Exploring the dynamics of adult Axin2 cell lineage integration into dentate gyrus granule neurons

The Wnt pathway plays critical roles in neurogenesis. The expression of Axin2 is induced by Wnt/β-catenin signaling, making this gene a reliable indicator of canonical Wnt activity. We employed pulse-chase genetic lineage tracing with the Axin2-CreERT2 allele to follow the fate of Axin2+ lineage in the adult hippocampal formation. We found Axin2 expressed in astrocytes, neurons and endothelial cells, as well as in the choroid plexus epithelia. Simultaneously with the induction of Axin2 fate mapping by tamoxifen, we marked the dividing cells with 5-ethynyl-2′-deoxyuridine (EdU). Tamoxifen induction led to a significant increase in labeled dentate gyrus granule cells three months later. However, none of these neurons showed any EdU signal. Conversely, six months after the pulse-chase labeling with tamoxifen/EdU, we identified granule neurons that were positive for both EdU and tdTomato lineage tracer in each animal. Our data indicates that Axin2 is expressed at multiple stages of adult granule neuron differentiation. Furthermore, these findings suggest that the integration process of adult-born neurons from specific cell lineages may require more time than previously thought

Mechanical and Transport Properties of the Poly(ethylene oxide)−Poly(acrylic acid) Double Network Hydrogel from Molecular Dynamic Simulations

We used atomistic molecular dynamics (MD) simulations to investigate the mechanical and transport properties of the PEO−PAA double network (DN) hydrogel with 76 wt % water content. By analyzing the pair correlation functions for polymer−water pairs and for ion−water pairs and the solvent accessible surface area, we found that the solvation of polymer and ion in the DN hydrogel is enhanced in comparison with both PEO and PAA single network (SN) hydrogels. The effective mesh size of this DN hydrogel is smaller than that of the SN hydrogels with the same water content and the same molecular weight between the cross-linking points (M_c). Applying uniaxial extensions, we obtained the stress−strain curves for the hydrogels. This shows that the DN hydrogel has a sudden increase of stress above ∼100% strain, much higher than the sum of the stresses of the two SN hydrogels at the same strain. This arises because PEO has a smaller M_c value than PAA, so that the PEO in the DN reaches fully stretched out at 100% strain that corresponds to 260% strain in the PEO SN (beyond this point, the bond stretching and the angle bending increase dramatically). We also calculated the diffusion coefficients of solutes such as d-glucose and ascorbic acid in the hydrogels, where we find that the diffusion coefficients of those solutes in the DN hydrogel are 60% of that in the PEO SN and 40% of that in the PAA SN due to its smaller effective mesh size

Extracellular microRNAs in blood differentiate between ischaemic and haemorrhagic stroke subtypes.

Rapid identification of patients suffering from cerebral ischaemia, while excluding intracerebral haemorrhage, can assist with patient triage and expand patient access to chemical and mechanical revascularization. We sought to identify blood-based, extracellular microRNAs 15 (ex-miRNAs) derived from extracellular vesicles associated with major stroke subtypes using clinical samples from subjects with spontaneous intraparenchymal haemorrhage (IPH), aneurysmal subarachnoid haemorrhage (SAH) and ischaemic stroke due to cerebral vessel occlusion. We collected blood from patients presenting with IPH (n = 19), SAH (n = 17) and ischaemic stroke (n = 21). We isolated extracellular vesicles from plasma, extracted RNA cargo, 20 sequenced the small RNAs and performed bioinformatic analyses to identify ex-miRNA biomarkers predictive of the stroke subtypes. Sixty-seven miRNAs were significantly variant across the stroke subtypes. A subset of exmiRNAs differed between haemorrhagic and ischaemic strokes, and LASSO analysis could distinguish SAH from the other subtypes with an accuracy of 0.972 ± 0.002. Further analyses predicted 25 miRNA classifiers that stratify IPH from ischaemic stroke with an accuracy of 0.811 ± 0.004 and distinguish haemorrhagic from ischaemic stroke with an accuracy of 0.813 ± 0.003. Blood-based, ex-miRNAs have predictive value, and could be capable of distinguishing between major stroke subtypes with refinement and validation. Such a biomarker could one day aid in the triage of patients to expand the pool eligible for effective treatment

Extracellular microRNAs in blood differentiate between ischaemic and haemorrhagic stroke subtypes

Rapid identification of patients suffering from cerebral ischaemia, while excluding intracerebral haemorrhage, can assist with patient triage and expand patient access to chemical and mechanical revascularization. We sought to identify blood-based, extracellular microRNAs 15 (ex-miRNAs) derived from extracellular vesicles associated with major stroke subtypes using clinical samples from subjects with spontaneous intraparenchymal haemorrhage (IPH), aneurysmal subarachnoid haemorrhage (SAH) and ischaemic stroke due to cerebral vessel occlusion. We collected blood from patients presenting with IPH (n = 19), SAH (n = 17) and ischaemic stroke (n = 21). We isolated extracellular vesicles from plasma, extracted RNA cargo, 20 sequenced the small RNAs and performed bioinformatic analyses to identify ex-miRNA biomarkers predictive of the stroke subtypes. Sixty-seven miRNAs were significantly variant across the stroke subtypes. A subset of exmiRNAs differed between haemorrhagic and ischaemic strokes, and LASSO analysis could distinguish SAH from the other subtypes with an accuracy of 0.972 +/- 0.002. Further analyses predicted 25 miRNA classifiers that stratify IPH from ischaemic stroke with an accuracy of 0.811 +/- 0.004 and distinguish haemorrhagic from ischaemic stroke with an accuracy of 0.813 +/- 0.003. Blood-based, ex-miRNAs have predictive value, and could be capable of distinguishing between major stroke subtypes with refinement and validation. Such a biomarker could one day aid in the triage of patients to expand the pool eligible for effective treatment.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Preclinical Models of Middle Cerebral Artery Occlusion: New Imaging Approaches to a Classic Technique

