Pharmacological Modeling and Regulation of Excitatory Amino Acid Transporters (EAATs)

L-Glutamate is the major excitatory neurotransmitter in the mammalian CNS that can mechanistically contribute to either neuronal signaling or neuronal pathology. Consequently, its concentration in the CNS must be carefully regulated, a critical need that is met by the excitatory amino acid transporters (EAATs). The presence of at least five isoforms of EAATs raises interesting questions as to potential structural and functional differences among the subtypes. We have investigated possible differences in the ligand binding domains of the EAATs through the development of computationally based pharmacophore models. An EAAT2-specific model was created with four potent and selective ligands that act as non-substrate inhibitors: cis-5-methyl-L-trans-2,3-pyrrolidine dicarboxylate, L-anti-endo-3,4-methano-pyrrolidine-3,4-dicarboxylate (L-anti-endo-3,4-MPDC), (2S,3R,4S)-2-(carboxy-cyclopropyl) glycine (L-CCG-IV) and L-B-threo-benzyloxy-aspartate (L-B-TBOA). This model predicts distinct regions that might influence the potency and selectivity of EAAT2 ligands, including: 1) a highly conserved positioning of the two carboxylate and the amino groups, 2) a nearby region that can accommodate selective modifications (e.g., cyclopropyl ring, CH3 groups, and O atoms), and 3) a region occupied by the benzyl ring of L-B-TBOA. This model was also used in conjunction with L-B-threo-benzyl aspartate (L-B-TBA), a recently characterized preferential inhibitor of EAAT3, to identify possible differences between EAAT2 and EAAT3. Functional studies on the EAATs also led to the identification of a putative modulatory mechanism that is specific for EAAT1. Thus, a series of sulfated neuroactive steroids, including pregnenolone sulfate (PREGS), were found to selectively increase the ability of EAAT1 to transport atypical substrates like D-aspartate and L-cysteine, but not L-glutamate. The effect was rapid, reversible, limited to a select group of sulfated steroids, and not observed with either EAAT2 or EAAT3. Interestingly, the action of PREGS could be blocked by the simultaneous addition of arachidonic acid, a previously recognized inhibitory modulator of EAAT1. The fact that this observed change in activity was produced by neurosteroids raises questions not only related to the regulatory mechanisms itself, but also to the possible role of neurosteroid in modulating glutamate transport

A Survey: Spider Monkey Optimization Algorithm

Swarm intelligence is a one of the areas for evaluating the optimization states. Many algorithms have been developed by simulating the swarming behaviour of various creatures like ants, honey bees, fishes, birds and their results are found as very motivating for solving optimization problems. In this paper, a new approach for optimization is proposed by modelling the social behaviour of spider monkeys. Spider monkeys have been categorized as fission-fusion social structure based animals. The animals which follow fission-fusion social systems, initially work in a large group and based on need after some time, they divide themselves in smaller groups led by an adult female for foraging. There- fore, the proposed strategy broadly classified as inspiration from the intelligent foraging behaviour of fission-fusion social structure based animals

Adenylyl Cyclase Isoform Specific Effects in Lipid Raft and Non-Lipid Raft Membrane Domains Regulate cAMP Compartmentation in Human Airway Smooth Muscle Cells

