bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiocentric glioma: elevated proliferation rate at clinical presentation does not preclude extended recurrence‐free survival
Authors: Li Jianyi , Langford Lauren A , Adesina Adekunle , Bodhireddy Surender R , Wang Min , Fuller Gregory N
Published: June 2021

Background: Angiocentric glioma (AG) is a recently described brain tumor with mixed features of ependymal and diffuse astrocytic differentiation that has been formally codified in 2007 WHO Classification of Tumours of the Central Nervous System. AGs characteristically exhibit low proliferation rates, with Ki‐67 labeling indices ranging from less than 1% to 5%. A single case with anaplastic recurrence and a labeling index of 10% has been reported. In the present study, we report a series of AGs that includes one case with an elevated proliferation rate at presentation that nonetheless has shown protracted recurrence‐free survival. Case reports: Case1, 4‐yo with a history of seizures and a frontal lobe mass. Case 2, 9‐yo with recent onset partial complex seizures and a temporal lobe mass. Case 3, 4‐yo with pervasive developmental delay, seizure disorder, and a frontal lobe mass. All three tumors showed classic histopathologic and immunophenotypic features of AG. The tumor from case 1 had 6 mitoses per 10 HPF, which were quickly and reliably quantitated using an anti‐phosphohistone H3 immunostain. Anti‐pHH3 immunostaining showed rare mitoses in cases 2 and 3. Postoperative recurrence‐free survivals for the three patients were 6 years, 9 years, and 5 months, respectively. Conclusion: Although anaplastic progression of AG has been reported, the presence of brisk mitotic activity at clinical presentation does not, per se, preclude extended recurrence‐free survival.
Incidence and extent of Lewy body‐related α‐synucleinopathy in human aging olfactory bulb
Authors: Sengoku Renpei , Saito Yuko , Ikemura Masako , Hatsuta Hiroyuki , Sakiyama Yoshio , Sawabe Motoji , Inoue Kiyoharu , Mochizuki Hideki
Published: June 2021

We investigated the incidence and extent of Lewy body (LB)‐related α synucleinopathy (LBAS) in the olfactory bulb (OB) in 320 consecutive autopsy cases from a general geriatric hospital (81.5±8.5‐years old). Paraffin sections were immunostained with anti‐phosphorylated α‐synuclein (psyn#64), tyrosine hydroxylase (TH), phosphorylated tau (AT8) and Aβ antibodies. Out of 102 LBAS cases (31.9%), OBs were involved in 85 cases (26.6%). Among them 14 were affected in the secondary olfactory structure (mitral, tufted, granule, and periglomerular cells and the surrounding neuropils) alone, 2 in the tertiary olfactory structure (anterior olfactory nucleus) and 69 in both. In five cases, intracytoplasmic aggregates were found exclusively in OB and three of the five accompanied Alzheimer disease (Braak NFT stage ≥IV and SP stage C). Only a few TH‐immunoreactive periglomerular cells presented LBAS. Thirty five LBAS cases with loss of pigmentation in substantia nigra all showed LBAS in OB. No correlation was found between LBAS and tauopathy or Aβ amyloidosis in OB, but AD changes significantly promoted LBAS in OB. This study demonstrated high incidence and extension from the second to tertiary olfactory structure of LBAS in human OB with aging.
Mode of cell death in C6 glial cell exposed to 1‐methyl‐4‐phenylpyridinum (MPP+)
Authors: Badisa Ramesh B , Williams Zakia R , Darling‐Reed Selina , Soliman Karam F
Published: June 2021

1‐methyl‐4‐phenylpyridinum (MPP+) is shown to cause selective death of nigrostriatal dopaminergic neurons in the brain, and is widely employed to induce Parkinson's disease symptoms in animal models. The present study was designed to examine if MPP+‐induced astrogliosis is related to apoptosis or necrosis in presence of 2 or 10 mM D‐glucose by using flowcytometry. For this purpose, the C6 glial cell line was used as an in vitro model to assess the MPP+‐induced astrogliosis by testing at 10, 30, 50, 70, 90, 100, 150, 250, 500 and 1000 μM for 12 h. The treated cells were stained with annexin V and propidium iodide (PI) and analyzed by flowcytometry within 1 h to assess the mode of cell death. In order to investigate the effect of MPP+on cell cycle stages, the rat C6 glial cells were treated with MPP+ at 100, 150, 250, 500 and 1000 μM for 12 h in the presence of 2 or 10 mM D‐glucose. These cells were stained with PI and analyzed by flow‐cytometry. The data indicate that exposure with MPP+ at 100 μM or above caused necrotic cell death predominantly when cells were grown in 2 mM D‐glucose, while MPP+atlower concentrations of 10, 30, 50, 70 and 90 μM caused significantly higher apoptotic cell death than necrotic. Interestingly, D‐glucose at 10 mM not only decreased the number of cells undergoing necrosis caused by MPP+ at 100 μM or higher but also increased the number of live cells at these concentrations. Cell cycle analysis indicate that glial cells in 2 mM D‐glucose were marginally (< 12%) arrested at G1 stage with 1000 μM MPP+, while 10 mM D‐glucose decreased the percentage of cell arrest (< 6%) with 1000 μM MPP+. Moreover, the cell cycle results confirm the protective role of D‐glucose against MPP+toxicity. (Supported by NIH Grant RR03020)
Chagas’ disease parasite neurotrophic factor ameliorates neurotoxin‐induced degeneration in vivo
Authors: Caradonna Kacey , PereiraPerrin Mercio
Published: June 2021

