ARIA 2016 : Care pathways implementing emerging technologies for predictive medicine in rhinitis and asthma across the life cycle

European Innovation Partnership on Active and Healthy Ageing Reference Site MACVIA-France, EU Structural and Development Fund Languedoc-Roussillon, ARIA.Peer reviewedPublisher PD

Recent advances in engineering microbial rhodopsins for optogenetics

Protein engineering of microbial rhodopsins has been successful in generating variants with improved properties for applications in optogenetics. Members of this membrane protein family can act as both actuators and sensors of neuronal activity. Chimeragenesis, structure-guided mutagenesis, and directed evolution have proven effective strategies for tuning absorption wavelength, altering ion specificity and increasing fluorescence. These approaches facilitate the development of useful optogenetic tools and, in some cases, have yielded insights into rhodopsin structure–function relationships

Engineering Novel Rhodopsins for Neuroscience

The overarching goal of my PhD research has been engineering proteins capable of controlling and reading out neural activity to advance neuroscience research. I engineered light-gated microbial rhodopsins, primarily focusing on the algal derived, light-gated channel, channelrhodopsin (ChR), which can be used to modulate neuronal activity with light. This work has required overcoming three major challenges. First, rhodopsins are trans-membrane proteins, which are inherently difficult to engineer because the sequence and structural determinants of membrane protein expression and plasma membrane localization are highly constrained and poorly understood (Chapter 3-5). Second, protein properties of interest for neuroscience applications are assayed using very low throughput patch-clamp electrophysiology preventing the use of high-throughput assays required for directed evolution experiments (Chapter 2, 5-6). And third, in vivo application of these improved tools require either retention or optimization of multiple protein properties in a single protein tool; for example, we must optimize expression and localization of these algal membrane proteins in mammalian cells while at the same time optimizing kinetic and functional properties (Chapter 5-6). These challenges restricted the field to low-throughput, conservative methods for discovery of improved ChRs, e.g., structure-guided mutagenesis and testing of natural ChR variants. I used an alternative approach: data-driven machine learning to model the fitness landscape of ChRs for different properties of interest and applying these models to select ChR sequences with optimal combinations of properties (Chapters 5-6). ChR variants identified from this work have unprecedented conductance properties and light sensitivity that could enable non-invasive activation of populations of cells throughout the nervous system. These ChRs have the potential to change how optogenetics experiments are done. This work is a convincing demonstration of the power of machine learning guided protein engineering for a class of proteins that present multiple engineering challenges. A component of the novel application of these new ChR tools relies on recent advances in gene delivery throughout the nervous system facilitated by engineered AAVs (Chapter 7). And finally, I developed a behavioral tracking system to monitor behavior and demonstrate sleep behavior in the jellyfish Cassiopea, the most primitive organism to have this behavior formally characterized (Chapter 8).</p

Effects of Altering the Sequence of a Combined Aerobic and Resistance Exercise Session on Energy Expenditure and Metabolism

Despite the known benefits of performing aerobic and resistance exercise independently, the metabolic effects of performing aerobic and resistance exercise in succession, remain unclear. Several studies suggest that the alteration of exercise sequence may influence carbohydrate and lipid oxidation and energy expenditure during exercise and in recovery. High intensity resistance exercise performed prior to a bout of aerobic exercise has been shown to augment fat oxidation during the subsequent bout of aerobic exercise. Changes in hormone and metabolite concentrations from prior resistance exercise could potentially influence substrate selection and energy expenditure in a subsequent bout of aerobic exercise. However, an exercise session whereby aerobic exercise is followed by a bout of resistance exercise has yet to be evaluated to determine the metabolic effects (specifically, the differences in substrate selection for energy provision) when exercise sequence is altered. It was hypothesized that when resistance exercise was performed prior to a bout of aerobic exercise, sympathetic nervous system activity would be elevated, leading to an increase in non-esterified fatty acid (NEFA) and glycerol concentrations and resultant increase in lipid oxidation during the aerobic portion of the exercise compared to the opposite sequence. It was also hypothesized that during recovery there would be an increased reliance on fat oxidation for energy provision with a resistance-aerobic exercise sequence compared to an aerobic-resistance exercise sequence. Additionally, the differences in metabolite concentrations and respiratory parameters between two identical bouts of aerobic exercise performed on separate days (~1 week apart) were measured and it was hypothesized that day-to-day variability would be non-significant (p>0.05). Plasma glucose, lactate, NEFA, glycerol, insulin, C-peptide, glucagon, epinephrine and norepinephrine concentrations in addition to oxygen consumption (VO2) and respiratory exchange ratio (RER) were measured in nine healthy, recreationally active males that participated in 3 different, randomized exercise trials (Trial A: aerobic exercise; Trial AR: aerobic exercise followed by a bout of resistance exercise; Trial RA: resistance exercise followed by an aerobic exercise bout). The aerobic exercise bout was performed at 60% VO2 max for 30 min while the resistance exercise bout consisted of 5 exercises (overhead squat, chest press, triceps extension, shoulder press, and dead-lift) performed for 3 sets of 8 repetitions at 70% 1-RM. Contrary to the primary hypothesis, NEFA concentrations and lipid oxidation rates were similar for the aerobic exercise bout of both the AR and RA trials. During recovery, lipid oxidation was elevated immediately post-exercise in the RA trial compared to the AR trial, however there were no differences between trials by 15 min post-exercise. Furthermore, only epinephrine, and not norepinephrine, concentrations were significantly higher after aerobic exercise in the RA trial compared to the AR trial. VO2 and energy expenditure values were similar for the duration of the 30 min recovery. These results suggest that while exercise sequence may influence carbohydrate and lipid oxidation immediately post exercise, substrate selection and utilization are similar during aerobic exercise bouts irrespective of the sequence in which aerobic and resistance exercise are performed. Thus, when resistance exercise is performed prior to aerobic exercise, compared to the opposite sequence, overall energy provision is not altered at the volume and intensity of exercise performed in this study

