bluuebunny/crossref_metadata_2025 huggingface dataset:
Brain Damage Following Smoke Inhalation and Skin Burn
Authors: Randolph Anita , Ihara Koji , Escobar Kenneth , Fukuda Satoshi , Salsbury John Randall , Prough Donald , Herndon David , Enkhbaatar Perenlei
Published: November 2023

RATIONALE

In the U.S., there is an estimated 300,000 to 500,000 patients hospitalized annually due to burn related injuries. Mortality and morbidity of burn patient's increase significantly when combined with smoke inhalation injury. Previous studies have shown combined burn and smoke inhalation results in systemic changes including hypermetabolic response, microvascular hyperpermeability and multiorgan dysfunctions. With the advanced technology and treatment, mortality of burn victims is remarkably reduced. However, there is no follow up study, among the survivors, on possible effects of burn and smoke on brain dysfunction. In this study, we tested the hypothesis that burn and smoke inhalation injury is sufficient to induce brain dysfunction by activating astrocytes and causing neuronal injury.

METHODS

To test our hypothesis, we used our well‐characterized ovine model of skin flame burn (40% TBSA, 3rd degree) and smoke inhalation (48 breaths of cooled cotton smoke). After the injury we studied the animals 48 hours in a conscious state under mechanical ventilation, recording hemodynamics and ventilator variables as well as taking blood samples every 6 hours. Postmortem, brains were taken and placed into 10% formalin for 4–6 weeks. We compared 3 different groups: n=3 sham (no injury), n=3 smoke only, n=3 combined burn and smoke. Brain tissue slides were prepared from the hippocampus and basal ganglial area (ten slides for each sheep) and stained with Luxol Fast Blue (LFB) to assess myelinated axon damage and Hematoxylin & Eosin (H&E) to assess astrocyte activation and neuronal damage/death.

RESULTS

Average carboxyhemoglobin levels were 2.1%, 67.3%, 60.4% in sham, smoke only and burn/smoke groups, respectively. No noticeable brain tissue changes were observed in sham, whereas there were clear evidences of astrocyte activation and neuron death in the smoke only group. The number of activated astrocytes per field and damaged neurons were significantly increased in the smoke only and burn/smoke groups. The percentage of damaged neurons in the smoke only and burn/smoke groups were 41.0% and 59.9% respectively compared to sham of 19.2%. Observed basophilic neuronal necrosis in smoke only vs. sham was increased as well. These changes were augmented by skin burn. In addition, vascular congestion and hemorrhage were observed in both smoke only and combined burn/smoke groups.

CONCLUSIONS

Smoke inhalation causes brain damage characterized by astrocyte activation, neuronal and myelinated axon damage and hemorrhage. Presence of skin burn exacerbates smoke‐induced brain injury.

Support or Funding Information

Shriners Hospital for Children SHC84050; SHC85220
microRNA profiling in rat model of neurofibrillary degeneration
Authors: Cente Martin , Kosikova Nina , Filipcik Peter , Novak Michal
Published: November 2023

Neurofibrillary tangles composed of post‐translationally modified protein tau are major hallmarks of neurodegenerative diseases collectively known as tauopathies. Pathogenesis of these disorders is characterized by gene expression changes resulting to altered signalling pathways. Moreover, deregulation of microRNA levels has been also associated with many neurodegenerative diseases, suggesting possible employment of miRNAs as diagnostic biomarkers. In order to identify differentially expressed microRNA molecules during neurofibrillary degeneration we have performed complex miRNA profiling in rat model of tauopathy (expressing human truncated Tau151‐391,4R) that recapitulates major features of neurodegeneration of AD type.

Transcriptomic analysis of brain tissue samples showed significantly increased expression of several particular miRNAs involved in the regulation of neuroinflammation (4,1‐fold), innate immune response (2,6‐fold), intracellular trafficking (2,4‐fold) and signal transduction (2,3‐fold). Interestingly, correlation analysis revealed strong positive relationship between dysregulated microRNA molecules.

The data suggest that expression of truncated tau protein leads to deregulation of signalling pathways during the neurodegeneration process manifested by altered miRNA expression.

Moreover, identified miRNA markers can be potentially applied for the monitoring of the neurodegenerative cascades in Alzheimer's disease and related tauopathies.

This work was supported by Research grants: APVV‐0677‐12 and VEGA 2/0141/15 and research grant from Axon Neuroscience SE.
Neural Development of Drosophila Homologs of Human Neurofibromatosis and Juvenile Neuronal Ceroid Lipofuscinosis
Authors: Bragg Jack , Balouch Bailey , Chu‐LaGraff Quynh
Published: November 2023

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is a juvenile onset neurodegenerative disease associated with the intracellular accumulation of ceroid lipofuscin in central and peripheral neural tissues. Loss of function mutation in the Ceroid Lipofuscinosis Neuronal‐3 (CLN3) gene is suspected to be the primary cause for JNCL, although the normal function of the protein product is yet to be understood. Neurofibromatosis type 1 (NF‐1) is a genetic disorder caused by the loss of the highly conserved Ras GTPase‐activating protein, Neurofibromin. Affecting an estimated 1 in 3000 individuals, NF‐1 is characterized by abnormal skin and bone pigmentation, and predisposition to peripheral nerve associated tumors. Although both genes are expressed in various regions of the developing brain, the underlying mechanisms of how alterations in these expressions lead to JNCL and NF1 diseases are not well known. In this study, we use Drosophila melanogaster as models to investigate the role of CLN3 and Neurofibromin during embryonic development of the nervous system. Using in situ immunostaining using various neural and axonal markers such as BP102 and FASII, axon guidance and ventral nerve cord organization was studied in embryos homozygous for the CLN3 and Neurofibromin genes at stage 16 of CNS and PNS embryonic development. We will present results to determine whether these genes have a developmental role during embryonic neurogenesis. If so, these studies could potentially uncover new mechanisms toward understanding the pathology of the human diseases.
Behavioral economic analysis of the effects of AHN 1‐055 and JHW 007, atypical dopamine uptake inhibitors, on cocaine self administration
Authors: Zanettini Claudio , Newman Amy H. , Wilkinson Derek , Katz Jonathan L.
Published: November 2023

Benztropine analogs and other atypical dopamine uptake inhibitors selectively decrease cocaine self‐administration in rats at doses that do not affect responding maintained by other reinforcers. In the current study a behavioral economic approach was employed to further characterize the behavioral mechanisms of effects of benztropine analogs (AHN 1‐055, JHW 007) on responding maintained by cocaine injection in rats. Behavioral economic analyses represent a translational approach that take into account the price of the reinforcer as an important determinant of consumption. Two groups of rats were trained to respond for food (45 mg pellet) or cocaine (0.32 mg/kg/inj) under a fixed‐ratio (FR) 5 schedule in experimental sessions comprising five 20‐min components. In selected sessions the FR value was increased (5, 10, 20, 40, 80) during successive components to determine demand curves, which plot consumption (reinforcers obtained) against price (FR value). An exponential function was fitted to the data to derive the consumption at zero price (Q0) and the rate of decrease in consumption (essential value). AHN 1‐055 (1.78–10 mg/kg, IP), JHW 007 (3.2–10 mg/kg, IP) or vehicle was administered immediately before each demand curve determination. Consumption of cocaine or food decreased as the price (FR requirement) increased. At low doses AHN 1‐055 shifted downward and to the right the cocaine and the food demand curves. A larger dose shifted the curves leftward and downward. Analysis of the demand curve parameters indicated that AHN 1‐055 had equivalent effects on the essential value of cocaine or food but was more effective in decreasing Q0 for cocaine (%Δ= −83%) compared to food (%Δ=−27%). The effects of JHW 007 were less than those of AHN 1‐055, with only trends for decreases in Q0 for cocaine, and no significant difference in essential value for cocaine or food. Therefore, AHN 1‐055, but not JHW 007, selectively decreased consumption at zero price. The current findings obtained using an approach that takes in account the price of the reinforcer, indicate that AHN 1‐055 might be more effective than JHW 007 in reducing selectively consumption of cocaine.

Support or Funding Information

This work was supported by NIDA Intramural Research Program
The effects of cocaine self‐administration and abstinence on dopamine D2/D3 receptor availability in female cynomolgus monkeys
Authors: Nader Michael A. , Nader Susan H. , Duke Angela N. , Solingapuram Sai Kiran K. , Mintz Akiva
Published: November 2023

Previous PET imaging studies have shown that individuals abusing many drugs of abuse, including cocaine, have lower measures of dopamine (DA) D2/D3 receptor availability compared to age‐matched controls; most of the human subjects in these studies are men. In monkeys, a within‐subjects design can be incorporated in which baseline measures of D2/D3 receptor availability can be obtained prior to any cocaine exposure and then assessed after a long cocaine self‐administration history and during abstinence. In male monkeys, we found intake‐dependent reductions in D2/D3 receptor availability over 1 year of self‐administration; recovery of receptor levels during abstinence was observed in approximately half the monkeys (Nader et al., 2006). The goal of the present study was to extend these findings to female monkeys. Ten experimentally naïve, individually housed, adult female cynomolgus macaques were trained to respond under a 4‐component multiple fixed‐interval (FI) schedule of food presentation. During this time, baseline measures of DA D2/D3 receptor availability using positron emission tomography and [11C]raclopride were obtained in each monkey. When the reinforcement schedule was changed to a multiple FI food (components 1 and 3) and cocaine (components 2 and 4) presentation, response rates maintained by a 0.2 mg/kg per injection cocaine did not correlate with baseline D2/D3 receptor availability. When monkeys (N=5) were rescanned after self‐administering 100 mg/kg and 1000 mg/kg cocaine, there were significant reductions in D2/D3 receptor availability in the caudate nucleus, putamen, and ventral striatum. This finding is consistent with those reported in male monkeys. These five female monkeys were also studied in abstinence with one showing recovery of D2/D3 receptor availability after 6 months and the other after 9 months of abstinence. In an effort to determine whether the reductions in D2/D3 receptor availability were permanent in the three monkeys that did not recover their baseline after 9 months of abstinence, they were treated them with raclopride (0.01 mg/kg per hour) via an osmotic pump for 30 days using a protocol that has been shown to increase D2/D3 receptor availability in male monkeys (Czoty et al., 2005). Chronic raclopride treatment increased D2/D3 receptor availability relative to their 9‐month abstinence levels by 10–20%. However, relative to their pre‐cocaine baselines, reductions were still apparent. These findings show that while the effects of long‐term cocaine self‐administration are similar in all monkeys, there are individual differences in rate of recovery and these reductions may be irreversible. Additional studies are necessary to determine whether there are behavioral characteristics or peripheral and central biomarkers predictive of DA receptor recovery.

