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"Aparicio, Gabriela"
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Heregulin Activity Assays for Residual Testing of Cell Therapy Products
Background
Heregulin is a ligand for the protooncogene product ErbB/HER that acts as a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a β1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells.
Results
Western blotting was used to measure the activity of three different β1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of β1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and reproducibility to detect β1-heregulin activity in the nanomolar range. Results from these assays showed that the culture medium from transplantable hSCs elicited no detectable activation of the aforementioned kinases in independent rounds of testing, indicating that the implemented measures can ensure that the final hSC product is devoid of bioactive β1-heregulin molecules prior to transplantation.
Conclusions
These assays may be valuable to detect impurities such as undefined soluble factors or factors for which other biochemical or biological assays are not yet available. Our workflow can be modified as necessary to determine the presence of ErbB/HER, ERK, and Akt activators other than β1-heregulin using native samples, such as fresh isolates from cell- or tissue extracts in addition to culture medium.
Journal Article
Mitochondrial Cardiolipin-Targeted Tetrapeptide, SS-31, Exerts Neuroprotective Effects Within In Vitro and In Vivo Models of Spinal Cord Injury
Spinal cord injury (SCI) affects millions globally, leading to severe motor and sensory deficits with no effective clinical treatment. Cardiolipin (CL), a mitochondria-specific phospholipid, plays a critical role in bioenergetics and apoptosis. Emerging evidence suggests that CL alterations contribute to secondary SCI pathology, but their precise role and underlying mechanisms remain fully understudied. In this study, we investigated the protective effects of SS-31 on CL alteration, neuronal death, tissue damage, and behavioral recovery after SCI using both in vitro and in vivo models, lipidomics analysis, histological evaluation, and behavioral assessments. In vitro investigations used primary spinal cord neuron cultures, challenged with either rotenone or glutamatergic excitotoxicity, with protective capabilities measured via cell death assays and neurite morphological analysis. In vivo investigations used female adult C57Bl/6 mice, challenged with a contusive SCI. The results showed that SS-31 reduced rotenone- and glutamate-induced mitochondrial dysfunction and neuronal death in a dose-dependent manner in vitro. Additionally, SS-31 attenuated rotenone- and glutamate-induced neurite degeneration in vitro. Lipidomics analysis revealed a reduction in CL at 24 h post-SCI in adult mice, which was attenuated by SS-31 in a dose-dependent manner. Consistent with this effect, SS-31 improved behavioral recovery after SCI in adult mice, although it had no significant effect on tissue damage. These findings suggest that CL alteration may play a key role in the pathogenesis of SCI, at least in the C57BL/6 mouse, and as such could be an attractive therapeutic target for ameliorating secondary SCI.
Journal Article
Endogenous Glycoprotein GPM6a Is Involved in Neurite Outgrowth in Rat Dorsal Root Ganglion Neurons
The peripheral nervous system (PNS) has a unique ability for self-repair. Dorsal root ganglion (DRG) neurons regulate the expression of different molecules, such as neurotrophins and their receptors, to promote axon regeneration after injury. However, the molecular players driving axonal regrowth need to be better defined. The membrane glycoprotein GPM6a has been described to contribute to neuronal development and structural plasticity in central-nervous-system neurons. Recent evidence indicates that GPM6a interacts with molecules from the PNS, although its role in DRG neurons remains unknown. Here, we characterized the expression of GPM6a in embryonic and adult DRGs by combining analysis of public RNA-seq datasets with immunochemical approaches utilizing cultures of rat DRG explants and dissociated neuronal cells. M6a was detected on the cell surfaces of DRG neurons throughout development. Moreover, GPM6a was required for DRG neurite elongation in vitro. In summary, we provide evidence on GPM6a being present in DRG neurons for the first time. Data from our functional experiments support the idea that GPM6a could contribute to axon regeneration in the PNS.
Journal Article
The Membrane Glycoprotein M6a Endocytic/Recycling Pathway Involves Clathrin-Mediated Endocytosis and Affects Neuronal Synapses
Single point mutations or variations in the expression of the gene encoding the neuronal glycoprotein M6a have been associated with psychiatric disorders such as Alzheimer's disease, depression and schizophrenia. In cultured neurons, M6a positively contributes to neurite extension, axon guidance, filopodia/spine outgrowth, and synapse formation. The endocytic processes of neuronal membrane proteins are linked to the differentiation, growth, signaling and plasticity of neurons. However, the roles of M6a and the precise mechanisms through which M6a internalizes and recycles back to the neuronal membrane are unknown. Here, by using a controlled
assay, we showed that if 30-40% of M6a is endocytosed, the number of synapses in hippocampal neurons decreases. When re-establishing the levels of M6a at the cell surface, the number of synapses returned to normal values. M6a internalization involves clathrin-coated pits, probably by association between the adaptor protein 2 and the 251YEDI254 \"tyrosine-based\" motif located within the C-tail of M6a. Upon endocytosis, M6a is sorted to early endosome antigen 1- and Rab5-positive endosomes and then sorted back to the cell surface via Rab11-positive endosomes or to degradation via Rab7 and, finally LAMP-1-positive endosomes. Our results demonstrated that the levels of M6a at the cell surface modified the formation/maintenance of synapses, without altering the protein levels of synaptophysin or
-methyl-D-aspartate receptor type-1. This novel mechanism might be relevant during neuronal development, pruning and/or many of the neurological disorders in which the number of synapses is affected.