Trypanosoma cruzi, the causative agent of Chagas’ disease, invades the central nervous system where it multiplies inside glial cells and neurons and yet, contrary to most other brain infections, it produces little or no tissue damage and symptoms. The molecular basis for this apparent paradox remains unknown but it may be that T. cruzi itself actively promotes neuroprotective mechanisms. Here we demonstrate that cultured neuronal cells infected with T. cruzi upregulate survival pathways and resist the deleterious effects of the mitochondrial toxin 3‐nitropropionic acid (3‐NP). In vivo 3‐NP induces Huntington's disease‐like striatal pathology and motor disorders, but these abnormalities develop to a much lesser extent or not at all in mice infected with T. cruzi. What's more, intranasal administration of parasite‐derived neurotrophic factor (PDNF), a T. cruzi surface protein normally shed into the extracellular milieu, reproduces neuroprotective responses and motor disorder prevention of the infection. These results suggest a unique symbiotic‐type mechanism in T. cruzi invasion of the nervous system and, additionally, a novel therapeutic strategy for Huntington's disease. (Supported by NIH NS40574 and NS429660)
Dense neurofibrillary degeneration of the posterior cortex, BA 19/37, in a cognitively normal monozygotic twin at high risk for Alzheimer's disease
Authors: McKee Ann Carolyn , Jefferson Angela , Barrup Melissa , Kowall Neil
Published: June 2021

We previously reported that 50% of cognitively intact elderly have dense neurofibrillary degeneration in Brodmann area (BA) 19/37. We now report the finding of focal tau pathology in the posterior cortex of a cognitively intact identical twin at high risk for AD. A male Caucasian chemical engineer developed AD symptoms at age 60, first manifest as the inability to read maps, followed by progressive loss of memory and intellect over 16 years until his death at age 76. Post‐mortem exam confirmed severe AD (NIA‐Reagan: high, CERAD plaque: frequent, Braak: VI). His identical twin, also a chemical engineer with an identical education, died at 79 years from prostate cancer. At the time of his death, he was cognitively intact, except for slight loss of his navigational skills. Post‐mortem exam revealed sparse neuritic plaques and entorhinal and transentorhinal NFTs (NIA‐Reagan: low, CERAD: sparse, Braak: II). Despite the mild non‐diagnostic AD changes, there were dense NFTs, neuropil threads and tau immunoreactive neuritic plaques in the posterior cortex, BA 19/37. The finding of dense BA 19/37 pathology in this monozygotic twin, at high risk for the development of AD, but in the preclinical stages of AD at the time of his death, further confirms the early neurofibrillary degeneration of the posterior cortex in some individuals prior to the development of AD. Supported by NIA P30 AG13846, and by the Dept of Veteran's Affairs.
Lipopolysaccharide toxicity to hippocampal intermediate neuronal progenitors is diminished in mice lacking prostaglandin E2 receptor subtype EP1
Authors: Keene C. Dirk , Chang Rubens , Hevner Robert F , Montine Thomas J
Published: June 2021

Innate immune activation is implicated in Alzheimer's and other neurodegenerative diseases, and is mediated in the central nervous system primarily by microglia via prostaglandin E2 (PGE2) signaling. Lipopolysaccharide (LPS) activates innate immunity, and leads to a microglia‐dependent reduction in hippocampal neurogenesis. We examined the effects of unilateral intraventricular LPS injection on hippocampal neuronal progenitor cells (hNPC) in wt mice and in mice lacking PGE2 receptor subtypes EP1 and EP2. Mice received post‐operative bromodeoxyuridine (BrdU) injections and were euthanized 72 hrs post‐surgery. Total Tbr2+ hNPC per section were significantly reduced in wt (−51%) LPS mice (79±15) compared with wt controls (164±28), and in EP2−/− (−32%) mice (97±9) compared with EP2−/− controls (142±21). Surprisingly, LPS EP1−/− mice (187±28) showed a significant increase (+47%) compared with EP1−/− controls (127±16). Total BrdU+ cells increased in LPS wt (+40%) and EP1−/− (+90%), while EP2−/− mice showed no significant difference (+2.1%). There were no significant differences in Tbr2+ hNPC between LPS and vehicle in wt and EP knockouts after 24 hours, between control wt and EP knockouts, or between ipsilateral and contralateral hippocampi. These results implicate PGE2 receptor EP1 activation in the regulation of hNPC cell proliferation and/or survival in response to innate immune activation.
Technical and practical limitations of whole slide digital imaging in the analysis of intracranial thrombectomies
Authors: Tsang Michael P , Zheng David D , Marder Victor J , Moya Antonio J , Starkman Sidney , Saver Jeffrey L , Liebeskind David , Yong William H
Published: June 2021

Whole slide digital imaging (WSDI) is a technology in which stained slides are entirely digitized, usually at 200X to 400X magnification, in an automated fashion. The advantage of this technique is that image analysis software can then be applied globally to the entire tissue area or focally as needed. With an Aperio ScanScope XT system, we digitized 125 hematoxylin and eosin stained slides from 52 patients who had intracranial intravascular thrombi removed by mechanical clot retrieval devices. In analyzing the WSDIs, we identified a number of challenges: (1) Public freeware such as Image J and Cellprofiler could not analyze large high quality image files (1‐5 gigabytes) (2) Tissue section thickness can change staining intensity and cellular overlap (3) Tissue folds can increase color intensity (4) Faded staining of archival specimens can decrease color intensity (5) Identification of some cellular features may require manipulations like removing the condenser that are not easily done with a digitizer. These findings suggest that upfront preparation of high quality tissue sections of the correct thickness and staining intensity is particularly important for WSDI analysis. Cropping large image files and assigning pseudocolors with Adobe Photoshop can reduce file sizes. Development of image analysis freeware that can handle large image files will be crucial for greater adoption of WSDIs as a research tool.
Eosinophilic intracytoplasmic inclusions within the cerebral cortex in chronic seizure disorder ‐ a report of two cases and a review of the literature
Authors: Cunliffe Clare Helen , Fischer Ingeborg , Zagzag David , Miller Douglas C
Published: June 2021

Brightly eosinophilic inclusions within astrocytes have been reported 6 times in the literature, occurring in maldevelopmental disorders, Aicardi Syndrome, and once in seizure disorder. We report 2 more cases of this histologic finding within the cortex in patients with chronic seizure disorder.

One of the cases was a 3 year old boy who developed seizures following routine vaccinations. The other was a 14 year old woman with seizures since infancy. The cortical inclusions were similar in both cases: rope‐like or globular and brightly eosinophilic on H&E stain, some resembling Rosenthal fibers, and bright red on Azocarmine and Masson‐Trichrome stains, labeled by an S‐100 immunostain, but not labeled by GFAP, synaptophysin, neurofilament protein, Neu‐N, alpha‐B‐crystallin, and alpha‐synuclein immunostains. Ultrastructurally, the inclusions in one of the cases consisted of granular osmiophilic material within the cytoplasm of astrocytes.

The nature and significance of the inclusions is unclear. The tinctorial and immunohistochemical properties of these inclusions is similar to those described in the literature. Whether these inclusions represent a reactive process to seizure activity, or are causative in nature remains to be elucidated. However, they represent an unusual change which may be present in resected seizure foci, and as such, it is helpful for neuropathologists to be aware of this phenomenon.
EV71 induces COX‐2 expression and PGE2 synthesis through MAPKs and NF‐κB pathways in SK‐N‐SH cells
Authors: Tung Wei‐Hsuan , Yang Chuen‐Mao
Published: June 2021

The infection of enterovirus 71 (EV71) in young children causes severe neurological diseases. Accumulating evidence has suggested that eicosanoids derived from cyclooxygenase‐2 (COX‐2) participate in a number of pathological processes in neurological diseases. However, the mechanisms underlying EV71‐initiated intracellular signaling pathways linked to COX‐2 expression remain unknown in SK‐N‐SH cells. We designed studies to determine whether (1) EV71 stimulates COX‐ 2 expression, (2) activation of mitogen‐activated protein kinase (MAPK) and NF‐κB involved in EV71‐induced COX‐2 expression, and (3) prostaglandin (PG)E2 synthesis in SK‐N‐SH cells. EV71‐induces COX‐2 promoter activity and mRNA and protein expression was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 (SB203580), JNK (SP600125), and NF‐κB (Bay11‐7085). Consistently, EV71‐stimulated translocation of NF‐κB into the nucleus and degradation of IκBα was blocked by U0126 and Bay11‐7085. Previous studies report that COX‐2 participates in the replication of virus. Our results confirmed this hypothesis indicating that expression of EV71 viral protein (VP)1 and VP2 in SK‐N‐SH was attenuated by COX‐2 inhibitor (celecoxib) determined by Western blotting, indicating that EV71‐induced COX‐2 expression supported replication and growth of EV71 in these cells. These results reveal more intact information of EV71 induced inflammatory responses and prove beneficial in therapeutic value in the EV71 infection.
Bleomycin Clearance from the murine lung after a single intratracheal administration
Authors: Wang Ping Ming , Martin William J
Published: June 2021

Intratracheal (IT) administration of bleomycin is a popular model for studying lung injury and fibrosis. However, the distribution of IT bleomycin and the time of clearance for bleomycin are poorly understood. To investigate this, we administrated bleomycin IT (1.944 mg/kg) into adult female C57BL/6J Mice. Lungs were harvested on days 0, 2, 4, 8, and 11, and infused with OCT and frozen in liquid nitrogen. Lungs were sectioned at 7 micron and immunostained with sheep anti‐bleomycin and mouse anti‐sheep IgG conjugated with FITC. We quantified immunoflurescence of bleomycin retrained in lung tissue using microscopy and image analysis software (MetaMorph). Fluorescence (in gray scale) for each lung was averaged from 6 randomly selected regions (0.9 × 0.7 mm^2 for each) in lung sections. Fluorescence of immunostained untreated lung served as the baseline. We found that bleomycin was distributed throughout the lung but patchy areas of variable intensity. The average immunoflurescence of bleomycin in the lung declined with time (t, day) from day 2 and reached a baseline at day 11 post bleomycin with a formula as: relative fluorescence (%) = 169.1 e^(−0.2505 t), R^2=0.9661. These results suggest that IT administrated bleomycin in the lung may be detectable for several days following administration. Strategies to enhance clearance may reduce bleomycin toxicity.
Increasing the availability of nitric oxide (NO) increases sensitivity of nitrous oxide (N2O)‐insensitive inbred mice to N2O‐induced antinociception
Authors: Chung Eunhee , Ohgami Yusuke , Quock Raymond M
Published: June 2021

N2O‐induced antinociception in mice is dependent on the neuromodulator NO. In contrast to C57BL/6 mice, DBA/2 mice fail to respond to N2O with a robust antinociceptive response or with an increase in brain nitric oxide synthase (NOS) enzyme activity, suggesting that failure of DBA/2 mice to respond to N2O might result from a deficit of NO function (Ishikawa and Quock, Brain Res. 976:262–263, 2003). Therefore, it was of interest to determine whether increasing the availability of NO might increased sensitivity of DBA mice to N2O. Male DBA/2 mice (20–25 g) were pretreated with sub‐antinociceptive intracerebroventricular doses of the NO donor 3‐morpholinosydnoimine (SIN‐1) or the NO precursor L‐arginine (L‐Arg) then assessed for responsiveness to N2O‐induced antinociception using the acetic acid abdominal constriction test. Both pretreatments increased the antinociceptive responsiveness of DBA/2 mice to N2O. These results show that the NOS enzyme in DBA/2 mice is functional and that the deficit in NO function that obstructs sensitivity to N2O‐induced antinociception may lie in some other component or function related to NO. (Supported in part by NIH Grant GM‐77153).
G protein gated potassium channels (GIRK/ Kir3) mediate the motor stimulatory effects of opiates
Authors: Pravetoni Marco , Wickman Kevin
Published: June 2021

Neuronal GIRK channels mediate the postsynaptic inhibitory effect of many neurotransmitters and drugs of abuse, including opioids. GIRK subunits are expressed in dopamine and GABA neurons of the VTA, a structure implicated in opioid‐induced behaviors. We measured the impact of genetic ablation of different GIRK subunits in mice on the motor stimulatory effects of morphine. While GIRK1 knockout and GIRK2 knockout mice showed higher motor activity levels than wild‐type controls, GIRK3 knockout mice showed decreased sensitivity to morphine. We next used a stereotaxic approach to probe the relevance of GIRK signaling in the VTA to the observed responses to systemic morphine. We challenged wild‐type and knockout mice with intra‐VTA administration of the MOR selective agonist DAMGO, and measured its motor inducing effects. Our findings mirrored phenotypes seen with systemic morphine administration. Differences in opioid‐induced behaviors seen in mice lacking different GIRK subunits likely relate to the differential distribution of GIRK subunits in GABA and dopamine neurons of the VTA. Our data suggest that GIRK channels in the VTA modulate the sensitivity to opiate‐induced behaviors in a subunit‐dependent manner.
Discriminative stimulus effects of hallucinogens and psychostimulants in S(+)‐MDMA, and R(−)‐MDMA trained mice
Authors: Murnane Kevin Sean , Howell Leonard L , Fantegrossi William E
Published: June 2021

3,4‐Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative with increasingly widespread abuse. MDMA has been difficult to classify as a stimulant or hallucinogen because it shares effects with both drug classes. Furthermore, racemic MDMA is composed of two isomers that vary in terms of behavioral effects rather than simple potency differences. Specifically, the R(−) isomer of MDMA has hallucinogen like effects whereas the S(+) isomer has stimulant like effects. In the present study, mice were trained under a liquid food reinforcer two lever operant paradigm to discriminate S(+) or R(−)‐MDMA from vehicle. Drugs that are hallucinogen like and either structurally similar (2,5‐dimethoxy‐4‐(n)‐propylthiophenethylamine ‐ 2C‐T‐7) or not (N,N‐Dipropyltryptamine ‐ DPT) and drugs that are stimulant like and either structurally similar (Amphetamine) or not (Cocaine) were substituted for the training drug in mice trained to discriminate each isomer. Within chemical class we have found apparent efficacy differences whereas within behavioral class we have found potency differences. These studies enlighten our understanding of the complex nature of the discriminative stimulus properties of MDMA.
The reinforcing and discriminative stimulus effects of self‐administered cocaine in monkeys: a within‐subject design
Authors: Martelle Jennifer L , Nader Michael A
Published: June 2021

Drug discrimination (DD) and drug self‐administration (SA) are preclinical assays used to assess abuse liability; however, studies have demonstrated that discriminative stimulus (SD) and reinforcing (SR) effects do not always share a predictive relationship. To date, all preclinical studies with cocaine have examined the SD and SR effects in separate groups of subjects. The use of group‐design studies may partially account for our limited understanding of the relationship between SD and SR effects. To this end, drug naïve rhesus macaques were trained to discriminate self‐administered cocaine from saline under a novel chained‐schedule to evaluate the mechanisms underlying SD effects and reinforcing efficacy using within‐subject analyses. Each subject acquired SA under a progressive‐ratio schedule with no prior SA training. Significant differences in breakpoint between saline and 0.1 mg/kg cocaine were evident by training session 5 in all subjects. DD dose‐effect curves (0.03–0.3 mg/kg) were determined for each subject and compared to SA dose‐effect curves. For each subject, there was at least one cocaine dose that did not engender drug‐appropriate responding during DD yet functioned as a reinforcer during SA, suggesting that SD and SR effects provide dissimilar information. This within‐subject model may provide novel information related to the neuropharmacology of both effects. DA021920, DA06634
Activation of muscarinic cholinergic receptors (mAChRs) in SH‐SY5Y neuroblastoma cells potentiates both the influx and efflux of potassium following hypotonic stress
Authors: Foster Daniel J , Heacock Anne M , Keep Richard F , Fisher Stephen K
Published: June 2021

Incubation of SH‐SY5Y neuroblastoma cells in buffers rendered increasingly hypotonic by a reduction in NaCl concentration resulted in an enhanced basal efflux of K+ (threshold of release, 200 mOsM), but had no effect on K+ influx. Addition of oxotremorine‐M (Oxo‐M) potently enhanced K+ efflux (EC50 = 0.45 μM) increasing the threshold of release to 280 mOsM, and elicited a similarly potent, volume‐independent, enhancement of K+ influx (EC50 = 1.35μM). When incubated under hypotonic conditions in which osmolarity was varied by the addition of sucrose to a fixed concentration of NaCl, both basal and Oxo‐M‐stimulated K+ influx and efflux were dependent upon osmolarity. Basal and Oxo‐M‐stimulated K+ influx (but not K+ efflux) were inhibited by inclusion of ouabain or furosemide. In addition to the mAChR, activation of other GPCRs previously implicated in osmolyte release also enhanced both K+ efflux and influx under hypotonic conditions. Oxo‐M had no effect on cellular K+ content under physiologically relevant reductions in osmolarity (0–15%) unless K+ influx was blocked by ouabain and furosemide. The results indicate that, although GPCR activation enhances K+ efflux under hypotonic conditions, it also stimulates K+ influx, and that this results in retention of K+by the cells. Supported by NS23831 (SKF) and GM007767 (DJF).
N‐terminus Regulation of Vesicular Monoamine Transporter 2 activity
Authors: Torres Brian , Pal Arindam , Ruoho Arnold
Published: June 2021