Support or Funding Information

DA017763
Active anti‐methamphetamine vaccination attenuates the effects of methamphetamine
Authors: Taffe Michael A , Nguyen Jacques D , Bremer Paul T , Vandewater Sophia A , Creehan Kevin M , Janda Kim D
Published: November 2023

Successful treatment remains elusive for methamphetamine (MA) addiction, which remains a serious public health concern. Immunopharmacotherapy has been shown to attenuate many effects of MA in rodent models. This study further investigated the efficacy of an anti‐MA vaccine in rats with locomotor and self‐administration assays. Male and female rats were vaccinated with either a control keyhole‐limpet hemocyanin vaccine (KLH) or a candidate anti‐MA vaccine (MH6‐KLH conjugate). MH6‐KLH vaccination blocked the homecage locomotor stimulant effects of MA (i.p.) in a dose dependent manner in female rats. The MH6‐KLH vaccine also interfered with a suppression of wheel running activity observed 30 min after vapor inhalation of MA in male rats. The MH6‐KLH vaccine also reduced the reinforcing efficacy of MA, but not alpha‐PVP, in intravenous self‐administration under a Progressive Ratio dose substitution procedure in male rats. This study further confirms the efficacy of the MH6‐KLH candidate vaccine and more generally that active immunopharmacotherapy can attenuate the effects of MA.

Support or Funding Information

NIH Grant R01 DA024705
Pharmacological Characterization of the Discriminative‐Stimulus Effects of the Nicotinic Agonist (+)‐Epibatidine in Squirrel Monkeys
Authors: Desai Rajeev I , Doyle Michelle R , Bergman Jack
Published: November 2023

The present studies were undertaken to characterize the discriminative‐stimulus (SD) effects of (+)‐epibatidine (EPI)–an α4β2 selective nicotinic agonist that is pharmacologically similar and structurally distinct from nicotine (NIC). Using a standard two‐lever drug discrimination procedure, squirrel monkeys (n=4) were trained to discriminate i.m. injections of 0.001 mg/kg (+)‐epibatidine (EPI) from saline on a 10‐response fixed‐ratio schedule of stimulus‐termination. Results show that high efficacy nicotinic agonists [(+)‐EPI, (−)‐EPI, NIC] substituted fully for (+)‐EPI, whereas the highest doses of other nicotinic agonists produced intermediate levels of (+)‐EPI‐like SD effects [varenicline (VAR), cytisine (CYT), isoarecolone (ISO)] or did not substitute for (+)‐EPI (lobeline). Drugs from other pharmacological classes (methamphetamine, atropine, citalopram, arecoline) did not generalize for (+)‐EPI's stimulus effects. Pretreatment studies with nicotinic antagonists show that: a) mecamylamine (non‐selective) insurmountably antagonized (+)‐EPI's effects; b) dihydro‐β‐erthroidine (α4β2‐selective) surmountably (>3‐fold rightward shift) blocked (+)‐EPI's effects; and c) methyllycaconitine (α7 selective) failed to modify the SD effects of (+)‐EPI. Interestingly, the peripherally‐restricted nicotinic antagonist hexamethonium also appeared to block (+)‐EPI's SD effects (approximately 3‐fold rightward shift) suggesting that a peripheral component may also play a role in 0.001 mg/kg (+)‐EPI's discriminable effects. In further studies, pretreatment with the partial nicotinic agonists VAR and CYT did not block the SD effects of (+)‐EPI, and in fact, CYT pretreatment shifted the (+)‐EPI dose‐effect curve to the left (>3‐10‐fold). These results suggest that the discriminative‐stimulus effects of (+)‐EPI are mediated through α4β2 nicotinic receptor subtypes at which the partial agonists VAR and CYT do not exert partial antagonist actions.

Support or Funding Information

Supported by NIH/NIDA DA031231
Expression of Nerve Membrane Proteins in a Naturally Occurring Ca2+ Channelopathy
Authors: Colón‐Carrion Nicole , Colón‐Rodriguez Alexandra , Atchison William D.
Published: November 2023

Neuronal voltage gated calcium channels (VGCC) are essential for numerous cellular functions including synaptogenesis and neurotransmitter release. Mutations in individual subunits of the VGCC are known to result in many neurological disorders. In tottering (tg) mice, the mutation in the α1A subunit of the P/Q calcium channel leads to ataxia and motor seizures that result in absence epilepsy in humans. Studies done with tg mice have also implied that GABAA receptor function is impaired. Consequently, we compared the expression of GABAA receptor subunits and VGCC subunits in the forebrain (FB), which is not a specific target of the tg mutation with that in the cerebellum (CB), which is a primary target. Previous functional studies have shown that Ca2+ channels subtypes such as the L and N type are upregulated and the inhibitory GABAA receptor expression is decreased in the CB of tg mice. Our hypothesis was that gene expression of L, N, R, & T type VGCCs will be upregulated and certain GABAA receptors subunits specific to CB will be downregulated in the CB and not the FB of tg mice. QPCR was performed on reverse transcript (cDNA) of RNA isolated from CB and FB tissue of 6mo old male wt, heterozygote (+/−) and homozygote (+/+) tg mice. VGCC α1A (P/Q type), α1B (N type), α1C (L type), α1E (R type), β1, β2, β3, and β4 subunits as well as GABAA α1, α6, β1, β3, γ2 and δ subunit expression was measured in CB and FB of mice. In the CB α1A, α1B, α1C, α1E subunit of the (+/−) tg mice were at control levels. The (+/+) tg mice showed a downregulation of the α1A, as expected, and an upregulation of the α1B. This suggests that the α1B subunit is compensating for the loss of α1A subunit expression as it has been shown previously. In the FB, (+/−) tg mice had all α1 subunits expressed similar to control, while the (+/+) tg mice displayed a downregulation. In the CB the β3 subunit for the (+/−) tg mice was upregulated. In the FB the β subunits were upregulated for the (+/−) tg mice, and no significant change was observed for the (+/+) tg mice. Expression of GABAA α1, α6, γ2 and δ was increased in the CB of (+/−) and (+/+) tg mice. However in the FB there was no significant difference in expression of GABAA receptor subunits in the (+/−) compared to control; there was a downregulation of the γ2 subunit in the (+/+) tg mice. Our results demonstrate that VGCC and GABAA receptor subunit expression is altered as a result of the homozygous mutation of the α1A subunit of the VGCC. These results suggest that alterations in expression of these nerve membrane proteins can be contributing to the observed alterations in synaptic transmission and phenotypic changes observed in the tg mouse model.