The formation of distinct macromolecular signaling complexes allows different G-protein coupled receptors to produce diverse functional responses, even while sharing a common second messenger such as cAMP. In human airway smooth muscle (HASM) cells, segregation of specific receptors into different membrane microdomains is thought to critically aid in generating compartmentalized cAMP responses. Whereas, E type prostaglandin receptors (EPRs) have been shown to be expressed in non-lipid raft domains of the plasma membrane, β-Adrenergic receptors (βARs) are predominantly expressed in caveolar lipid rafts. In the present study, we tested the hypothesis that adenylyl cyclase type 2 (AC2) preferentially couples to EPRs in a non-lipid raft domain, while adenylyl cyclase type 6 (AC6) selectively couples to βARs in lipid rafts. To do this, we examined the effect of overexpressing AC2 and AC6 on cAMP responses detected using genetically-encoded FRET-based biosensors targeted to lipid raft and non-lipid raft domains of the plasma membrane, as well as the bulk cytosolic compartment in HASM cells. This approach has the advantage of measuring the kinetics of cAMP production in living cells without the use of PDE inhibitors. Overexpression of AC2 enhanced the cAMP response to EPR activation associated with non-lipid raft domains, without significantly affecting responses detected elsewhere. AC2 overexpression also had no effect on cAMP responses to βAR activation detected in any subcellular location. These data confirm the hypothesis that AC2 couples exclusively with EPRs in a non-lipid raft membrane compartment. Overexpression of AC6, on the other hand, actually decreased the response to βAR stimulation associated with lipid rafts, without significantly affecting responses elsewhere. AC6 overexpression also had no effect on the responses to EPR activation detected anywhere in the cell. The ability of AC6 overexpression to inhibit βAR production of cAMP in lipid raft domains was reversed by inhibition of PDE4 activity with rolipram. It was also reversed by overexpression of Ht31 peptide, which disrupts the interaction of protein kinase A with A-kinase anchoring proteins (AKAPs). These results suggests that AC6 overexpression upregulates and/or recruits PKA and PDE4 activity, which then reduces βAR production of cAMP associated specifically with lipid raft domains

Surgical Excision of Heterotopic Ossification Leads to Re‐Emergence of Mesenchymal Stem Cell Populations Responsible for Recurrence

Trauma‐induced heterotopic ossification (HO) occurs after severe musculoskeletal injuries and burns, and presents a significant barrier to patient rehabilitation. Interestingly, the incidence of HO significantly increases with repeated operations and after resection of previous HO. Treatment of established heterotopic ossification is challenging because surgical excision is often incomplete, with evidence of persistent heterotopic bone. As a result, patients may continue to report the signs or symptoms of HO, including chronic pain, nonhealing wounds, and joint restriction. In this study, we designed a model of recurrent HO that occurs after surgical excision of mature HO in a mouse model of hind‐limb Achilles’ tendon transection with dorsal burn injury. We first demonstrated that key signaling mediators of HO, including bone morphogenetic protein signaling, are diminished in mature bone. However, upon surgical excision, we have noted upregulation of downstream mediators of osteogenic differentiation, including pSMAD 1/5. Additionally, surgical excision resulted in re‐emergence of a mesenchymal cell population marked by expression of platelet‐derived growth factor receptor‐α (PDGFRα) and present in the initial developing HO lesion but absent in mature HO. In the recurrent lesion, these PDGFRα+ mesenchymal cells are also highly proliferative, similar to the initial developing HO lesion. These findings indicate that surgical excision of HO results in recurrence through similar mesenchymal cell populations and signaling mechanisms that are present in the initial developing HO lesion. These results are consistent with findings in patients that new foci of ectopic bone can develop in excision sites and are likely related to de novo formation rather than extension of unresected bone. Stem Cells Translational Medicine 2017;6:799–806Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136492/1/sct312067.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136492/2/sct312067-sup-0001-suppinfo1.pd

Targeting of ALK2, a Receptor for Bone Morphogenetic Proteins, Using the Cre/lox System to Enhance Osseous Regeneration by Adipose‐Derived Stem Cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135224/1/sct320143111375.pd

Hair follicle specific ACVR1/ALK2 critically affects skin morphogenesis and attenuates wound healing

The bone morphogenic protein signaling (BMP) is intricately involved in the quiescence and regulation of stem cells through activation of BMP receptors. Hair follicle stem cells play a critical role in cutaneous homeostasis and regeneration. Here, we utilize a novel mouse model with targeted overexpression of the BMP receptor ALK2/ACVR1 in hair follicle stem cells, to characterize its role in skin development and postnatal wound healing. Initial histologic evaluation demonstrated significant dysregulation in hair follicle morphogenesis in mutant mice. These demonstrated increased numbers of individual hair follicles with altered morphology and localization. Mutant follicles were found to exhibit elevated proliferative activity as well as increased prevalence of CD34 and ITGA6 positive follicle stem cells. Interestingly, constitutive overexpression of ALK2 resulted in attenuation of cutaneous wound healing. These findings demonstrate that hair follicle specific ALK2 is intricately involved in maintenance of the stem cell niche and wound healing.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138367/1/wrr12549_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138367/2/wrr12549.pd