The 20 amino acid N‐terminus of the vesicular monoamine transporter 2 (VMAT2) was examined as an enabler and regulator of transport activity. Relative to accumulation of 3H 5HT substrate by WT VMAT2 in transfected SH‐SY5Y cells, accumulation by truncants, lacking the first 16 or 19 N‐terminus amino acids, was reduced by over 50%. The N‐terminus contains 2 putative protein kinase C (PKC) phosphorylation sites at serines 15 and 18. A construct of the N‐terminus N‐terminally linked to GST underwent specific incorporation of 32P in the presence of 32P‐γ‐ATP and PKC at serines 15 and 18. A peptide of VMAT2 N‐terminus residues 1–20 inhibited transport by WT VMAT2 in digitonin permeabilized COS cells with a Ki of 100μM. However, peptides comprised of residues 1–11 or 12–20, a randomized peptide as well as a peptide in which serines 15 and 18 were completely phosphorylated, all failed to inhibit transport. Phospho‐mimetic mutations of serines 15 and 18 to aspartate resulted in a 50% loss in transport activity by the full length VMAT2 molecule in transfected permeabilized COS cells, whereas mutation to alanine retained full transport activity. Antisera raised against the phosphorylated N‐terminus showed specific immunoreactivity toward denucleated membranes obtained from WT VMAT2 transfected COS cells. These findings suggest the N‐terminal domain enables and regulates VMAT2 transport. supported by NIH R01NS033650‐08
Characterization of novel selective positive allosteric modulators (PAMS) of the M4 muscarinic acetylcholine receptor (mAChR)
Authors: Brady Ashley Elizabeth , Bridges Tom M , Jones Carrie K , Shirey Jana K , Lindsley Craig W , Conn P Jeffrey
Published: June 2021

Resent clinical studies have revealed that the M1/M4 preferring mAChR agonist xanomeline induces a robust antipsychotic effect in schizophrenic patients, suggesting that mAChR agonists may be efficacious in reducing psychotic symptoms in patients suffering from a variety of neurodegenerative and psychiatric disorders. Nevertheless, a lack of highly selective compounds has made it impossible to conclusively verify whether one or both of these receptor subtypes is responsible for mediating these observed clinical effects. Previous compounds developed to selectively activate mAChR receptors have failed in clinical development due to a lack of true specificity and adverse effects associated with activation of peripheral M2 and M3 mAChR subtypes. Here we report characterization of a series of novel, selective M4 PAMs which do not activate the receptor directly, but dramatically potentiate activation of the receptor by its natural agonist, acetylcholine. These novel M4‐selective agents can now be utilized as tools to ascertain whether selective potentiation of the M4 mAChR will mimic the in vivo effects of traditional orthosteric agonists and, more importantly, to rigorously test the hypothesis that activation of M4 has effects in animal models predictive of antipsychotic efficacy in humans. This work supported by NIMH grants MH073676‐01A1,1 F32 MH079678‐01, 1 F31 MH80559‐01 and 5T90DA022873‐02.
Identification of residues lining the entrance of the human serotonin transporter substrate permeation pathway using homology modeling
Authors: Torres‐Altoro Melissa I. , Nichols David E. , Barker Eric L.
Published: June 2021

Appropriate removal of monoamine neurotransmitters after release into the synapse is mediated by presynaptic transporters. Many neurological disorders have been implicated in the dysfunction of these important drug targets. A high‐resolution crystal structure for the monoamine transporters is yet to be resolved. We have developed a homology model for the serotonin transporter (SERT) based on the crystal structure of the leucine transporter (LeuTAa) from Aquifex aeolicus. The objective of my studies is to identify the structural determinants to the entrance into the substrate permeation pathway based on predictions drawn from the homology model. Taking advantage of the substituted cysteine accessibility method (SCAM), we identified residues that were sensitive and accessible to different MTS (methanethiosulfonate) reagents. These residues were protected against MTS reactivity with different SERT ligands. Moreover, those residues with significant changes in MTS reactivity after ligand protection were further analyzed by determining their rate of inactivation. Furthermore, bifunctional MTS cross‐linkers were used to assess the proximity of the helices predicted to be forming the entrance into the protein pore. Our studies provide the opportunity to refine the model and further extend SERT structural characterization.

This research supported by NIH grant R01DA018682.
Serotonin and Supermodels: Model‐guided exploration of hSERT TM6
Authors: Field Julie R. , Kaufmann Kristian W. , Henry L. Keith , Dawson Eric S. , Meiler Jens , Blakely Randy D.
Published: June 2021

The serotonin (5HT) transporter (SERT) is a clinically important target for serotonin selective reuptake inhibitor (SSRI) antidepressants, as well as MDMA (“ecstasy”) and cocaine. The determination of a crystal structure of the leucine transporter LeuTAa, a SERT homolog, has allowed us to model SERT in a serotonin bound state and to predict aspects of substrate selectivity and antagonist recognition. Our model predicts that specific residues associated with transmembrane domains (TMs) 1, 3, 6, and 8 comprise the 5HT (and MDMA) binding pocket. TM6 is predicted to be comprised of a set of inner and outer alpha helices connected by a flexible linker that can support TM6 reorientations during the transport cycle. Use of the substituted cysteine accessibility method (SCAM) identifies key residues in TM6 that relocate during the SERT transport cycle. Subsequent mutagenesis studies have refined our SERT models, predicting residues that govern specificity for serotonin and SSRI medications and establishing a path for the rational design of novel SERT‐targeted medications. J.R.F. is supported by NIDA 1 F31 DA023337. R.D.B is supported by NIDA Award DA07390.
Role of Toll‐like receptors in ischemic stroke induced endothelial cell dysfunction
Authors: Tiwari Samata , Mattson Mark P , Arumugam Thiruma V
Published: June 2021

Toll‐like receptors (TLRs) regulate host responses to pathogens and inflammatory responses. Activation of TLRs initiates signal transduction cascades involving kinases and transcription factors activator protein‐1 and NF‐kappa B. Recently studies have shown that TLR2 and −4 may play a role in ischemic stroke induced brain injury. Our recent findings showed that TLR2 and −4 are expressed in cerebral cortical neurons where their levels and downstream signaling via JNK are increased in response to energy deprivation in cell culture and ischemia in vivo. Here we use human microvascular endothelial cells (HMECs), mouse brain endothelial cells (MBECs) and in vivo models of ischemic stroke to elucidate roles of endothelial TLR2, −3 and −4 signaling in ischemic stroke induced endothelial dysfunction. Preliminary data shows that TLR2, −3 and −4 mRNA expression were increased during the first few hours in glucose deprivation (GD) and oxygen and glucose deprivation (OGD) conditions in both HMECs and MBECs. We also observed increased levels of TLR signaling adaptor molecule MYD88 mRNA in response to GD and OGD. Immunoblot analysis also showed increased expression of TLR proteins and MyD88 in response to OGD in MBECs. Preliminary results indicate a role for endothelial TLR signaling in pathogenesis of stroke.

Research Support: TTUHSC School of Pharmacy Start up Fund.
Validation of effect of Pioglitazone on acute phase changes induced by partial global cerebral ischemia in mice
Authors: Medhi Bikash , Aggrawal Raman , Chakrabarty Amitava
Published: June 2021

Objective: Present study was carried out to investigate the possible neuroprotective effect of pioglitazone on acute phase changes in mice model of cerebral ischemia.

Materials and Methods: Male mice weighing 20–30 gm were used in the study. BCCAO model was used to induce partial global cerebral ischemia. Animals were divided into 3 groups and following parameters were assessed (infarct size, brain edema, MDA, SOD, Catalase, GPx &TNF (). Postischemic seizure susceptibility was assessed using subconvulsive dose of PTZ (30 mg/kg i.p.).

Results: BCCAO induced significant brain infarct size and edema in control group with increase oxidative stress. Pioglitazone (20 mg/kg orally) has shown neuroprotective effects by reducing cerebral infarct size significantly as compared to control group. Postischemic seizure susceptibility was also reduced as number of positive responders decreased to a significant number. Brain edema was subsided to a significant level. Pioglitazone has reduced the plasma TNF‐ a and improved antioxidant parameters levels as compared to ischemia group significantly.

Conclusion: Pioglitazone showed neuroprotection against ischemic insult suggesting the role of PPAR ? agonist as neuroprotective agents. (Supported by PGIMER, Chandigarh, India.) Key words: Cerebral ischemia, pioglitazone, PPAR‐gamma, neuroprotection
PEGylated phospholipid nanomicelles protect Swedish mutants APP695 and sweΔ9 N2a mouse neuroblastoma cells from β‐amyloid induced apoptosis
Authors: Pai Ashwini S. , Onyuksel Hayat , Rubinstein Israel
Published: June 2021

The purpose of this study was to determine whether β‐amyloid evokes cell death in Swedish mutants APP695 and sweΔ9 mouse N2a neuroblastoma cells and, if so, whether sterically stabilized phospholipid nanomicelles (SSM) inhibit this process. Mouse N2a neuroblastoma cells stably expressing Swedish mutant APP695 and exon‐9 deletion mutant PS1 (sweΔ9) (2 × 103cells/well) were incubated in the absence or presence of SSM (10–100 μM) overnight. Thereafter, LDH release from and caspase3/7 activity in these cells were determined. We found that β‐amyloid had no significant effects on LDH release from N2a cells either in the absence or presence of SSM (n=3; p>0.5). By contrast, exposure to β‐amyloid was associated with significantly higher caspase 3/7 activity in comparison to control that was significantly attenuated in the presence of SSM in a concentration‐dependent fashion (n=3; p<0.05). Collectively, these data indicate that SSM protect Swedish mutants APP695 and sweΔ9 N2a mouse neuroblastoma cells from β‐amyloid induced apoptosis. W e suggest that SSM should be further developed as a novel nanomedicine for Alzheimer's disease.

Supported by IDPH # 73280001, NIH RO1 5R01CA121797, AG024026, VA merit Review and was conducted in a facility supported from NIH RFIP 06RR15482.Ashwini S. Pai is a recipient of Deans Scholar Award UIC 2007–2008 and American Foundation For Aging research (AFAR) fellowship 2007–2008.
Activation of p38 MAP kinase is required for acrylamide‐inhibited NCAM expression in SK‐N‐SH neuroblastoma cells.
Authors: Lee Hyerim , Son Eunwha , Rhee Dong‐Kwon , Pyo Suhkneung
Published: June 2021

Acrylamide is a well‐known industrial neurotoxic chemical that can induce neurodegeneration. Neural cell adhesion molecule (NCAM) is cell surface macromolecules that belong to the immunoglobulin(Ig) superfamily, which is expressed as three main isoforms‐NCAM‐120, NCAM‐140 and NCAM‐180. We investigated the effect of acrylamide on NCAM expression in human neuroblastoma cell (SK‐N‐SH), assessed by RT‐PCR, EMSA and western blot analysis. The results show that treatment of cells with acrylamide resulted in the decrease of protein and mRNA levels of NCAM in a time‐ and dose‐dependent manner. We next examined the possible roles of the MAP kinase family on acrylamide‐treated cells, because these kinases family have been described as playing a critical role in cellular signal pathways induced by a variety of environmental stresses. Western blot analysis showed that the activation of p38 was induced in response to acrylamide in a dose‐dependent manner, whereas it did not affect JNK and ERK activity. In addition, electrophoretic mobility shift assay indicated that acrylamide dose‐dependently decreased the DNA binding affinity of the Ikaros transcription factor. Taken together, these results indicate that activation of p38 but not of ERK and JNK is essential for acrylamide‐inhibited NCAM expression of SK‐N‐SH cells and acrylamide influences transcriptional stage of NCAM expression via ikros activation.
In vitro screening of acetylcholinesterase reactivating potency and oxime assisted organophosphate hydrolysis for a library of novel oxime reactivators synthesized by “click‐chemistry”.
Authors: Radic Zoran , Kalisiak Jaroslaw , Zhang Limin , Kovarik Zrinka , Calic Maja , Amitai Gabi , Fokin Valery V , Sharpless Barry K , Taylor Palmer
Published: June 2021