Support or Funding Information

Supported by NIH NIEHS grants R25ES025060, T32ES007255 and R01ES024064.
Homo‐Oligomerization of Regulator Of G Protein Signaling 7 (RGS7)
Authors: Slepak Vladlen Z , Tayou Junior , Wang Qiang , Pronin Alexey
Published: November 2023

RGS proteins belonging to the R7 subfamily (RGS6, 7, 9 and 11) are highly expressed in the CNS neurons and to a much lesser extent in glands and heart where they regulate many physiological processes. R7 proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5, which is essential for the stability of these complexes. They also interact with other proteins such as R7BP (R7‐binding protein), R9AP (R9‐anchoring protein) and the orphan G protein coupled receptors, GPR158 and GPR179. These interactions modulate the function of R7 proteins, facilitate their stability and targeting to plasma membranes. In neurons and transfected cells, RGS7 localizes not only to the plasma membrane but also to cytoplasmic granules where its function is unknown. Here, using chemical cross‐linking, immunoprecipitation and mass spectrometry approaches, we sought to identify new binding partners of granular RGS7. We found that RGS7 can exist as a homo‐oligomer that after cross‐linking has the apparent molecular weight of approximately 150 kDa upon resolution by SDS PAGE. Formation of this oligomeric complex was confirmed by co‐immunoprecipitation assays with or without cross‐linking. The RGS7‐RGS7 interaction requires the DEP domain, but not the RGS, DHEX domains or the obligatory subunit of RGS7, Gβ5. We also demonstrated that oligomerization of RGS7 was inhibited in the presence of R7BP or constitutively activated Gαo. However, GPR158 could bind to the oligomeric form of RGS7. While oligomerization of other proteins in G protein signaling (e.g., GPCRs or arresting) is known, or results for the first time show the existence of RGS protein homo‐oligomers.

Support or Funding Information

This work is supported by NIH Grant R01DK105427 (VZS)
The IBMX‐insensitive PDE8A is expressed in human airway smooth muscle cells and selectively regulates signaling through AC6
Authors: Hill Kaitlyn , Li Fengying , Bogard Amy S. , Ostrom Rennolds S.
Published: November 2023

The second messenger cAMP is produced by several different adenylyl cyclase (AC) isoforms in human airway smooth muscle (HASM). We previously defined two distinct cAMP signaling compartments in these cells, one containing AC6 and another containing AC2 (Naunyn‐Schmiedebergs Arch Pharmacol, 387(4):329–39, 2014). We hypothesized that phosphodiesterase (PDE) isozymes may also be compartmentized in HASM. We detected mRNA and protein expression of the IBMX‐insensitive PDE8A in cultured HASM cells. Using lentiviral delivery of shRNA we knocked down PDE8A expression and measured cAMP levels stimulated by forskolin. The broad‐spectrum PDE inhibitor, IBMX, was included in these assays to eliminate contributions by most other PDE isoforms. 1 μM forskolin stimulated 2‐fold higher cAMP in HASM with PDE8A knockdown than in cells treated with control lentivirus. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased this response an additional 80%. We used TaqMan PCR to quantify the expression of AREG (a gene whose expression is regulated by cAMP from both AC2 and AC6) and NR4A2 (a gene regulated only by cAMP from AC6). PDE8A knockdown increased forskolin‐stimulated AREG and NR4A2 expression in AC6 overexpressing HASM but had no effect on either of these genes when AC2 was overexpressed. Quantitative phosphoproteomic analysis of whole cell lysates show that forskolin treatment causes PDE8A phosphorylation in HASM but that only AC6 overexpression increased this phosphorylation. To determine receptor‐mediated signaling regulated by PDE8A, we examined cAMP production in response to isoproterenol (β2AR, which couple primarily to AC6) and butaprost (EP2R, which couple to only AC2). PDE8A knockdown did not alter butaprost‐stimulated cAMP in HASM overexpressing AC2, consistent with no role of this PDE isoform in AC2‐mediated cAMP signaling. Isoproterenol‐stimulated cAMP was reduced by 70% in HASM overexpressing AC6, as effect opposite of what was predicted based on forskolin‐mediated responses. We conclude that PDE8A selectively hydrolyzes cAMP produced by AC6 with no effect on cAMP emanating from AC2. However, knockdown of PDE8A expression causes an uncoupling of β2AR to cAMP production through an undetermined mechanism.

