Collective Behavior of Drosophila Melanogaster Neural Progenitor and Imaginal Disc Cells within Controlled Microenvironments

Regenerative therapies for the damaged visual system have introduced stem-derived cells to recapitulate developmental processes and initiate functional regeneration in different components of the eye. The developing visual system in Drosophila Melanogaster offers a model in which to analyze the associated processes in retinogenesis. The optic nerve is critical to vision and is developmentally preceded in Drosophila by a structure called the Optic Stalk (OS). Collective migration of neural and retinal progenitor cells (RPCs) from the developing brain lobes (DBL) to the Imaginal Disc (ID), through the OS, is a fundamental part of regenerative strategies in retina. Developmental signals governing retinal cell fate and migration have been well-studied using Drosophila Melanogaster. While conserved signaling pathways are known to drive retinogenesis across invertebrate and vertebrate species, the role(s) of diffusible signaling molecules in the collective migratory processes critical to eye development remain incompletely understood. Invertebrate models remain largely underutilized for in vitro study of cell response to controlled stimuli. In this thesis, the collective behavior and migration of primary Drosophila-derived neural progenitor cells (NPC) and Drosophila imaginal disc cells were analyzed on different extracellular matrices and coatings, poly-L-lysine (PLL), laminin (LM) and Concanavalin- A (Con-A), when exposed to exogenous gradients of growth factors and within microfluidic systems in order to propose an animal model for retinogenesis. The formation of single, small clusters and large clusters of NPCs were observed on all matrices and within the μLane, a bridged microchannel system. Furthermore, small and large clusters were demonstrated chemotaxis, directed cell migration, to gradients of fibroblast growth factor 8 (FGF8), while single cells demonstrated chemokinesis, non- directed cell migration within the μLane. A microfluidic system called the Micro Optic Stalk (μOS), that recapitulated in vivo geometric constraints seen during Drosophila retinal development, was designed, fabricated and validated. When NPCs were cultured and exposed to FGF8 within the μOS, the formation of single cells, small clusters and large clusters was observed. Furthermore, clusters demonstrated chemotaxis and gradient-dependent migration patterns. Imaginal disc cells were studied in order to look at the behavior of the secondary structure involved in retinogenesis. Dm-D17-c3 (D17) cells were examined as they have been utilized for motility studies in literature. D17 cells demonstrated two cell populations, rounded and elongated cells, on PLL and Con- A, while they did not adhere and grew in suspension on LM. Utilizing Boyden Chamber Assays, D17 cells showed significant migration toward brain-derived neurotrophic factor (BDNF), concentration-dependent migration toward Insulin (In), and no significant migration toward FGF8. Furthermore, D17 cells showed high viability when cultured within the μLane when seeded at higher cell densities (7.5 ́105 cells and 1 ́106 cells). Although D17 cells were shown to not be suitable for examination in a retinogenesis model centering on the role of FGF8, they show promise for use in other developmental models and microfluidic systems. Future work will utilize FGFR receptor knock outs in Drosophila within the μOS, in order to further understand FGF8’s role in retinogenesis

Invertebrate Retinal Progenitors as Regenerative Models in a Microfluidic System

Regenerative retinal therapies have introduced progenitor cells to replace dysfunctional or injured neurons and regain visual function. While contemporary cell replacement therapies have delivered retinal progenitor cells (RPCs) within customized biomaterials to promote viability and enable transplantation, outcomes have been severely limited by the misdirected and/or insuffcient migration of transplanted cells. RPCs must achieve appropriate spatial and functional positioning in host retina, collectively, to restore vision, whereas movement of clustered cells differs substantially from the single cell migration studied in classical chemotaxis models. Defining how RPCs interact with each other, neighboring cell types and surrounding extracellular matrixes are critical to our understanding of retinogenesis and the development of effective, cell-based approaches to retinal replacement. The current article describes a new bio-engineering approach to investigate the migratory responses of innate collections of RPCs upon extracellular substrates by combining microfluidics with the well-established invertebrate model of Drosophila melanogaster. Experiments utilized microfluidics to investigate how the composition, size, and adhesion of RPC clusters on defined extracellular substrates affected migration to exogenous chemotactic signaling. Results demonstrated that retinal cluster size and composition influenced RPC clustering upon extracellular substrates of concanavalin (Con-A), Laminin (LM), and poly-L-lysine (PLL), and that RPC cluster size greatly altered collective migratory responses to signaling from Fibroblast Growth Factor (FGF), a primary chemotactic agent in Drosophila. These results highlight the significance of examining collective cell-biomaterial interactions on bio-substrates of emerging biomaterials to aid directional migration of transplanted cells. Our approach further introduces the benefits of pairing genetically controlled models with experimentally controlled microenvironments to advance cell replacement therapies

A Micro-Optic Stalk (muOS) System to Model the Collective Migration of Retinal Neuroblasts