Stroke remains a major burden on patients, families, and healthcare professionals, despite major advances in prevention, acute treatment, and rehabilitation. Preclinical basic research can help to better define mechanisms contributing to stroke pathology, and identify therapeutic interventions that can decrease ischemic injury and improve outcomes. Animal models play an essential role in this process, and mouse models are particularly well-suited due to their genetic accessibility and relatively low cost. Here, we review the focal cerebral ischemia models with an emphasis on the middle cerebral artery occlusion technique, a gold standard in surgical ischemic stroke models. Also, we highlight several histologic, genetic, and in vivo imaging approaches, including mouse stroke MRI techniques, that have the potential to enhance the rigor of preclinical stroke evaluation. Together, these efforts will pave the way for clinical interventions that can mitigate the negative impact of this devastating disease

Preclinical models of middle cerebral artery occlusion: new imaging approaches to a classic technique

Stroke remains a major burden on patients, families, and healthcare professionals, despite major advances in prevention, acute treatment, and rehabilitation. Preclinical basic research can help to better define mechanisms contributing to stroke pathology, and identify therapeutic interventions that can decrease ischemic injury and improve outcomes. Animal models play an essential role in this process, and mouse models are particularly well-suited due to their genetic accessibility and relatively low cost. Here, we review the focal cerebral ischemia models with an emphasis on the middle cerebral artery occlusion technique, a “gold standard” in surgical ischemic stroke models. Also, we highlight several histologic, genetic, and in vivo imaging approaches, including mouse stroke MRI techniques, that have the potential to enhance the rigor of preclinical stroke evaluation. Together, these efforts will pave the way for clinical interventions that can mitigate the negative impact of this devastating disease

Extracellular RNAs: development as biomarkers of human disease

Ten ongoing studies designed to test the possibility that extracellular RNAs may serve as biomarkers in human disease are described. These studies, funded by the NIH Common Fund Extracellular RNA Communication Program, examine diverse extracellular body fluids, including plasma, serum, urine and cerebrospinal fluid. The disorders studied include hepatic and gastric cancer, cardiovascular disease, chronic kidney disease, neurodegenerative disease, brain tumours, intracranial haemorrhage, multiple sclerosis and placental disorders. Progress to date and the plans for future studies are outlined

Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium

Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field

Structure and Function Studies of the Human Dopamine Receptors

Dopamine neurotransmitter and its receptors play a critical role in cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's and schizophrenia would be significantly enhanced with the availability of the three-dimensional (3-D) structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. In this thesis, I report the 3-D structures of the 5 subtypes of the human dopamine receptors, predicted from primary sequence using first principles theoretical and computational techniques. I use the term "first principles" to mean that we do not use the high resolution crystal structure of rhodopsin as a template, nor do we use homology modeling or threading of any kind to determine the structure. Predicting the binding sites, and the relative binding affinities of endogenous ligands and various pharmaceuticals to the 5 receptors validates the predicted structures. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies and give relative binding affinities that correlate well with experiment. The predicted binding site for dopamine and agonists is located between transmembrane helices (TM) 3, 4, 5, and 6, while the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM 5. We identify characteristic differences between the binding sites of agonists and antagonists, as well as factors that cause differential binding to the 5 subtypes of the human dopamine receptors. This thesis consists of five chapters that have, or will shortly result in publications. The first chapter is a brief introduction to the field, the motivation for the project, my scientific contributions, and contribution of others on the team. Chapter two introduces the methods and their successes at reproducing experimentally known results for the human D2 dopamine receptor; it discusses, in great detail, the active site of pharmaceutical agonists and antagonists to the human D2 dopamine receptor, and highlights the strengths and shortcomings of homology modeling for membrane bound proteins; this chapter will be submitted for publication to the Journal of Molecular Biology. Chapter three reports the results of a blind study performed in collaboration with Aventis Pharmaceuticals. For this study, we were provided with the two-dimensional structure of 9 antagonists and were asked to predict their binding sites, binding affinities, and to explain the differential binding of the ligands to the human D2 and D3 dopamine receptors and the human a1A adrenergic receptor. The results of this study are in preparation for submission to the Journal of Medicinal Chemistry. Chapters four and five of the thesis give preliminary results of comparative studies of the agonist and antagonist binding sites of the five subtypes of the human dopamine receptors. Chapter 6 contains results of another blind study on the G2A receptor with Professor Owen Witte. In addition to the six main chapters, this thesis contains 6 independent appendices that report results of similar studies in other systems. The first 2 appendices are work that has already been published. The remaining 4 appendices will shortly result in publications, but at this time, they are not publication worthy; these appendices represent data that has been analyzed but has not been written in paper format. In addition, I would like to make note of the studies that I have conducted on the 9 subtypes of the human adrenergic receptors with Mr. Peter Freddolino, the 4 human histamine receptors that were conducted with Mr. Freddolino and Mr. Maziyar Kalani, and the 4 G2A-like lipid receptors conducted with Mr. Rene Trabanino, Dr. Radu, Dr. Yang, and Professor Owen Witte of the Howard Hughes Medical Institute at the David Geffen School of Medicine at the University of California, Los Angeles.</p