Support or Funding Information

Supported by NIH (GM107094) and AHA (14GRNT20380762)
Evaluating the Specificity and Potency of Activators and Inhibitors of the cAMP Sensors PKA, Epac2/Rapgef4, and NCS/Rapgef2 Using Cell‐Based Assays for Activation of CREB, p38 MAPK, and ERK
Authors: Emery Andrew C , Eiden Maribeth V , Eiden Lee E
Published: November 2023

Cyclic AMP elevation is well known to cause dividing neuroendocrine (e.g. PC12) cells to differentiate through a process that includes growth arrest, neurite extension, and induction of neuronal genes. Using the PC12 subclone NS‐1, we have previously shown that downstream of cAMP, signaling for differentiation is mediated by three insulated signaling pathways, initiated by cAMP activation of Epac2/Rapgef4, NCS/Rapgef2, and PKA, which regulate the activation of p38 MAP kinase, ERK, and CREB, respectively. These, in turn, are individually necessary for growth arrest (Epac2/Rapgef4), neurite extension (NCS/Rapgef2), and neuron‐specific gene expression (PKA) (Emery et al., 2014, J Biol Chem, 289:10126–39). Specific inhibitors and activators of PKA, Epac, and NCS/Rapgef2 would be useful to further explore the unique physiological functions of these three cAMP sensors in the CNS. Several compounds that show differential activity with respect to PKA and Epac exist, however, the selectivity of these agents across all three neuroendocrine cAMP sensors is not yet known. The goal of the work reported here was to identify cAMP analogs that provide specific activation and inhibition of each of these cAMP sensors. To this end, we have used a medium‐throughput assay platform that allows for quantitative measurement of the phosphorylation status of ERK, CREB, and p38 MAPK, which serve as read‐outs for activation of NCS/Rapgef2, PKA, and Epac2, respectively. 8‐CPT‐cAMP was found to be a full agonist with a similar potency at each sensor, while 8‐CPT‐2′‐O‐Me‐cAMP was highly specific for Epac relative to PKA (as reported by Enserink et al., 2002, Nat Cell Biol, 4:901–6) and also NCS/Rapgef2. We examined previously characterized PKA site‐specific agonists, i.e. compounds that stimulate PKA holoenzyme activity only when applied in combination, to determine if any of these provide SAR entree to further development of Epac or NCS/Rapgef2‐specific agonists. Our preliminary evidence suggests that several B site‐specific compounds, when administered singly, show significant activity at NCS/Rapgef2 without stimulating either Epac or the PKA holoenzyme. None of the cyclic nucleotide antagonists (Rp diastereomeric cAMP analogs) that we tested effectively inhibited cAMP‐dependent NCS/Rapgef2 signaling. Based on our previous work indicating that the nucleoside adenylate cyclase inhibitor SQ22,536 inhibits NCS/Rapgef2 (Emery et al., 2013, Mol Pharmacol, 83:95–105), along with new insights into structure‐activity relationships for NCS/Rapgef2 gleaned from our survey of B‐site‐specific activators, we propose that the development of a cell‐permeant, highly specific inhibitor of NCS/Rapgef2 is feasible.

Support or Funding Information

Supported by NIMH‐IRP project ZO1‐MH002386
Evaluations of the Inhibitory effects of Psoralea Corylifolia Benzopyrone Derivatives in Human Monoamine Oxidase A and B
Authors: Zarmouh Najla O , Soliman Karam F.
Published: November 2023

The classical inhibitors of monoamine oxidases (MAOs) are effectively used in the therapeutic management of neurological disorders with rare, but serious, side‐effects. In our investigation for safe and natural MAO‐B inhibitors, Psoralea corylifolia Linn seeds (PCS) ethanolic extract (PCSEE) and seven of its benzopyrone constituents were investigated for their recombinant human (h) MAO‐A and B inhibition and selectivity. The extensively extracted PCSEE selectively inhibited MAO‐B (IC50 of 3.03 μg/ml) more than A. PCS benzopyrones showed weak to potent hMAO‐A and B hydrogen peroxide (H2O2) byproduct inhibition. MAO‐A inhibition potencies of PCSEE common compounds were in biochanin‐A (BIO‐A) > isopsoralen > genistein > 6‐prenylnaringenin > neobavaisoflavone > psoralen. Meanwhile, MAO‐B inhibition potencies were in BIO‐A > genistein > 6‐prenylnaringenin > isopsoralen > neobavaisoflavone > psoralen. Psoralidin was inactive in both isozymes. Compared to PCSEE and its tested benzopyrones, BIO‐A and genistein, showed the most potent hMAO‐A and B inhibition, equal or more potent than PCSEE. Furthermore, the most potent MAOI, BIO‐A, reversibly inhibited both isozymes. BIO‐A reversibility was supported by docking and kinetics studies. BIO‐A was predicted to form reversible interactions with FAD of MAO‐A, and with threonine 201 and p phenylalanine 168 in MAO‐B active site. Michaelis‐Menten kinetics studies also showed a competitive mode of BIO‐A inhibition on both isozymes. The obtained data indicate that PCSEE benzopyrones may contribute to its MAOs inhibitory activities and could be utilized as a natural neuroprotective source in neurological disorders.

Support or Funding Information

Supported by NIH grants from NIMHD G12 MD007582 and P20 MD006738
Effect of Hibiscus sabdariffa Calyx Extract on Erythrocytic Indices in Sickle Cell Anemia in Vitro
Authors: Mojiminiyi Frank Babatunde , Pereye Blessing , Ndakotsu Mohammed , Ndodo Darlington , Ikhuenbor Dorcas , Igbokwe Vincent
Published: November 2023