Contemporary regenerative therapies have introduced stem-like cells to replace damaged neurons in the visual system by recapitulating critical processes of eye development. The collective migration of neural stem cells is fundamental to retinogenesis and has been exceptionally well-studied using the fruit fly model of Drosophila Melanogaster. However, the migratory behavior of its retinal neuroblasts (RNBs) has been surprisingly understudied, despite being critical to retinal development in this invertebrate model. The current project developed a new microfluidic system to examine the collective migration of RNBs extracted from the developing visual system of Drosophila as a model for the collective motile processes of replacement neural stem cells. The system scales with the microstructure of the Drosophila optic stalk, which is a pre-cursor to the optic nerve, to produce signaling fields spatially comparable to in vivo RNB stimuli. Experiments used the micro-optic stalk system, or μOS, to demonstrate the preferred sizing and directional migration of collective, motile RNB groups in response to changes in exogenous concentrations of fibroblast growth factor (FGF), which is a key factor in development. Our data highlight the importance of cell-to-cell contacts in enabling cell cohesion during collective RNB migration and point to the unexplored synergy of invertebrate cell study and microfluidic platforms to advance regenerative strategies

Green Production of Anionic Surfactant Obtained from Pea Protein

A pea protein isolate was hydrolyzed by a double enzyme treatment method in order to obtain short peptide sequences used as raw materials to produce lipopeptides-based surfactants. Pea protein hydrolysates were prepared using the combination of Alcalase and Flavourzyme. The influence of the process variables was studied to optimize the proteolytic degradation to high degrees of hydrolysis. The average peptide chain lengths were obtained at 3–5 amino acid units after a hydrolysis of 30 min with the mixture of enzymes. Then, N-acylation in water, in presence of acid chloride (C12 and C16), carried out with a conversion rate of amine functions of 90%, allowed to obtain anionic surfactant mixtures (lipopeptides and sodium fatty acids). These two steps were performed in water, in continuous and did not generate any waste. This process was therefore in line with green chemistry principles. The surface activities (CMC, foaming and emulsifying properties) of these mixtures were also studied. These formulations obtained from natural renewable resources and the reactions done under environmental respect, could replace petrochemical based surfactants for some applications

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome

Purpose Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. Methods We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. Results Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. Conclusion Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome

Actin Nemaline Myopathy Mouse Reproduces Disease, Suggests Other Actin Disease Phenotypes and Provides Cautionary Note on Muscle Transgene Expression

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations

Relationship of Weather Types on the Seasonal and Spatial Variability of Rainfall, Runoff, and Sediment Yield in the Western Mediterranean Basin

Rainfall is the key factor to understand soil erosion processes, mechanisms, and rates. Most research was conducted to determine rainfall characteristics and their relationship with soil erosion (erosivity) but there is little information about how atmospheric patterns control soil losses, and this is important to enable sustainable environmental planning and risk prevention. We investigated the temporal and spatial variability of the relationships of rainfall, runoff, and sediment yield with atmospheric patterns (weather types, WTs) in the western Mediterranean basin. For this purpose, we analyzed a large database of rainfall events collected between 1985 and 2015 in 46 experimental plots and catchments with the aim to: (i) evaluate seasonal differences in the contribution of rainfall, runoff, and sediment yield produced by the WTs; and (ii) to analyze the seasonal efficiency of the different WTs (relation frequency and magnitude) related to rainfall, runoff, and sediment yield. The results indicate two different temporal patterns: the first weather type exhibits (during the cold period: autumn and winter) westerly flows that produce the highest rainfall, runoff, and sediment yield values throughout the territory; the second weather type exhibits easterly flows that predominate during the warm period (spring and summer) and it is located on the Mediterranean coast of the Iberian Peninsula. However, the cyclonic situations present high frequency throughout the whole year with a large influence extended around the western Mediterranean basin. Contrary, the anticyclonic situations, despite of its high frequency, do not contribute significantly to the total rainfall, runoff, and sediment (showing the lowest efficiency) because of atmospheric stability that currently characterize this atmospheric pattern. Our approach helps to better understand the relationship of WTs on the seasonal and spatial variability of rainfall, runoff and sediment yield with a regional scale based on the large dataset and number of soil erosion experimental stations.Spanish Government (Ministry of Economy and Competitiveness, MINECO) and FEDER Projects: CGL2014 52135-C3-3-R, ESP2017-89463-C3-3-R, CGL2014-59946-R, CGL2015-65569-R, CGL2015-64284-C2-2-R, CGL2015-64284-C2-1-R, CGL2016-78075-P, GL2008-02879/BTE, LEDDRA 243857, RECARE-FP7, CGL2017-83866-C3-1-R, and PCIN-2017-061/AEI. Dhais Peña-Angulo received a “Juan de la Cierva” postdoctoral contract (FJCI-2017-33652 Spanish Ministry of Economy and Competitiveness, MEC). Ana Lucia acknowledge the "Brigitte-Schlieben-Lange-Programm". The “Geoenvironmental Processes and Global Change” (E02_17R) was financed by the Aragón Government and the European Social Fund. José Andrés López-Tarazón acknowledges the Secretariat for Universities and Research of the Department of the Economy and Knowledge of the Autonomous Government of Catalonia for supporting the Consolidated Research Group 2014 SGR 645 (RIUS- Fluvial Dynamics Research Group). Artemi Cerdà thank the funding of the OCDE TAD/CRP JA00088807. José Martínez-Fernandez acknowledges the project Unidad de Excelencia CLU-2018-04 co-funded by FEDER and Castilla y León Government. Ane Zabaleta is supported by the Hydro-Environmental Processes consolidated research group (IT1029-16, Basque Government). This paper has the benefit of the Lab and Field Data Pool created within the framework of the COST action CONNECTEUR (ES1306)

Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI