Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry

The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry–mass spectrometry (IMS–MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human ​islet amyloid polypeptide (​hIAPP) aggregation or ​amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS–MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of ​hIAPP amyloid assembly

Cerebral Infarcts and Vasculopathy in Tanzanian Children With Sickle Cell Anemia

BACKGROUND: Cerebral infarcts and vasculopathy in neurologically asymptomatic children with sickle cell anemia (SCA) have received little attention in African settings. This study aimed to establish the prevalence of silent cerebral infarcts (SCI) and vasculopathy and determine associations with exposure to chronic hemolysis, anemia, and hypoxia. METHODS: We prospectively studied 224 children with SCA with transcranial Doppler (TCD), and magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA). Regressions were undertaken with contemporaneous hemoglobin, reticulocyte count, mean prior hemoglobin, oxygen content, reticulocyte count, and indirect bilirubin. RESULTS: Prevalence of SCI was 27% (61 of 224); cerebral blood flow velocity was abnormal (>200 cm/s) in three and conditional (>170<200 cm/s) in one. Vasculopathy grades 2 (stenosis) and 3 (occlusion) occurred in 16 (7%) and two (1%), respectively; none had grade 4 (moyamoya). SCI was associated with vasculopathy on MRA (odds ratio 2.68; 95% confidence intervals [95% CI] 1.32 to 5.46; P = 0.007) and mean prior indirect bilirubin (odds ratio 1.02, 95% CI 1.00 to 1.03, P = 0.024; n = 83) but not age, sex, non-normal TCD, or contemporaneous hemoglobin. Vasculopathy was associated with mean prior values for hemoglobin (odds ratio 0.33, 95% CI 0.16 to 0.69, P = 0.003; n = 87), oxygen content (odds ratio 0.43, 95% CI 0.25 to 0.74, P = 0.003), reticulocytes (odds ratio 1.20, 95% CI 1.01-1.42, P = 0.041; n = 77), and indirect bilirubin (odds ratio 1.02, 95% CI 1.01 to 1.04, P = 0.009). CONCLUSIONS: SCI and vasculopathy on MRA are common in neurologically asymptomatic children with SCA living in Africa, even when TCD is normal. Children with vasculopathy on MRA are at increased risk of SCI. Longitudinal exposure to anemia, hypoxia, and hemolysis appear to be risk factors for vasculopathy

Conclusive quantum steering with superconducting transition edge sensors

Quantum steering allows two parties to verify shared entanglement even if one measurement device is untrusted. A conclusive demonstration of steering through the violation of a steering inequality is of considerable fundamental interest and opens up applications in quantum communication. To date all experimental tests with single photon states have relied on post-selection, allowing untrusted devices to cheat by hiding unfavourable events in losses. Here we close this "detection loophole" by combining a highly efficient source of entangled photon pairs with superconducting transition edge sensors. We achieve an unprecedented ~62% conditional detection efficiency of entangled photons and violate a steering inequality with the minimal number of measurement settings by 48 standard deviations. Our results provide a clear path to practical applications of steering and to a photonic loophole-free Bell test.Comment: Preprint of 7 pages, 3 figures; the definitive version is published in Nature Communications, see below. Also, see related experimental work by A. J. Bennet et al., arXiv:1111.0739 and B. Wittmann et al., arXiv:1111.076

Reciprocal relationship between expression of hypoxia inducible factor 1α (HIF-1α) and the pro-apoptotic protein Bid in ex vivo colorectal cancer

Hypoxia inducible factor 1 (HIF-1) represses the transcription of pro-apoptotic bid in colorectal cancer cells in vitro. To assess the clinical relevance of this observation, HIF-1α and Bid were assessed in serial sections of 39 human colorectal adenocarcinomas by immunohistochemistry. In high HIF-1α nuclear-positive cell subpopulations, there was a significant reduction in Bid expression (ANOVA, P=0.04). Given the role of Bid in drug-induced apoptosis, these data add impetus to strategies targeting HIF-1 for therapeutic gain

An in vivo platform to select and evolve aggregation-resistant proteins

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve ‘manufacturable’ biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease

Cue-Reactors: Individual Differences in Cue-Induced Craving after Food or Smoking Abstinence