Organophosphorus (OP) pesticides and nerve warfare agents stoichiometrically inactivate acetylcholinesterase (AChE; EC ) by phosphylation of the active serine 203. Catalytic activity of the covalently inhibited AChE can be effectively reclaimed only by nucleophiles stronger than water. In this study we analyzed reactivating potency of close to 100 novel oximes synthesized using [3+2] cycloaddition between alkyne and azide building blocks, the “Click reaction”. Human AChE (hAChE) covalent adducts resulting from inhibition by nerve agents soman, sarin, cyclosarin, VX and tabun and pesticide derived paraoxon were used for reactivation experiments. We were able to identify several oximes with significantly improved reactivating efficacy against larger spectrum of offending OPs. We furthermore measured rates of hydrolysis of fluorescent derivatives of nerve agents soman, sarin, cyclosarin and VX assisted by oxime reactivators, for the wt AChE and Tyr337Ala mutant of hAChE, and found that in the presence of selected oximes rates of OP hydrolysis increased up to forty times with wt AChE and more with the Tyr337Ala hAChE mutant.
Astragalus Polysaccharides (APS) improves Free Fatty acids induced insulin resistance
Authors: Song Jie , Ouyang Jingping , Zou Feng , Liu Jian
Published: June 2021

The recent increase in the prevalence of obesity has been associated with a coincident rise in the prevalence of Type 2 diabetes, whereas weight loss has been shown to decrease the risk of Type 2 diabetes. The aim of the prestent study was to examine the effects of Astragalus Polysaccharides (APS) on FFAs treated C2C12 cells, thus to reveal its further protective mechanisms. AMP‐activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy and is therefore a target for type 2 diabetes treatment. The C2C12 cells which were pretreated with FFAs (200μmol/L) for 24h, were treated with APS(100μg/ml). The MTT experiment indicated that APS could improve the condition of C2C12 cells which were pretreated with FFAs. The expressions of total AMPK, activation of AMPK (p‐AMPK) and the activited acetyl CoA carboxylase (p‐ACC) were examined by Western blotting. Our results suggest that the total AMPK expression of C2C12 cells change little between APS group and FFAs group (p>0.05).But p‐AMPK expression increased in APS group(p<0.05) and p‐ACC expression decreased in the same group(p<0.05). This study demonstrated that APS availability affected C2C12 cells AMPK activity. The lower expression of p‐ACC could result in increasing of Fatty Acid Oxidation. Meanwhile, GLUT4 protein expression was elevated 40% by 2 days treatment with APS. In short, p‐AMPK can indirectly activate ACC and GLUT4. And AMPK can be activated by APS. Therefore, in C2C12 cells, APS improves FFAs induced insulin resistance via a AMPK‐dependent mechanism. This subject has a financial assistance by NSFC(30370673).
Prostacyclin receptor regulates contraction‐associated proteins and contractile proteins in pregnant human myometrium via PKA/cAMP and the transcription factors GATA‐6 and myocardin
Authors: Fetalvero Kristina , Zhang Pei‐Sheng , Shyu Maureen , Young Benjamin , Hwa John , Young Roger C. , Martin Kathleen A.
Published: June 2021

Objective: Levels of the smooth muscle relaxant prostacyclin increase prior to labor. We investigated the role of prostacyclin in myometrial activation.

Methods: Myometrial tissue was obtained from term pregnant women during cesarean delivery and treated with vehicle or the prostacyclin receptor (IP) agonist iloprost in organ or cell culture models. siRNA was transfected using a Nucleofector. Contraction‐associated protein (CAP) and contractile protein expression were analyzed by immunoblotting or RT‐PCR.

Results: Iloprost induced expression of the CAPs COX‐2 and connexin‐43, and the contractile proteins smooth muscle‐myosin heavy chain, h‐caldesmon, and calponin in organ culture and/or cell culture. COX‐2 activity increased as measured by PGF2αrelease. Iloprost activated cAMP/PKA, and siRNA against PKA reduced iloprost‐induced CAP and contractile protein expression, while 8‐Br‐cAMP mimicked the effect. siRNA against the transcription factors GATA‐6 or myocardin inhibited iloprost‐induced SM‐MHC and COX‐2.

Conclusions: IP activation of cAMP/PKA induces CAPs and contractile proteins in human myometrium, perhaps by regulating GATA‐6 and myocardin. Prostacyclin may provide positive feedback, linking CAPs and contractile proteins, by increasing COX‐2 activity. These mechanisms may be involved in the conversion of the human uterus from quiescent to an actively contracting labor phenotype.

This work was supported by grants from the NIH NHLBI and an NIH NCI training grant
Direct detection of intracellular superoxide generation from freshly isolated rat aorta: a novel approach
Authors: Goel Aditya , Matkar Smita , Rahimian Roshanak
Published: June 2021

Although superoxide (O2−) plays an important role in vascular diseases, direct measurement of O2− in viable tissue remains a challenge. The aim of this study was to design and validate a procedure to measure O2− generation using freshly isolated aorta.

Method:

The 8 mm fresh aortic segments from male and female rats were cut longitudinally in Krebs buffer at 37°C and mounted on the silicon elastomer surface with the lumenal surface facing upward. The segments were incubated with the nitric oxide probe 4,5‐diaminofluorescein diacetate (DAF‐2DA, 5 μM) or O2− probe dihydroethidium (DHE, 2.5 μM), followed by fluorescent recoding at longitudinal edge using Leica DMIRE2 microscope. Acetylcholine‐induced increased DAF fluorescence was taken as evidence for the endothelial integrity. To examine the effect of O2− generator, DHE fluorescence was quantified before and after incubation of segments with phorbol myristate acetate (PMA, 0.1 μM), or PMA in the presence of O2− scavenger (tempol, 100 μM) for 30 min.

Results:

The extent of PMA‐induced O2− increase was greater in female tissues (9.2 fold) than that in males (2.8 fold), and this was prevented by the presence of tempol. This data was comparable to our recent report on gender difference in PMA‐induced O2− production in rat aorta using isometric tension study.

Conclusion:

This procedure will allow us to detect O2− generation in translucent viable vessels. (Supported by NHLBI)
Trait and state variables associated with social rank and cocaine abuse in female cynomolgus monkeys
Authors: Riddick Natallia V. , Czoty Paul W. , Kaplan Jay R. , Gage H. Don , Icenhower Michelle , Nader Michael A.
Published: June 2021

Social rank has been shown to affect susceptibility to many diseases including drug addiction. In the present studies we examined several behavioral and neurochemical markers as possible predictors of eventual social rank (i.e. trait variables) in 16 female cynomolgus monkeys and assessed whether these markers changed after social housing (i.e., state variables). Body weight, cortisol levels and locomotor activity did not predict social rank. In contrast, eventual subordinate monkeys were more impulsive as shown by lower latencies to approach a novel object in the home cage and had significantly higher levels of cerebrospinal fluid serotonin (5‐HT), but not dopamine (DA), metabolites (5‐HIAA and HVA, respectively). Using positron emission tomography, neither 5‐HT transporter nor DA D2 receptor availability was predictive of eventual social rank. However, as was observed in earlier studies with male monkeys, D2 receptor availability increased in monkeys that became dominant. Low D2 receptor availability and low social rank have been associated with heightened vulnerability to reinforcing effects of cocaine (Morgan et al. 2002; Nader et al. 2006); this association is currently being investigated. DA017763.
The glucose moiety of uridine 5′–diphosphoglucose is structurally permissive in activation of the human P2Y14 receptor
Authors: Jacobson Kenneth A. , Fricks Ingrid , Melman Artem , Carter Rhonda , Ivanov Andrei A. , Harden T. Kendall , Ko Hyojin
Published: June 2021

The P2Y14 receptor is a signaling protein in the eight member P2Y nucleotide receptor family that is activated by UDP‐glucose (UDPG) and other uridine nucleotides. The P2Y14 receptor plays a role in the neuroimmune system, with expression in T cells, dendritic cells, and hematopoietic stem cells, and also is expressed in other tissues. We have systematically explored the structure activity relationships of UDPG analogues at this receptor. With the exception of 2‐ and 4‐thio modifications, most modifications of the nucleobase or ribose moieties diminished activity. UDPG also activates the P2Y2 receptor, but its 2‐thio analogue (MRS2690) was selective for the P2Y14 receptor and activated this receptor with 7‐fold greater potency than UDPG. Molecules in which the glucose moiety was replaced with a simple β‐methyloxy group retained agonist activity at the P2Y14 receptor. Replacement of the ribose moiety with a rigid methanocarba group, either in the North or South conformation, abolished agonist activity. Many sugar moieties, such as mannose and fructose, may be substituted for the glucose with retention of activity. Thus, the glucose moiety is the most structurally permissive region of the ligand for derivatization.
Antibacterial Activity of New Pyrimidinylamino‐ and 4‐Benzothiazolylamino‐3‐nitro‐2H[1]‐Benzopyran‐2‐ones
Authors: Vehapi Idriz , Hoti Ramiz , Mulliqi‐Osmani Gjyle , Plakolli Mujë , Kurteshi Kemajl
Published: June 2021

Synthesized heterocyclic compounds 4‐(5‐bromopyrimidinylamino)‐3‐nitro‐ 2H[1]‐benzopyran‐2‐one 3a , 4‐(4‐Chloropyrimidinylamino)‐3‐nitro‐ 2H[1]‐benzopyran‐2‐one 3b , 4‐(4‐Methylbenzothiazolylamino)‐3‐nitro‐2H[1]‐benzopyran‐2‐one 3c and 4‐(6‐fluorobenzothiazolylamino)‐3‐nitro‐2H[1]‐benzopyran‐2‐one 3d are tested for their antibacterial activity against Staphylococcus aureus, Clebsiella and Escherichia coli. Compounds 3 (a–d) showed considerable bactericide activity against these microorganisms. The observed antibacterial activity were in subordination of their structure and concentration. These compounds were synthesized by condensation reactions of 4‐Chloro‐3‐nitro‐2H[1]‐benzopyran‐2‐one 1 and corresponding heteroarylamines 2(a–d) and characterized on the basis of their spectroscopic data.
Advantage of 2D‐QSAR in the discovery of novel protein geranylgeranyltransferase inhibitor (GGTI) scaffolds
Authors: Peterson Yuri Karl , Wang Simon X , Casey Patrick J , Tropsha Alexander
Published: June 2021

Prenyltransferases selectively modify CaaX motif proteins (including Rho, Rap1, Rac, and Cdc42) with an isoprene lipid critically important for localization, function, and degradation. Selective GGTI's have been reported in the literature; however, all publicly available inhibitors are derived from essentially only two chemical scaffolds. We have extended quantitative structure‐activity relationship (QSAR) modeling traditionally reserved for lead optimization towards virtual screening. A series of 48 GGTI's were analyzed by 2D‐QSAR models using k‐nearest neighbor (kNN), automated lazy learning (ALL), and the partial least square (PLS) method. Validated QSAR models were used to mine ∼9.5 million compounds from public chemical databases. These searches resulted in 79 consensus hits that featured several novel scaffolds. Comparing structural similarity of database hits to the training set indicate many of the QSAR derived GGTI's are structurally divergent from the training set. Seven of these compounds with divergent backbones and high predicted GGTI activity were tested in vitro and all were found to be bona fide and selective inhibitors. These data demonstrate that validated QSAR models can serve as reliable virtual screening tools, providing higher hit rates as compared to chemical similarity searches. This strategy can be extended to any chemical biological system. Supported by F32‐GM073420