Site-directed spin labeling for the evaluation of amyloid and non-amyloid aggregation mechanisms of β-lactoglobulin using electron paramagnetic resonance spectroscopy and mass spectrometry

The approach of site-directed spin labeling can be implemented for the food protein β lactoglobulin (β lg), by adjusting the pH value and the spin label/β lg ratio, although it contains five cysteine residues, and used to characterize the amyloid aggregation mechanism of β lg. Further, the surface activity of the amyloid aggregate system is determined by the smaller aggregates, peptides, and monomeric sized protein of the non-amyloid fraction due to faster migration to the interface and a higher adsorption rate. The results of this work are mainly based on measurements using electron spin resonance spectroscopy and mass spectrometry. MTSSL and IPSL bind covalently and reproducibly to all five cysteine residues with different preferences. The best labelling conditions for MTSSL are pH 7.5 and an equimolar MTSSL/β-lg ratio. Whereby MTSSL dimers are already formed at a pH value of 7.5. The bonding of the spin labels to β-lg is temperature (90°C) and acid (pH 2) resistant, whereas it is assumed the biradicals are reduced to monoradical dimers during heat incubation. Spin labeling with IPSL reduces the intra- and intermolecular β-sheets at pH 3.5. However, SDSL does not change the morphology and building blocks of amyloid aggregates but interferes with the thioflavin-T measurement. Impact of the label on the secondary protein structure is lower for MTSSL compared to IPSL because of its flexible bond. MTSSL is most sensitive to changes in the arrangement of β-sheets, whereas IPSL is most sensitive to changes in random coil at position Cys160. The amyloid aggregates formed at pH 3.5 could be characterized using SDSL. The monomer-like β-lg aggregates had the form of a pearl chain. However, the spin labeled side chains were not incorporated into amyloid structures formed at pH 2. Fibrils and non-amyloid material act synergistically at the air-water interface because non-amyloid material is embedded into fibrillar network. But the adsorption behavior of the amyloid aggregate system is dominated by the non-amyloid fraction. Only small amounts of amyloid aggregates were necessary to stabilize foams. The airflow used for foaming resulting in gas bubbles caused sufficient shearing to partly degrade fibrils

SCAN Testbed, Overview and Opportunity for Experiments

No abstract availabl

Soil warming alters nitrogen cycling in a New England forest : implications for ecosystem function and structure

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Oecologia 168 (2012): 819-828, doi:10.1007/s00442-011-2133-7.Global climate change is expected to affect terrestrial ecosystems in a variety of ways. Some of the more well-studied effects include the biogeochemical feedbacks to the climate system that can either increase or decrease the atmospheric load of greenhouse gases such as carbon dioxide and nitrous oxide. Less well-studied are the effects of climate change on the linkages between soil and plant processes. Here, we report the effects of soil warming on these linkages observed in a large field manipulation of a deciduous forest in southern New England, USA, where soil was continuously warmed 5°C above ambient for 7 years. Over this period, we have observed significant changes to the nitrogen cycle that have the potential to affect tree species composition in the long term. Since the start of the experiment, we have documented a 45% average annual increase in net nitrogen mineralization and a three-fold increase in nitrification such that in years 5 through 7, 25% of the nitrogen mineralized is then nitrified. The warming-induced increase of available nitrogen resulted in increases in the foliar nitrogen content and the relative growth rate of trees in the warmed area. Acer rubrum (red maple) trees have responded the most after 7 years of warming, with the greatest increases in both foliar nitrogen content and relative growth rates. Our study suggests that considering species-specific responses to increases in nitrogen availability and changes in nitrogen form is important in predicting future forest composition and feedbacks to the climate system.This work was supported by the National Institute for Climate Change Research (DOE-DE-FCO2-06-ER64157), DOE BER (DE-SC0005421) and the Harvard Forest Long-Term Ecological Research program (NSF-DEB-0620443)

AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine

International audienceAGuIX® are sub-5 nm nanoparticles made of a polysiloxane matrix and gadolinium chelates. This nanoparticle has been recently accepted in clinical trials in association with radiotherapy. This review will summarize the principal preclinical results that have led to first in man administration. No evidence of toxicity has been observed during regulatory toxicity tests on two animal species (rodents and monkeys). Biodistributions on different animal models have shown passive uptake in tumours due to enhanced permeability and retention effect combined with renal elimination of the nanoparticles after intravenous administration. High radiosensitizing effect has been observed with different types of irradiations in vitro and in vivo on a large number of cancer types (brain, lung, melanoma, head and neck…). The review concludes with the second generation of AGuIX nanoparticles and the first preliminary results on human

The role of inflammation in epilepsy.