ARIA-EAACI care pathways for allergen immunotherapy in respiratory allergy

Rinitis al·lèrgica; Asma; ImmunoteràpiaRinitis alérgica; Asma; InmunoterapiaAllergic rhinitis; Asthma; ImmunotherapyARIA, Grant/Award Number: N/

Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins

Integral membrane proteins (MPs) are key engineering targets due to their critical roles in regulating cell function. In engineering MPs, it can be extremely challenging to retain membrane localization capability while changing other desired properties. We have used structure-guided SCHEMA recombination to create a large set of functionally diverse chimeras from three sequence-diverse channelrhodopsins (ChRs). We chose 218 ChR chimeras from two SCHEMA libraries and assayed them for expression and plasma membrane localization in human embryonic kidney cells. The majority of the chimeras express, with 89% of the tested chimeras outperforming the lowest-expressing parent; 12% of the tested chimeras express at even higher levels than any of the parents. A significant fraction (23%) also localize to the membrane better than the lowest-performing parent ChR. Most (93%) of these well-localizing chimeras are also functional light-gated channels. Many chimeras have stronger light-activated inward currents than the three parents, and some have unique off-kinetics and spectral properties relative to the parents. An effective method for generating protein sequence and functional diversity, SCHEMA recombination can be used to gain insights into sequence–function relationships in MPs

Implementation of the MASK-Air® App for Rhinitis and Asthma in Older Adults: MASK@Puglia Pilot Study

Introduction: MASK-air® is an app whose aim is to reduce the global burden of allergic rhinitis and asthma. A transfer of innovative practices was performed to disseminate and implement MASK-air® in European regions. The aim of the study was to examine the implementation of the MASK-air® app in older adults of the Puglia TWINNING in order to investigate (i) the rate of acceptance in this population, (ii) the reasons for refusal and (iii) the evaluation of the app after its use. Methods: All consecutive geriatric patients aged between 65 and 90 years were included by the outpatient clinic of the Bari Geriatric Immunoallergology Unit. After a 1-h training session, older adults used the app for 6 months. A 6-item questionnaire was developed by our unit to evaluate the impact of the app on the management of the disease and its treatment. Results: Among the 174 recruited patients, 102 accepted to use the app (mean age, SD: 72.4 ± 4.6 years), 6 were lost to follow-up, and 63 had a low education level. The reasons given not to use the app included lack of interest (11%), lack of access to a smartphone or tablet (53%), low computer literacy (28%), and distrust (8%). At follow-up, the overall satisfaction was high (89%), the patient considered MASK-air® “advantageous” (95%), compliance to treatment was improved (81%), and the rate of loss to follow-up had decreased to 6%. Conclusion: Older adults with a low level of education can use the MASK-air® app after a short training session

Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo

Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering

Survey of the needs of patients with spinal cord injury: impact and priority for improvement in hand function in tetraplegics\ud

Objective: To investigate the impact of upper extremity deficit in subjects with tetraplegia.\ud \ud Setting: The United Kingdom and The Netherlands.\ud \ud Study design: Survey among the members of the Dutch and UK Spinal Cord Injury (SCI) Associations.\ud \ud Main outcome parameter: Indication of expected improvement in quality of life (QOL) on a 5-point scale in relation to improvement in hand function and seven other SCI-related impairments.\ud \ud Results: In all, 565 subjects with tetraplegia returned the questionnaire (overall response of 42%). Results in the Dutch and the UK group were comparable. A total of 77% of the tetraplegics expected an important or very important improvement in QOL if their hand function improved. This is comparable to their expectations with regard to improvement in bladder and bowel function. All other items were scored lower.\ud \ud Conclusion: This is the first study in which the impact of upper extremity impairment has been assessed in a large sample of tetraplegic subjects and compared to other SCI-related impairments that have a major impact on the life of subjects with SCI. The present study indicates a high impact as well as a high priority for improvement in hand function in tetraplegics.\ud \u

Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons

Probing the neural circuit dynamics underlying behaviour would benefit greatly from improved genetically encoded voltage indicators. The proton pump ​Archaerhodopsin-3 (​Arch), an optogenetic tool commonly used for neuronal inhibition, has been shown to emit voltage-sensitive fluorescence. Here we report two ​Arch variants with enhanced radiance (Archers) that in response to 655 nm light have 3–5 times increased fluorescence and 55–99 times reduced photocurrents compared with ​Arch WT. The most fluorescent variant, Archer1, has 25–40% fluorescence change in response to action potentials while using 9 times lower light intensity compared with other ​Arch-based voltage sensors. Archer1 is capable of wavelength-specific functionality as a voltage sensor under red light and as an inhibitory actuator under green light. As a proof-of-concept for the application of ​Arch-based sensors in vivo, we show fluorescence voltage sensing in behaving Caenorhabditis elegans. Archer1’s characteristics contribute to the goal of all-optical detection and modulation of activity in neuronal networks in vivo