Journal Article
Urban growth and spatial transition in nepal
Nepal is undergoing two momentous transformations-from a rural to an urbanizing economy and from a unitary to a federal state. This book aims at understanding the first of these two transitions: Nepal's journey toward becoming a predominantly urban economy. The study carries out an initial assessment of Nepal's transition from a predominantly rural to an urbanizing economy. This assessment aims at strengthening our understanding of the demographic and economic dimensions of the transition, and exploring the links between urbanization and economic growth in the context of Nepal. This book has five chapters. Chapter one presents an overview of the urban and economic transition in Nepal. Chapter two discusses the spatial patterns of Nepal's rapid urbanization and internal migration-a driving force of urban change from both a demographic and an economic perspective. Chapter three presents an initial assessment of the challenges facing Nepal's cities in urban planning and the delivery of infrastructure and services. And it discusses the spatial distribution of public expenditure for local infrastructure based on the results of a public expenditure survey carried out as part of the study. Chapter four presents a scoping assessment of the growth drivers of Nepal's urban economies and the main constraints to turning these comparative advantages into competitive advantages. And chapter five draws the main conclusions and proposes strategic directions and actions to accelerate urban-based economic growth and foster sustainable urban development.
eBook
Bangladesh
Bangladesh seeks to attain middle-income status by 2021, the 50th anniversary of its independence. To accelerate growth enough to do so, it will need to undergo a structural transformation that will change the geography of economic production and urbanization. Critical to its transformation will be the creation of a globally competitive urban space, defined here as a space that has the capacity to innovate, is well connected internally and to external markets, and is livable (Organization for Economic Cooperation and Development, or OECD 2006; World Bank 2010). This study identifies what is unique about Bangladesh s process of urbanization and examines the implications for economic growth. Through the lens of Bangladesh s most successful industry, the garment sector, it describes the drivers of and constraints to urban competitiveness. Based on the findings, it provides policy directions to strengthen the competitiveness of Bangladesh s urban space in ways that will allow Bangladesh to reach middle-income status by 2021.
eBook
Initial dysnatremia and clinical outcomes in pediatric traumatic brain injury: a multicenter observational study
PurposeWe aimed to investigate the association between initial dysnatremia (hyponatremia and hypernatremia) and in-hospital mortality, as well as between initial dysnatremia and functional outcomes, among children with traumatic brain injury (TBI).MethodWe performed a multicenter observational study among 26 pediatric intensive care units from January 2014 to August 2022. We recruited children with TBI under 18 years of age who presented to participating sites within 24 h of injury. We compared demographics and clinical characteristics between children with initial hyponatremia and eu-natremia and between those with initial hypernatremia and eu-natremia. We defined poor functional outcome as a discharge Pediatric Cerebral Performance Category (PCPC) score of moderate, severe disability, coma, and death, or an increase of at least 2 categories from baseline. We performed multivariable logistic regression for mortality and poor PCPC outcome.ResultsAmong 648 children, 84 (13.0%) and 42 (6.5%) presented with hyponatremia and hypernatremia, respectively. We observed fewer 14-day ventilation-free days between those with initial hyponatremia [7.0 (interquartile range (IQR) = 0.0–11.0)] and initial hypernatremia [0.0 (IQR = 0.0–10.0)], compared to eu-natremia [9.0 (IQR = 4.0–12.0); p = 0.006 and p < 0.001]. We observed fewer 14-day ICU-free days between those with initial hyponatremia [3.0 (IQR = 0.0–9.0)] and initial hypernatremia [0.0 (IQR = 0.0–3.0)], compared to eu-natremia [7.0 (IQR = 0.0–11.0); p = 0.006 and p < 0.001]. After adjusting for age, severity, and sex, presenting hyponatremia was associated with in-hospital mortality [adjusted odds ratio (aOR) = 2.47, 95% confidence interval (CI) = 1.31–4.66, p = 0.005] and poor outcome (aOR = 1.67, 95% CI = 1.01–2.76, p = 0.045). After adjustment, initial hypernatremia was associated with mortality (aOR = 5.91, 95% CI = 2.85–12.25, p < 0.001) and poor outcome (aOR = 3.00, 95% CI = 1.50–5.98, p = 0.002).ConclusionAmong children with TBI, presenting dysnatremia was associated with in-hospital mortality and poor functional outcome, particularly hypernatremia. Future research should investigate longitudinal sodium measurements in pediatric TBI and their association with clinical outcomes.
Journal Article