Epilepsy is the third most common chronic brain disorder, and is characterized by an enduring predisposition to generate seizures. Despite progress in pharmacological and surgical treatments of epilepsy, relatively little is known about the processes leading to the generation of individual seizures, and about the mechanisms whereby a healthy brain is rendered epileptic. These gaps in our knowledge hamper the development of better preventive treatments and cures for the approximately 30% of epilepsy cases that prove resistant to current therapies. Here, we focus on the rapidly growing body of evidence that supports the involvement of inflammatory mediators-released by brain cells and peripheral immune cells-in both the origin of individual seizures and the epileptogenic process. We first describe aspects of brain inflammation and immunity, before exploring the evidence from clinical and experimental studies for a relationship between inflammation and epilepsy. Subsequently, we discuss how seizures cause inflammation, and whether such inflammation, in turn, influences the occurrence and severity of seizures, and seizure-related neuronal death. Further insight into the complex role of inflammation in the generation and exacerbation of epilepsy should yield new molecular targets for the design of antiepileptic drugs, which might not only inhibit the symptoms of this disorder, but also prevent or abrogate disease pathogenesis

Shifting the limits in wheat research and breeding using a fully annotated reference genome

Introduction: Wheat (Triticum aestivum L.) is the most widely cultivated crop on Earth, contributing about a fifth of the total calories consumed by humans. Consequently, wheat yields and production affect the global economy, and failed harvests can lead to social unrest. Breeders continuously strive to develop improved varieties by fine-tuning genetically complex yield and end-use quality parameters while maintaining stable yields and adapting the crop to regionally specific biotic and abiotic stresses. Rationale: Breeding efforts are limited by insufficient knowledge and understanding of wheat biology and the molecular basis of central agronomic traits. To meet the demands of human population growth, there is an urgent need for wheat research and breeding to accelerate genetic gain as well as to increase and protect wheat yield and quality traits. In other plant and animal species, access to a fully annotated and ordered genome sequence, including regulatory sequences and genome-diversity information, has promoted the development of systematic and more time-efficient approaches for the selection and understanding of important traits. Wheat has lagged behind, primarily owing to the challenges of assembling a genome that is more than five times as large as the human genome, polyploid, and complex, containing more than 85% repetitive DNA. To provide a foundation for improvement through molecular breeding, in 2005, the International Wheat Genome Sequencing Consortium set out to deliver a high-quality annotated reference genome sequence of bread wheat. Results: An annotated reference sequence representing the hexaploid bread wheat genome in the form of 21 chromosome-like sequence assemblies has now been delivered, giving access to 107,891 high-confidence genes, including their genomic context of regulatory sequences. This assembly enabled the discovery of tissue- and developmental stage–related gene coexpression networks using a transcriptome atlas representing all stages of wheat development. The dynamics of change in complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. Aspects of the future value of the annotated assembly for molecular breeding and research were exemplarily illustrated by resolving the genetic basis of a quantitative trait locus conferring resistance to abiotic stress and insect damage as well as by serving as the basis for genome editing of the flowering-time trait. Conclusion: This annotated reference sequence of wheat is a resource that can now drive disruptive innovation in wheat improvement, as this community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding. Importantly, the bioinformatics capacity developed for model-organism genomes will facilitate a better understanding of the wheat genome as a result of the high-quality chromosome-based genome assembly. By necessity, breeders work with the genome at the whole chromosome level, as each new cross involves the modification of genome-wide gene networks that control the expression of complex traits such as yield. With the annotated and ordered reference genome sequence in place, researchers and breeders can now easily access sequence-level information to precisely define the necessary changes in the genomes for breeding programs. This will be realized through the implementation of new DNA marker platforms and targeted breeding technologies, including genome editing

Identification of an optimized ratio of amyloid and non-amyloid fractions in engineered fibril solutions from whey protein isolate for improved foaming

Engineered fibril solutions from whey protein beta-lactoglobulin are known for their excellent foaming capacity. These amyloid aggregates solutions (AAS) usually contain a polydisperse mixture of different protein structures. An optimized ratio of amyloid (AF, fibrils) and non-amyloid fractions (n-AF) in AAS may improve foaming, particularly by interactions at the air-water interface. Foamability, surface activity, and monolayer phase behavior at the air-water interface of isolated AF and n-AF as well as AAS with different AF/n-AF-ratios were investigated using drop tensiometry, Langmuir trough and foam analysis. N-AF exhibited faster migration, twice as fast adsorption and thus faster spreading at the air-water interface than the fibrils (AF). N-AF required less energy to assemble in a liquid-expanded phase in a monolayer, i.e., they were more compressible in the monolayer than AF. This resulted in rapid stabilization of lamellae in foam. High surface hydrophobicity of AF results in faster adsorption and formation of capillary forces between adsorbed fibrils, improving the attraction for additional fibrils. Orientation of semi-flexible and larger fibrils in AF consumes high energy. In combination with n-AF, the energy needed for orientation and assembly of fibrils is equivalent, however, the yield of AF in AAS was only 20 %, indicating the interplay of amyloid and non-amyloid proteins at the air-water interface. N-AF can be incorporated into a fibrillar film, which increase the network's density and stiffness and the interfacial film stability. Consequently, in AAS fibrils and non-amyloid material acted synergistically at the air-water interface, whereby only small amounts of amyloid aggregates are required to stabilize foams