Background: Pavlovian conditioning plays a critical role in both drug addiction and binge eating. Recent animal research suggests that certain individuals are highly sensitive to conditioned cues, whether they signal food or drugs. Are certain humans also more reactive to both food and drug cues? Methods: We examined cue-induced craving for both cigarettes and food, in the same individuals (n = 15 adult smokers). Subjects viewed smoking-related or food-related images after abstaining from either smoking or eating. Results: Certain individuals reported strong cue-induced craving after both smoking and food cues. That is, subjects who reported strong cue-induced craving for cigarettes also rated stronger cue-induced food craving. Conclusions: In humans, like in nonhumans, there may be a ‘‘cue-reactive’ ’ phenotype, consisting of individuals who are highly sensitive to conditioned stimuli. This finding extends recent reports from nonhuman studies. Further understanding this subgroup of smokers may allow clinicians to individually tailor therapies for smoking cessation

Emplacement of inflated Pāhoehoe flows in the Naude’s Nek Pass, Lesotho remnant, Karoo continental flood basalt province: use of flow-lobe tumuli in understanding flood basalt emplacement

Physical volcanological features are presented for a 710-m-thick section, of the Naude’s Nek Pass, within the lower part of the Lesotho remnant of the Karoo Large Igneous Province. The section consists of inflated pāhoehoe lava with thin, impersistent sedimentary interbeds towards the base. There are seven discreet packages of compound and hummocky pāhoehoe lobes containing flow-lobe tumuli, making up approximately 50% of the section. Approximately 45% of the sequence consists of 14 sheet lobes, between 10 and 52-m-thick. The majority of the sheet lobes are in two packages indicating prolonged periods of lava supply capable of producing thick sheet lobes. The other sheet lobes are as individual lobes or pairs, within compound flows, suggesting brief increases in lava supply rate. We suggest, contrary to current belief, that there is no evidence that compound flows are proximal to source and sheet lobes (simple flows) are distal to source and we propose that the presence of flow-lobe tumuli in compound flows could be an indicator that a flow is distal to source. We use detailed, previously published, studies of the Thakurvadi Formation (Deccan Traps) as an example. We show that the length of a lobe and therefore the sections that are ‘medial or distal to source’ are specific to each individual lobe and are dependent on the lava supply of each eruptive event, and as such flow lobe tumuli can be used as an indicator of relative distance from source

Evaluation of in vitro cross-reactivity to avian H5N1 and pandemic H1N1 2009 influenza following prime boost regimens of seasonal influenza vaccination in healthy human subjects: A randomised trial

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Regulation of Neuronal APL-1 Expression by Cholesterol Starvation

Background: Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the deposition of b-amyloid plaques composed primarily of the amyloid-b peptide, a cleavage product of amyloid precursor protein (APP). While mutations in APP lead to the development of Familial Alzheimer’s Disease (FAD), sporadic AD has only one clear genetic modifier: the e4 allele of the apolipoprotein E (ApoE) gene. Cholesterol starvation in Caenorhabditis elegans leads to molting and arrest phenotypes similar to loss-of-function mutants of the APP ortholog, apl-1 (amyloid precursor-like protein 1), and lrp-1 (lipoprotein receptor-related protein 1), suggesting a potential interaction between apl-1 and cholesterol metabolism. Methodology/Principal Findings: Previously, we found that RNAi knock-down of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. Here we find the same defect is recapitulated during lrp-1 knock-down and by cholesterol starvation. A cholesterol-free diet or loss of lrp-1 directly affects APL-1 levels as both lead to loss of APL-1::GFP fluorescence in neurons. However, loss of cholesterol does not affect global transcription or protein levels as seen by qPCR and Western blot. Conclusions: Our results show that cholesterol and lrp-1 are involved in the regulation of synaptic transmission, similar to apl-1. Both are able to modulate APL-1 protein levels in neurons, however cholesterol changes do not affect global apl-1 transcription or APL-1 protein indicating the changes are specific to neurons. Thus, regulation of synaptic transmission an

Trioctylphosphine as Both Solvent and Stabilizer to Synthesize CdS Nanorods

High quality CdS nanorods are synthesized reproducibly with cadmium acetate and sulfur as precursors in trioctylphosphine solution. The morphology, crystalline form and phase composition of CdS nanorods are characterized by transmission electron microscopy (TEM), high-resolution TEM and X-ray diffraction (XRD). CdS nanorods obtained are uniform with an aspect ratio of about 5:1 and in a wurtzite structure. The influence of reaction conditions on the growth of CdS nanorods demonstrates that low precursor concentration and high reaction temperature (260 °C) are favorable for the formation of uniform CdS nanorods with 85.3% of product yield