Extracellular vesicles (EVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential ...diagnostic biomarkers. Human serum is a rich source of readily accessible EVs; however, the separation of EVs from serum proteins and non-EV lipid particles represents a considerable challenge. In this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 µl of human serum and their separation from non-EV protein and lipid particles present in serum. The size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of EVs from soluble protein was determined by calculating the ratio of EV particle count to protein concentration. Finally, lipoprotein particles co-isolated with EVs was determined by Western blot analysis of lipoprotein markers APOB and APOE. Overall, this study reveals that the choice of EV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants.
Bacterial colonisation of the intestine has a major role in the post-natal development and maturation of the immune and endocrine systems. These processes are key factors underpinning central nervous ...system (CNS) signalling. Regulation of the microbiome-gut-brain axis is essential for maintaining homeostasis, including that of the CNS. However, there is a paucity of data pertaining to the influence of microbiome on the serotonergic system. Germ-free (GF) animals represent an effective preclinical tool to investigate such phenomena. Here we show that male GF animals have a significant elevation in the hippocampal concentration of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, its main metabolite, compared with conventionally colonised control animals. Moreover, this alteration is sex specific in contrast with the immunological and neuroendocrine effects which are evident in both sexes. Concentrations of tryptophan, the precursor of serotonin, are increased in the plasma of male GF animals, suggesting a humoral route through which the microbiota can influence CNS serotonergic neurotransmission. Interestingly, colonisation of the GF animals post weaning is insufficient to reverse the CNS neurochemical consequences in adulthood of an absent microbiota in early life despite the peripheral availability of tryptophan being restored to baseline values. In addition, reduced anxiety in GF animals is also normalised following restoration of the intestinal microbiota. These results demonstrate that CNS neurotransmission can be profoundly disturbed by the absence of a normal gut microbiota and that this aberrant neurochemical, but not behavioural, profile is resistant to restoration of a normal gut flora in later life.
Question Repetitive transcranial magnetic stimulation (rTMS) is now a well-established new treatment modality for patients with depressive disorders that has been assessed across a large range of ...controlled studies. However, limited research has explored the basic parameters used in the design of rTMS treatment protocols especially in regards to variables determining the dose of treatment. Over time, dose has generally increased without the impact of this being systematically assessed. Therefore, we have been exploring aspects of treatment dose and how they relate to treatment outcomes. Methods We have conducted several studies designed to address rTMS dose related issues. First, we have conducted several studies using TMS-EEG and TMS-NIRS methods to explore the effects of stimulation at increasing intensities on the prefrontal cortex. Second, we have conducted a large multi-site trial investigating the effect of pulse number as a dose variable on clinical response to rTMS in depression. Results TMS-NIRS studies have indicated that there is a clearly bimodal response to increasing TMS stimulation intensity which is likely to have implications for the determination of optimal dosing for clinical outcomes. Our randomised controlled data do not suggest that increasing pulse number in an rTMS treatment protocol above standard levels is associated with improved outcomes. Conclusions Improvements in the application of rTMS treatment are likely to be possible but ‘more does not always seem to be better’. Improved outcomes may be achieved but optimising stimulation intensity but not increasing pulse number in individual treatment sessions.
Recently proposed concepts regarding the nature and assessment of the thyroid state have provided a model more consistent with empiric evidence. It now appears likely that there are no such entities ...as thyroid set points and individual euthyroidism. Rather than there being discrete thyroid states, peripheral organ parameters are associated with thyroid function in a continuous manner. Thyroid hormone levels and, in particular, levels of free thyroxine now appear to be superior to thyrotropin levels as indicators of the thyroid state. Complicating the assessment of the correlations of the thyroid state with pregnancy outcomes are the contribution of the placenta to maternal thyroid function, fetal thyroid development, the multiple potential pathways to any particular outcome, the likely presence of small critical periods of time, the differing genetics of fetal and maternal tissues, and the unreliability of thyroid hormone assays. Nevertheless, there is no apparent reason for there to be a change in pregnancy to the basic principles of thyroid hormone action. The relationships between mild abnormalities of the thyroid state and pregnancy outcomes and the value of treating such mild abnormalities remain uncertain and controversial. The evidence suggests that further investigation of these clinical questions might better be based on thyroid hormone, particularly free thyroxine, levels. In the investigation of borderline low thyroid states, the categories of subclinical hypothyroidism and isolated hypothyroxinemia might both be abandoned with attention being directed to low free thyroxine levels regardless of the thyroid-stimulating hormone (TSH) levels. For these changes to occur, there would ideally be improvements in the assays for free thyroxine in pregnancy. The evidence suggests that, just as in the non-pregnant situation, pregnancy guidelines based on thyrotropin levels may need revision.
Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex ...(the 'necrosome'), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.
Objective
We review the published cases of clozapine‐induced myocarditis and describe reasons for the higher incidence in Australia (>1%) than elsewhere (<0.1%).
Method
Medline was searched to ...September 2014 using ‘clozapine’ as the sole term.
Results
A total of around 250 cases of clozapine‐induced myocarditis have been published. Fever among patients commencing clozapine has been reported internationally, and very few of these cases were investigated for myocarditis. The time to onset of fever is consistent with its being part of a prodrome of undiagnosed myocarditis, and the risk factors are similar to those for myocarditis. In more severe cases, clozapine is discontinued, avoiding fatalities which may occur with myocarditis. Furthermore, cases of sudden death and respiratory illness may well have been undiagnosed myocarditis. The diagnosis of myocarditis is confounded by the non‐specific nature of the signs and symptoms, and it depends on appropriate investigations being conducted at the time of myocardial involvement or, for fatal cases, the affected area of the myocardium being sampled for histology.
Conclusion
It is likely that the incidence of myocarditis is around 3%. Implementation of monitoring procedures will increase case ascertainment and result in more patients benefiting from this valuable medication.
Highlights • This review is an up-date document on basic aspects of non-invasive stimulation of brain, spinal cord and nerve roots. • The main physiological, theoretical and methodological features ...of transcranial magnetic stimulation (TMS) are described. • Instructions for practical use of non-invasive stimulation in clinical applications and research are provided.
There is a current lack of consensus on defining metabolically healthy obesity (MHO). Limited data on dietary and lifestyle factors and MHO exist. The aim of this study is to compare the prevalence, ...dietary factors and lifestyle behaviours of metabolically healthy and unhealthy obese and non-obese subjects according to different metabolic health criteria.
Cross-sectional sample of 1,008 men and 1,039 women aged 45-74 years participated in the study. Participants were classified as obese (BMI ≥ 30 kg/m(2)) and non-obese (BMI < 30 kg/m(2)). Metabolic health status was defined using five existing MH definitions based on a range of cardiometabolic abnormalities. Dietary composition and quality, food pyramid servings, physical activity, alcohol and smoking behaviours were examined.
The prevalence of MHO varied considerably between definitions (2.2% to 11.9%), was higher among females and generally increased with age. Agreement between MHO classifications was poor. Among the obese, prevalence of MH was 6.8% to 36.6%. Among the non-obese, prevalence of metabolically unhealthy subjects was 21.8% to 87%. Calorie intake, dietary macronutrient composition, physical activity, alcohol and smoking behaviours were similar between the metabolically healthy and unhealthy regardless of BMI. Greater compliance with food pyramid recommendations and higher dietary quality were positively associated with metabolic health in obese (OR 1.45-1.53 unadjusted model) and non-obese subjects (OR 1.37-1.39 unadjusted model), respectively. Physical activity was associated with MHO defined by insulin resistance (OR 1.87, 95% CI 1.19-2.92, p = 0.006).
A standard MHO definition is required. Moderate and high levels of physical activity and compliance with food pyramid recommendations increase the likelihood of MHO. Stratification of obese individuals based on their metabolic health phenotype may be important in ascertaining the appropriate therapeutic or intervention strategy.
Periodic arrays of plasmonic nanostructures are able to strongly confine light at the nanometer scale because of surface lattice resonances. These resonances are the result of electromagnetic ...coupling between single-particle localized surface plasmon resonances and Bragg resonances of the periodic lattice. Here, we investigate the effect of a finite size refractive index environment on the formation of surface lattice resonances by increasing the thickness of a polymer coating in nanometer-scale increments. Wet-chemically synthesized, spherical silver and gold nanoparticles with soft hydrogel shells are self-assembled into macroscopic, hexagonally ordered arrays on glass substrates using an interface-assisted approach. The resulting periodic plasmonic monolayers are subsequently coated by a polymer matching closely the refractive index of the glass support. The optical response of the plasmonic arrays is studied using far-field extinction spectroscopy and supported by numerical simulations. We show the formation of surface lattice resonances as well as higher order resonances in finite thickness polymer coatings. The resonance positions are determined by the interparticle spacing as well as the plasmonic material. Additionally, we demonstrate that a coating thickness of 450 nm is sufficient to support strong in-plane surface lattice resonances. This enables us to prepare macroscopic, free-standing polymer films with embedded plasmonic nanoparticle arrays, which feature strong surface lattice resonances.
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