This study tested the antisickling properties of Hibiscus sabdariffa calyx extract (HS) in vitro. Following ethical approval, 5 ml of venous blood was collected from SCA patients and baseline haematological indices were determined. The blood was divided into 4 groups. To the first group, sodium metabisulphite (Na2S2O5) was added to the blood and then incubated with 0.l, 1 and 10mg/ml of HS (reversal assay). To the second group, the blood was incubated with different concentrations (0.l, 1 and 10mg/ml) of HS extract before adding Na2S2O5, and then haematological indices were determined (protective assay). The third group was incubated with 0.5 mg/ml Hydroxyurea and the fourth with 0.9% NaCl. Experiments were performed in accordance with the Principles of the Declaration of Helsinki. Results are expressed as mean±SEM. Statistical Analyses were done using ANOVA, and a post hoc Dunnett's test. P<0.05 was considered significant. In the protective assay, there was a dose dependent significant decrease in MCHC in the HS treated groups (36.64±0.47g/dL in both 0.1mg/ml and 1mg/ml and 31.57±1.65g/dL 10mg/ml) compared to control (37.76±0.35g/dL; P=0.0017). In the reversal assay, HS had a dose dependent decrease in MCHC (34.98±0.61g/dL, 35.22±0.65g/dL and 33.08±2.08g/dL in 0.1,1 and 10mg/ml respectively) compared to control (37.76±0.35g/dL; P=0.0021). However, HS had no significant effect on MCV, RDW and MCH. These results suggest that HS may have antisickling properties.
Natural compounds ursolic acid and isoliquiritigenin target GRP78 to enhance human gastric cancer cell chemosensitivity by 5‐fluorouracil
Authors: Lin Mingwei , Wu Dengchyang , Huang Yawbin
Published: November 2023

Gastric cancer is one of the most common malignancies worldwide, accounting for nearly half of cancer‐related mortality. Chemotherapy is the treatment of choice for gastric cancer, but the currently available therapeutic drugs for the treatment have limited efficacy. Recent studies suggested that gastric cancer stem cells may play an important role in drug resistance of chemotherapy. Therefore, new agents that selectively target gastric cancer stem cells in gastric tumor are urgently needed. Regarding to this concept, natural products may gain considerable important in this area for both cancer cell and cancer stem cell targeting. Ursolic acid (UA), a pentacyclic triterpenoid isolated from Catharanthus trichophyllus roots, and isoliquiritigenin (ISL), a chalconetype dietary compound derived from licorice, have been reported to process anti‐tumor properties. However, the potential role of UA and ISL in the improvement of chemotherapy sensitivity of gastric cancer has not been evaluated. The data from our study indicated that UA and ISL promoted human gastric cancer MKN45 cell apoptosis, and enhanced chemosensitivity with combination of 5‐fluorouracil by elimination of endoplasmic reticulum chaperone protein, glucose‐regulated protein 78 (GRP78), and down‐regulation of cancer stem cell surface marker, adenosine triphosphate‐binding cassette subfamily G2, LGR5, CD24 and CD44. UA not only induced excessive ER stress in cancer cell, but also inhibited the expression of GRP78 by down‐regulation of the transcription factor YY1. Taken together, UA and ISL may be suggested as promising agents in anti‐gastric tumor therapy.

Support or Funding Information

This work was supported by the slow aging program, “Aim for the Top Universities Grant” (KMU‐TP104G00 & KMU‐TP104G01) at Kaohsiung Medical University, and the Ministry of Science and Technology of Taiwan, MOST, 104‐2314‐B‐037‐003.
Biochemical and cellular monitoring of the activity of the ecto‐5′‐nucleotidase (CD73), a key cancer modulator using HTS‐formatted Bioluminescent Technology
Authors: Goueli Said
Published: November 2023

The ecto‐5′‐nucleotidase (CD73) is glycosyl phosphatidylinositol (GPI) anchored cell surface protein that is involved in switching on adenosinergic signaling. Its enzymatic and nonenzymatic activities (via its interaction with extracellular matrix components) are involved in cancer associated processes and not completely independent of each other. It catalyzes the hydrolysis of AMP into adenosine and phosphate, where adenosine plays important role in tumor immune escape. It is overexpressed in many types of cancer cell lines and patient's biopsies including breast, colon, ovarian, gastric, ovarian, etc. In addition to being important as clinical and prognostic marker in cancer patients, overexpression of CD73 is associated with resistance to antitumor agents, and inhibition of CD73 activity or knocking it down by SiRNA reversed chemoresistant phenotype of glioblastoma multiforme cells. Because of the significant role of CD73, it is of importance to develop an assay that monitor the activity of CD73 in order to develop modulators of its activity. Towards this goal, we have developed a bioluminescent assay to monitor the activity of the enzyme in biochemically pure as well cellular anchored enzyme forms. The assay is homogenous and formatted for HTS screening research, very sensitive, and robust as indicated by the high Z’. We also show that the use of inhibitors such as adenosine 5′‐(a‐b‐methylene) diphosphate (APCP) generates data with biochemically pure enzyme similar to the cellular bound form. We also demonstrate that cells that are enriched in CD73 can be easily identified from those that have low CD73. Thus, the current assay is robust, sensitive, and easy to use making it an ideal assay for HTS screening for new modulators of CD73 and generation of potential novel cancer therapeutics.

Support or Funding Information

Promega Corp.
Enhancing Molecular Targets in Cancer through the use of Very Low Level Radiation
Authors: Branly Rolando , Oseni Saheed O , Kumi‐Diaka James , Tulp Orien
Published: November 2023

Some recent studies show increased levels of apoptosis in cancer cell cultures utilized for phytonutrient effect studies when a low dose of X‐rays is initially given. We consider some of the effects of electromagnetic radiation in the production of intra‐cellular acoustical waves and the implications for altered probabilities of Biochemical Pathways. Monte Carlo simulation methods are employed with initial coarse grid models based on in vitro studies using LNCaP and PC3 cancer cell lines in combination therapy studies with X‐rays. Very Low Dose Radiation with X‐rays appears to enhance apoptosis mechanisms in the laboratory. The models presented serve as useful tools for temporal effect studies.

