflammation. The method presented offers simplicity, speed, robustness, reproducibility, efficiency, and proven utility in clinical scenarios. UNCR fluctuations underline the importance of longitudinal monitoring, vs a single time point, to capture a more representative estimate of an individual's inflammatory state over time.

Acute exposure to diesel exhaust induces central nervous system stress and altered learning and memory in honey bees.

For effective foraging, many insect pollinators rely on the ability to learn and recall floral odours, behaviours that are associated with a complex suite of cellular processes. Here, we investigated how acute exposure to a high-dose of diesel exhaust (containing 19.8 and 17.5 ppm of NO and NO2, respectively) affected associative learning behaviour of honey bees (Apis mellifera) and expression of a ubiquitous heat shock protein, HSP70, in their central nervous system (CNS). To determine whether exposure to diesel exhaust would alter their tolerance to a subsequent abiotic stress, we further subjected individuals to heat stress. Diesel exhaust exposure decreased honey bees' ability to learn and recall a conditioned odour stimulus. Whilst there was no significant difference in CNS HSP70 expression between honey bees exposed to either diesel exhaust or clean air across the entire duration of the experiment (3.5 h), there was a significant effect of time and a significant interaction between exposure treatment and time. This interaction was investigated using correlation analyses, which demonstrated that only in the diesel exhaust exposed honey bees was there a significant positive correlation between HSP70 expression and time. Furthermore, there was a 44% reduction in honey bee individuals that were able to recall the odour 72 h after diesel exposure compared with clean air control individuals. Moreover, diesel exhaust affected A. mellifera in a way that reduced their ability to survive a second subsequent stressor. Such negative effects of air pollution on learning, recall, and stress tolerance has potential to reduce foraging efficiency and pollination success of individual honey bees.

A convenient protocol for establishing a human cell culture model of the outer retina.

The retinal pigment epithelium (RPE) plays a key role in the pathogenesis of several blinding retinopathies. Alterations to RPE structure and function are reported in Age-related Macular Degeneration, Stargardt and Best disease as well as pattern dystrophies. However, the precise role of RPE cells in disease aetiology remains incompletely understood. Many studies into RPE pathobiology have utilised animal models, which only recapitulate limited disease features. Some studies are also difficult to carry out in animals as the ocular space remains largely inaccessible to powerful microscopes. In contrast, in-vitro models provide an attractive alternative to investigating pathogenic RPE changes associated with age and disease. In this article we describe the step-by-step approach required to establish an experimentally versatile in-vitro culture model of the outer retina incorporating the RPE monolayer and supportive Bruch's membrane (BrM). We show that confluent monolayers of the spontaneously arisen human ARPE-19 cell-line cultured under optimal conditions reproduce key features of native RPE. These models can be used to study dynamic, intracellular and extracellular pathogenic changes using the latest developments in microscopy and imaging technology. We also discuss how RPE cells from human foetal and stem-cell derived sources can be incorporated alongside sophisticated BrM substitutes to replicate the aged/diseased outer retina in a dish. The work presented here will enable users to rapidly establish a realistic in-vitro model of the outer retina that is amenable to a high degree of experimental manipulation which will also serve as an attractive alternative to using animals. This in-vitro model therefore has the benefit of achieving the 3Rs objective of reducing and replacing the use of animals in research. As well as recapitulating salient structural and physiological features of native RPE, other advantages of this model include its simplicity, rapid set-up time and unlimited scope for detailed single-cell resolution and matrix studies.

Polymersome nanoparticles for delivery of Wnt-activating small molecules.

Spatiotemporal control of drug delivery is important for a number of medical applications and may be achieved using polymersome nanoparticles (PMs). Wnt signalling is a molecular pathway activated in various physiological processes, including bone repair, that requires precise control of activation. Here, we hypothesise that PMs can be stably loaded with a small molecule Wnt agonist, 6-bromoindirubin-3'-oxime (BIO), and activate Wnt signalling promoting the osteogenic differentiation in human primary bone marrow stromal cells (BMSCs). We showed that BIO-PMs induced a 40% increase in Wnt signaling activation in reporter cell lines without cytotoxicity induced by free BIO. BMSCs incubated with BIO-PMs showed a significant up-regulation of the Wnt target gene AXIN2 (14 ±â€¯4 fold increase, P < 0.001) and a prolonged activation of the osteogenic gene RUNX2. We conclude that BIO-PMs could represent an innovative approach for the controlled activation of Wnt signaling for promoting bone regeneration after fracture.

Exploiting Routine Clinical Measures to Inform Strategies for Better Hearing Performance in Cochlear Implant Users.

Neuroprostheses designed to interface with the nervous system to replace injured or missing senses can significantly improve a patient's quality of life. The challenge remains to provide implants that operate optimally over several decades. Changes in the implant-tissue interface may precede performance problems. Tools to identify and characterize such changes using existing clinical measures would be highly valuable. Modern cochlear implant (CI) systems allow easy and regular measurements of electrode impedance (EI). This measure is routinely performed as a hardware integrity test, but it also allows a level of insight into the immune-mediated response to the implant, which is associated with performance outcomes. This study is a 5-year retrospective investigation of MED-EL CI users at the University of Southampton Auditory Implant Service including 176 adult ears (18-91) and 74 pediatric ears (1-17). The trend in EI in adults showed a decrease at apical electrodes. An increase was seen at the basal electrodes which are closest to the surgery site. The trend in the pediatric cohort was increasing EI over time for nearly all electrode positions, although this group showed greater variability and had a smaller sample size. We applied an outlier-labeling rule to statistically identify individuals that exhibit raised impedance. This highlighted 14 adult ears (8%) and 3 pediatric ears (5%) with impedance levels that deviated from the group distribution. The slow development of EI suggests intra-cochlear fibrosis and/or osteogenesis as the underlying mechanism. The usual clinical intervention for extreme impedance readings is to deactivate the relevant electrode. Our findings highlight some interesting clinical contradictions: some cases with raised (but not extreme) impedance had not prompted an electrode deactivation; and many cases of electrode deactivation had been informed by subjective patient reports. This emphasizes the need for improved objective evidence to inform electrode deactivations in borderline cases, for which our outlier-labeling approach is a promising candidate. A data extraction and analysis protocol that allows ongoing and automated statistical analysis of routinely collected data could benefit both the CI and wider neuroprosthetics communities. Our approach provides new tools to inform practice and to improve the function and longevity of neuroprosthetic devices.

Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE.

The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch's membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells. Here, we evaluate how two different types of BrM substrates; (1) a commercially-available polyester transwell membrane, and (2) a novel electrospun scaffold developed in our laboratory, could support the generation of realistic RPE tissues in culture. Our findings reveal that both substrates were capable of supporting long-lasting RPE monolayers with structural and functional specialisations of in-situ RPE cells. These cultures were used to study autofluorescence and barrier formation, as well as activities such as outer-segment internalisation/trafficking and directional secretion of key proteins; the impairment of which underlies retinal disease. Hence, both substrates fulfilled important criteria for generating authentic in-vitro cultures and act as powerful tools to study RPE pathophysiology. However, RPE grown on electrospun scaffolds may be better suited to studying complex RPE-BrM interactions such as the formation of drusen-like deposits associated with early retinal disease.

PEGylated liposomes associate with Wnt3A protein and expand putative stem cells in human bone marrow populations.

AIM: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow. METHODS: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes. RESULTS: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis. CONCLUSION: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.

Quantification of intracellular payload release from polymersome nanoparticles.

Polymersome nanoparticles (PMs) are attractive candidates for spatio-temporal controlled delivery of therapeutic agents. Although many studies have addressed cellular uptake of solid nanoparticles, there is very little data available on intracellular release of molecules encapsulated in membranous carriers, such as polymersomes. Here, we addressed this by developing a quantitative assay based on the hydrophilic dye, fluorescein. Fluorescein was encapsulated stably in PMs of mean diameter 85 nm, with minimal leakage after sustained dialysis. No fluorescence was detectable from fluorescein PMs, indicating quenching. Following incubation of L929 cells with fluorescein PMs, there was a gradual increase in intracellular fluorescence, indicating PM disruption and cytosolic release of fluorescein. By combining absorbance measurements with flow cytometry, we quantified the real-time intracellular release of a fluorescein at a single-cell resolution. We found that 173 ± 38 polymersomes released their payload per cell, with significant heterogeneity in uptake, despite controlled synchronisation of cell cycle. This novel method for quantification of the release of compounds from nanoparticles provides fundamental information on cellular uptake of nanoparticle-encapsulated compounds. It also illustrates the stochastic nature of population distribution in homogeneous cell populations, a factor that must be taken into account in clinical use of this technology.

Transient Canonical Wnt Stimulation Enriches Human Bone Marrow Mononuclear Cell Isolates for Osteoprogenitors.

Activation of the canonical Wnt signaling pathway is an attractive anabolic therapeutic strategy for bone. Emerging data suggest that activation of the Wnt signaling pathway promotes bone mineral accrual in osteoporotic patients. The effect of Wnt stimulation in fracture healing is less clear as Wnt signaling has both stimulatory and inhibitory effects on osteogenesis. Here, we tested the hypothesis that transient Wnt stimulation promotes the expansion and osteogenesis of a Wnt-responsive stem cell population present in human bone marrow. Bone marrow mononuclear cells (BMMNCs) were isolated from patients undergoing hip arthroplasty and exposed to Wnt3A protein. The effect of Wnt pathway stimulation was determined by measuring the frequency of stem cells within the BMMNC populations by fluorescence-activated cell sorting and colony forming unit fibroblast (CFU-F) assays, before determining their osteogenic capacity in in vitro differentiation experiments. We found that putative skeletal stem cells in BMMNC isolates exhibited elevated Wnt pathway activity compared with the population as whole. Wnt stimulation resulted in an increase in the frequency of skeletal stem cells marked by the STRO-1(bright) /Glycophorin A(-) phenotype. Osteogenesis was elevated in stromal cell populations arising from BMMNCs transiently stimulated by Wnt3A protein, but sustained stimulation inhibited osteogenesis in a concentration-dependent manner. These results demonstrate that Wnt stimulation could be used as a therapeutic approach by transient targeting of stem cell populations during early fracture healing, but that inappropriate stimulation may prevent osteogenesis.

The Effects of Diesel Exhaust Pollution on Floral Volatiles and the Consequences for Honey Bee Olfaction.

There is growing evidence of a substantial decline in pollinators within Europe and North America, most likely caused by multiple factors such as diseases, poor nutrition, habitat loss, insecticides, and environmental pollution. Diesel exhaust could be a contributing factor to this decline, since we found that diesel exhaust rapidly degrades floral volatiles, which honey bees require for flower recognition. In this study, we exposed eight of the most common floral volatiles to diesel exhaust in order to investigate whether it can affect volatile mediated plant-pollinator interaction. Exposure to diesel exhaust altered the blend of common flower volatiles significantly: myrcene was considerably reduced, ß-ocimene became undetectable, and ß-caryophyllene was transformed into its cis-isomer isocaryophyllene. Proboscis extension response (PER) assays showed that the alterations of the blend reduced the ability of honey bees to recognize it. The chemically reactive nitrogen oxides fraction of diesel exhaust gas was identified as capable of causing degradation of floral volatiles.