Amyloid aggregation of spin-labeled β-lactoglobulin. Part II : Identification of spin-labeled protein and peptide sequences after amyloid aggregation

Site-directed spin labeling (SDSL) of natural β-lactoglobulin (β-lg) was established. Combined electron paramagnetic resonance (EPR) and mass spectrometric analysis following tryptic digestion demonstrated that spin labels bind site-specifically but are not directed to all five cysteine residues to various preferred and reproducible extents. MTSSL and iodoacetamido-proxyl spin label (IPSL) were 80 and 60% reliably bound to the H strand, respectively, and combined in one spectral component and buried in the protein core. After heat incubation at pH 2 and fractionation, all labeled side chains (peptides) were part of the amyloid and non-amyloid fractions, even if they could not detect amyloid structures. It was assumed that the IPSL-labeled side chains of peptides with Cys160 from random coil were incorporated into small non-amyloid aggregates in non-polar environments. After heating at pH 3.5, a rearrangement of the previous α-helix was assumed to shift from the autonomous folding domain during partial unfolding, which improved the accessibility of β-sheets to the water/DMSO-environment. β-sheets were likely densely packed by the accumulation of intermolecular β-sheets, which suggests that amyloid-like structures can be formed from building blocks of the entire primary β-lg structure. Double electron-electron resonance (DEER) confirmed that the spatial distribution of labels within the amyloid-like fraction in a one-dimensional arrangement of the entire protein aggregates was similar to a string of pearls. Thus, SDSL of proteins containing several cysteine residues can be used to gain deep insights into the aggregation mechanism of proteins under food processing conditions.</p

Evaluation de l’effet sensibilisant des nanoparticules AGuIX® associées à une irradiation photonique dans un modèle de chondrosarcome

International audienceLes chondrosarcomes (CHS), tumeurs malignes du cartilage chimio- et radio-résistantes, représentent environ 20% des tumeurs osseuses primitives de l’adulte. Les nanoparticules AGuIX® associées à l’irradiation photonique ayant conduit à la radiosensibilisation de plusieurs types de tumeurs radiorésistantes (cancer de la tête et du cou, du col utérin, mélanome …), cette stratégie a été testée dans un modèle de CHS in vitro sur cultures 3D en conditions normoxique et physioxique et in vivo dans des souris avec xenogreffe tumorale de CHS (ANR CHONDRAD). L’effet radiosensibilisant des AGuIX® associées à une irradiation photonique a été analysé sur la lignée HEMC-SS dérivée d’un chondrosarcome extra squelettique myxoïde humain.La microscopie en temps réel (IncuCyte®) a permis de suivre l’évolution des sphéroïdes fluorescents HEMC-SS-mKate2 après irradiation associée ou non aux AGuIX®. La combinaison d’une irradiation unique de 4Gy ou d’une irradiation fractionnée (1Gy x 4 jours consécutifs) avec un traitement par AGuIX® a conduit à une radiosensibilisation que ce soit en condition normoxique ou physioxique (2% O2).Pour soutenir l’application clinique potentielle en radiothérapie, des études in vivo ont été réalisées sur un modèle de souris nude avec des xénogreffes HEMC-SS irradiées par des photons avec une dose unique de 4 Gy après une injection intratumorale d’AGuIX®. Les résultats (croissance tumorale et survie) montrent un effet radiosensibilisant significatif des AGuIX®.L’ensemble de ces résultats apportent la preuve de concept de l’effet radiosensibilisant des AGuIX® dans le chondrosarcome

Reframing Multilingualism in the Classroom: A Poetic Celebration of Diversity [2015]

Presented on April 9, 2015 from 11:00 a.m. - 12:00 p.m. in the Stephen C. Hall Building, room 102 on the Georgia Tech campus.The event opened with remarks by Jacqueline J. Royster, Dean of Georgia Tech's Ivan Allen College of Liberal Arts, and concluded with presentations by featured student speakers and faculty: •Dr. Thomas Lux •Meghan Popick •Melissa Aberle-Grasse"The Sea of Clouds in Jiri Mountain" read by Yesol Do (South Korea, Industrial Engineering, ’18). Written by Hyo-guen Bok."Look at the Light" read by Vedant Pradeep (India, Biomedical Engineering, ’18). Written by Anonymous."Welcome to America" read by Harrison Williams (United States, Civil Engineering, ‘18). Written by Lecrae.Runtime: 47:42 minutesThe event featured international Georgia Tech students reading poetry from their home countries, both in the original language and in English, and concluded with a question and answer panel discussion of innovative teaching methods that incorporate the diverse cultural and linguistic backgrounds of Georgia Tech students