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We are thankful and acknowledge institutional support
Overcoming Chemoresistance in Pancreatic Cancer by Targeting the Anti‐Apoptotic Mcl‐1 Protein
Authors: Badejo Adeleke , McCreary Jordan , True Heather , Weisenburger Alexis , Davis Madison , Malhotra Ashim
Published: November 2023

Pancreatic cancer is highly aggressive with high mortality and poor 5‐year prognosis. Treatment is challenging due to substantial chemoresistance. The ability of cancers to limit the apoptotic response to drug treatment emphasizes the urgent need to develop drugs that can overcome chemoresistance. Overexpression of the anti‐apoptotic Mcl‐1 protein is associated with the development of chemoresistance in some cancers. Using the human pancreatic cancer cell line MIA PaCa‐2, we investigated the effect of the drug raloxifene on the regulation of Mcl‐1 in this model. The rationale for the use of raloxifene was the observation that it can cause apoptosis in cancer cell lines. We treated MIA PaCa‐2 cells with 0.05, 0.5, 5, and 20 μM raloxifene for 72 hours, followed by the preparation of whole cell lysates, which we used for Western Blot analysis. Our data show that: 1) raloxifene disrupted the protein‐protein interaction between the anti‐apoptotic Mcl‐1 and the pro‐apoptotic Bax, suggesting Bax homodimerization at the mitochondria, and possible activation of the apoptotic or autophagy response. To explain this, we investigated 1) the effect of raloxifene on the protein expression of the alternative splice variants of Mcl‐1, Mcl‐1L or Mcl‐1S; and, 2) whether raloxifene‐induced protein mistrafficking of Mcl‐1. Our data showed no changes in Mcl‐1L and Mcl‐1S, although levels decreased in the Bax/Mcl‐1 complex, as indicated by co‐immunoprecipitation experiments. 4) Additionally, we investigated the effect of raloxifene on global transcription by screening its effect on NFκB, Nrf‐2, eif‐2α, phospho‐cdc2, and cyclin proteins. While raloxifene treatment did not induce any of these transcription factors, at high doses it induced the expression of SRSF‐5, but had no effect on the expression of SRSF‐1, which are proteins that regulate the differential splicing of the Mcl‐1 gene. It also induced cyclin proteins. To further investigate the specific role of raloxifene in the turnover of Mcl‐1 protein, we pre‐incubated MIA PaCa‐2 cells with 10 μM cycloheximide, a global translation inhibitor. Our data showed a reduction in Mcl‐1 in raloxifene treated samples compared to control, suggesting a role for estrogen receptor β in the de novo expression of Mcl‐1. 6) Additionally, raloxifene treatment induced the autophagy markers Beclin‐1 and LCIII‐B, indicating a correlation between the differential regulation of Mcl‐1 and autophagy. Taken together, these data suggest that the main mechanism for the action of raloxifene is competitive displacement of Mcl‐1 from the Bax/Mcl‐1 protein complex.

Support or Funding Information

This research was supported in part by Pacific University Research Incentive Grant and Collins Medical Trust grant to AM.
Pathomimetic Avatars for Live‐Cell Imaging of Tumor Proteolysis and Drug Screening
Authors: Sloane Bonnie F. , Ji Kyungmin , Chalasani Anita , Moin Kamiar , Mattingly Raymond R , Xu Yong
Published: November 2023

To define protease‐related druggable pathways involved in malignant progression of cancer, our laboratory has pioneered novel techniques for functional live‐cell imaging of protease activity, initially concentrating on pathomimetic avatars for breast cancer. We analyze proteolysis in the context of proliferation and formation of structures by breast cells (benign and cancerous) in 3‐D cultures over time (4D). In order to recapitulate the cellular composition and architecture of breast tissue, we include other tumor‐associated cells (e.g., fibroblasts, myoepithelial cells, lymphatic endothelial cells). We also model non‐cellular aspects of the tumor microenvironment, e.g., an acidic pericellular pH. Use of these pathomimetic avatars in concert with various types of imaging probes has allowed us to image, quantify and follow the dynamics of proteolysis in the tumor microenvironment and to test interventions that impact directly or indirectly on proteolytic pathways. To facilitate use of the pathomimetic avatars for drug screening, we have designed culture chambers with multiple wells that are either individual or connected by a bridge to allow cells to migrate between wells. Optical glass microscope slides underneath an acrylic plate allow the cultures to be imaged with an inverted microscope. Fluid ports in the acrylic plate are at a level above the 3D cultures to allow introduction of culture media and test agents such as drugs into the wells and the harvesting of media conditioned by the cultures for immunochemical and biochemical analyses. Covers contain integrated gas exchange ports and sensors to monitor oxygen levels, pH and temperature over the extended time periods in culture and to insure maintenance of such experimental conditions as hypoxia and/or a pericellular acidic pH. We predict that the pathomimetic avatars will accelerate identification of druggable pathways, screening of drug and natural product libraries and the entry of validated drugs or natural products into clinical trials.

Support or Funding Information

Supported in part by National Institute of Health R21 CA 131990 (BFS) and the President's Research Enhancement Fund of Wayne State University (BFS and YX). Live‐cell imaging was performed in the Microscopy, Imaging and Cytometry Resources Core of Wayne State University, which was supported in part by National Institutes of Health P30 CA22453 to the Karmanos Cancer Institute and by the Perinatology Research Branch of the National Institute of Child Health and Human Development.
Transglutaminase 2 Plays a Critical, but Context‐Specific Role in Glioblastoma Proliferation
Authors: Monteagudo Alina , Gundemir Soner , Akbar Abdullah , Keillor Jeffrey , Johnson Gail
Published: November 2023

Glioblastoma multiforme (GBM) is the most aggressive and common form of adult primary brain tumors. GBMs are characterized by resistance to chemotherapy and radiation, and exhibit a high propensity for reoccurrence. Therefore, it is imperative to define molecular mechanisms that contribute to GBM tumor survival and proliferation. One possible protein that may contribute to GBM proliferation is the multifunctional protein transglutaminase 2 (TG2). TG2 has been shown to play a critical role in proliferation in other types of cancers, and therefore we investigated its role in GBMs. Given the heterogeneity of GBM tumors, we used various GBM cell lines as well as non‐serum containing GBM neurospheres to determine if the role of TG2 in GBMs was broadly applicable, or just relevant to certain populations. Additionally, the neurospheres used represent three different GBM subtypes: mesenchymal, proneural and classical. By the use of novel inhibitors and genetic manipulations of TG2, results show that in most, but not all, cell lines TG2 plays a critical role in the proliferative process of GBM cells. Given the heterogeneity of GBM tumors, it is not surprising that TG2 facilitates the proliferative process in only certain GBM subtypes. Nonetheless, as the understanding of signaling mechanisms that contribute to GBM proliferation increases, TG2 inhibitors may in the future be effective therapeutic agents for certain GBMs, as determined by specific genetic determinants.

Support or Funding Information

NIH RO1 NS065825
Pharmacological inhibition of soluble epoxide hydrolase ameliorates experimental acute pancreatitis in mice
Authors: Bettaieb Ahmed , Chahed Samah , Bachaalany Santana , Griffey Stephen , Hammock Bruce D. , Haj Fawaz G.
Published: November 2023

Background

Acute pancreatitis (AP) is an inflammatory disease, and is one of the most common gastrointestinal disorders worldwide. Soluble epoxide hydrolase (sEH; encoded by Ephx2) deficiency and pharmacological inhibition have beneficial effects in inflammatory diseases. Ephx2 whole‐body deficiency mitigates experimental AP in mice but the suitability of sEH pharmacological inhibition for treating AP remains to be determined.

Results

We investigated the effects of sEH pharmacological inhibition on cerulein‐ and arginine‐induced AP using the selective sEH inhibitor 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) urea (TPPU) which was administered before and after induction of pancreatitis. Serum amylase and lipase levels were lower in TPPU‐treated mice compared with controls. In addition, pancreatic mRNA of the inflammatory cytokines TNFα, IL‐1β and IL‐6 was reduced in TPPU‐treated mice. Moreover, sEH pharmacological inhibition before and after induction of pancreatitis was associated with decreased cerulein‐ and arginine‐induced NF‐κB inflammatory response and endoplasmic reticulum stress.

Conclusions

sEH pharmacological inhibition before and after induction of pancreatitis mitigated cerulein‐ and arginine‐induced AP. This work suggests that sEH pharmacological inhibition may be of value in the therapy of acute pancreatitis.

Support or Funding Information

This work was supported by research grants from the JDRF (1‐2009‐337) and NIH (R56DK084317, R01DK090492 and RO1DK095359) to F.G.H.; K99DK100736 to A.B. and by NIH ES02710 and Superfund Basic Research Program Grant P42 ES04699 from NIEHS, and HL059699 Grant from NHLBI and U24DK097154 (to B.D.H.).
The Cholesterol‐Lowering Effect of Statins is Potentiated by Whole Grains Intake. The Polish Norwegian Study (PONS)
Authors: Vaidean Georgeta , Manczuk Marta , Vansal Sandeep , Griffith Jacqueline
Published: November 2023

Introduction

Adequate whole grains (WG) dietary intake is advocated for cardiovascular health. Statins represent the cornerstone of medical control of hypercholesterolemia. Whether there is an interaction between WG intake and statins, has not been fully explored in ‘real life’ settings. Our objective was to assess the interaction between statin use and whole grains fiber intake on plasma lipids.

Methods

We used cross‐sectional data of 12754 participants, ages 45 to 64 years, in an ongoing cohort study in Poland. Statin use during the last 30 days was recorded by trained nurses. WG intake was estimated from a food frequency questionnaire assessing participants’ habitual food intake in the past year. Plasma total, LDL, HDL cholesterol, triglycerides and glucose were measured in a fasting state. Multivariable linear and logistic regression models were used for analyses.

Results

There were 1728 (13.55%) statin users. High WG grain intake (at or above the recommended levels of ≥3 oz/d) was achieved by 2835 (22.23%) participants. Those taking statins, had lower mean (95%CI) plasma lipid levels if they had a high WG intake, even after adjusting for age, sex, education, total caloric intake, various dietary nutrients intake, physical activity, smoking, glucose levels, BMI and prevalent CVD: Cholesterol: 185.13 mg/dL (181.65–188.60) vs. 190.14 mg/dL (188.02–192.27); non‐HDL cholesterol: 127.26 mg/dL (123.83–130.71) vs. 133.02 mg/dL (130.92–135.12); LDL: 104.42 mg/dL (101.37–107.45) vs. 108.54 mg/dL(106.69–110.40). High vs. low WG intake did not have an effect among those not taking statins, nor on triglyceride or HDL levels. In fully adjusted models, statin users with a high WG intake had 33% lower odds of having a cholesterol level ≥ 240 mg/dL and 38% lower odds of having a non‐HDL cholesterol ≥130 mg/dL, compared to statin users with low WG intake.

Conclusion

We found a potentiation of effect of statins by whole grain dietary intake on plasma lipid levels. Given that the current paradigm on controlling plasma lipids is that “lower is better” and that the uptitration of statins can be potentially toxic, the interaction with the dietary intake of WG has potential clinical significance.

Support or Funding Information

FINANCIAL DISCLOSURES: The data collection was supported by a grant from the Polish‐Norwegian Research Fund (PNRF‐228‐AI‐1/07).