Aerobic Exercise and Scaffolds with Hierarchical Porosity Synergistically Promote Functional Recovery Post Volumetric Muscle Loss

Volumetric muscle loss (VML) is a composite loss of skeletal muscle tissue (greater than 20%) that heals with minimal muscle regeneration, substantial fibrosis, and subsequent functional deficits. Standard treatment, involving free functional muscle transfer and physical therapy, cannot restore full muscle function following VML. Tissue engineered scaffolds, 3D structural templates that mimic native extracellular matrix, are promising to enhance functional muscle formation and recovery. Bioprinted 3D scaffolds are engineered using bioinks, created from scaffolding material, cells, and growth factors, to replicate skeletal muscle architecture with precise control over their spatial deposition. METHODS: The present study evaluates a 3D-printed foam-like scaffold for the treatment of VML in a murine model. This colloidal foam-like scaffold was developed to have high porosity to improve tissue ingrowth, in contrast to dense polymeric scaffolds that routinely resulted in very poor tissue ingrowth, and sufficient stiffness to maintain its shape

Diminished Chondrogenesis and Enhanced Osteoclastogenesis in Leptin-Deficient Diabetic Mice (ob/ob) Impair Pathologic, Trauma-Induced Heterotopic Ossification

Diabetic trauma patients exhibit delayed postsurgical wound, bony healing, and dysregulated bone development. However, the impact of diabetes on the pathologic development of ectopic bone or heterotopic ossification (HO) following trauma is unknown. In this study, we use leptin-deficient mice as a model for type 2 diabetes to understand how post-traumatic HO development may be affected by this disease process. Male leptin-deficient (ob/ob) or wild-type (C57BL/6 background) mice aged 6?8 weeks underwent 30% total body surface area burn injury with left hind limb Achilles tenotomy. Micro-CT (?CT) imaging showed significantly lower HO volumes in diabetic mice compared with wild-type controls (0.70 vs. 7.02?mm3, P?Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140207/1/scd.2015.0135.pd

Comparative efficacy of topical tetraVisc versus lidocaine gel in cataract surgery

<p>Abstract</p> <p>Background</p> <p>To compare the clinical efficacy of lidocaine 2% with tetracaine 0.5% for cataract surgery.</p> <p>Methods</p> <p>In a randomized, multi-surgeon, controlled clinical trial,122 consecutive cataract cases eligible for topical anesthesia, were randomly assigned to receive lidocaine 2% gel (1 ml) or tetracaine solution 0.5% (TetraVisc, 0.5 ml) before clear corneal phacoemulsification. Main outcome measure was visual analog scale (0 to 10), which was used to measure intra-operative pain. Secondary outcome measures included patients' discomfort due to tissue manipulation and surgeon graded patients' cooperation. Duration of surgery and intra-operative complications were also recorded.</p> <p>Results</p> <p>The mean age in TetraVisc (TV) group was 70.4 years and in the lidocaine gel group (LG) it was 70.6 years (p = 0.89). Patient reported mean intra-operative pain scores by visual analog scale were 0.70 ± 0.31 in TV group and 1.8 ± 0.4 in LG group (<it>P </it>< 0.001). Mean patient cooperation was also marginally better in the TV group (8.3 ± 0.3) compared to LG group (8.4 ± 0.6) (P = 0.25). 96% of patients in TV group showed intra-operative corneal clarity compared to 91% in LG group. TV group had less (1 out of 61 patients, 1.6%) intra-operative complications than LG group (3 out of 61 patients, 4.8%). No anesthesia related complications were noted in either group</p> <p>Conclusion</p> <p>Topical TetraVisc solution was superior to lidocaine 2% gel for pain control in patients undergoing clear corneal phacoemulsification. Lidocaine 2% gel is similar to TetraVisc in patient comfort and surgeon satisfaction.</p> <p>Trial Registration</p> <p><b>Clinical trials number</b>: ISRCTN78374774</p