bluuebunny/crossref_metadata_2025 huggingface dataset:
The Impact of Virgin Coconut and Safflower Oils on Lipids and Cytokines in Postmenopausal Women
Authors: Harris Margaret , Fryda Lisa , Hutchins Andrea
Published: June 2021

12 women (58.8+3.7 yr, 26.4+4.6 kg/m2) were recruited for a randomized cross‐over study. They consumed 30g virgin coconut oil (CO) or high‐oleic safflower oil (SO) for 28 days (28‐d washout period between oils, 28 d of food records). Diet was not altered. A lipid panel and cytokines (proinflammatory: IL‐1β, IL‐6, and TNF‐α & anti‐inflammatory: IL10) were obtained pre/post each oil intervention after 12‐hr fast. Data were analyzed using SPSS. MIXED ANOVA showed no intervention effect. Comparisons were analyzed using paired t‐test. Results showed CO significantly raised total cholesterol, TC (+17.0+22.0 mg/dL), low‐density lipoprotein, LDL (+12.1+16.0 mg/dL) & high‐density lipoprotein, HDL (+6.3+7.1 mg/dL) (all p<0.05) but lowered triglycerides, TG (‐8.6+32.1 mg/dL, p=NS). SO lowered TC (‐5.2+15.7 mg/dL), LDL (‐4.9 + 12.5 mg/dL), HDL (‐1.9+5.3 mg/dL), and increased TG (+8.0+48.4) but changes were not significant. TC and HDL were significantly different between test oils, p<0.05. The TC/HDL ratio change showed a neutral effect of both CO and SO, Δ pre/post each= 0 mg/dL. Cytokines results varied. One person had adverse reactions to CO & increased inflammation. CO decreased IL‐1β (cytokine indicative for neurological degeneration) for each person who had a detected sample. CO and SO varied in their impact on other cytokines on an individual basis, some showing increased inflammation, others decreasing inflammation in no consistent manner. Both CO & SO had a neutral impact on lipids. The varied nature of inflammatory markers suggest that there may be epigenetic interactions. More research needs to be conducted to evaluate LDL particle size. Larger sample sizes are needed to determine impact on cytokines before recommendations can be made.
Phenotypic Diversity of Anthocyanins in 25 Sorghum Accessions
Authors: Su Xiaoyu , Rhodes Davina , wang weiqun , Yu Miao , Katz Benjamin , Tomich John
Published: June 2021

Anthocyanins, a sub‐class of flavonoids, are natural pigments known as functional health benefits. Sorghum (Sorghum bicolor) is a rich source of various phytochemicals including anthocyanins. In this study, 25 sorghum accessions with a wide variety of pigmented pericarp were grown in Florence, South Carolina during 2012. The profile of anthocyanins were identified and quantitated by HPLC‐MS/MS. A total of 11 anthocyanins were identified and detected in sorghum bran. The predominant anthocyanins found in sorghums were 3‐deoxyanthocyanidins including the unique apigeninidin and leuteolinidin. The high levels of anthocyanins were found in eight accessions with purple or red pericarp (PI221723, PI297139, PI35038, PI229830, PI221610, PI533902, PI229838, and PI533957), while anthocyanins are undetectable in the sorghums with white‐yellow bran (PI65007, PI561072 and PI565121). Furthermore, total anthocyanin contents at apigeninidin equivalent in each accession were quantitated. Establishing a database of anthocyanin profile in various sorghum accessions may lead to the development of novel functional sorghum products for human health benefits (supported by USDA Cooperative KS511‐1001903).
Effect of Green and Black Tea Extracts on Intestinal Microbiota and Body Composition in Mice Fed a High Fat/High Sucrose/Western Diet
Authors: Henning Susanne , Yang Jieping , Hsu Mark , Heber David , Li Zhaoping
Published: June 2021

The gut microbiota is an important contributor to human health and has been implicated in the development of obesity and obesity‐related diseases such as diabetes and cardiovascular disease. The consumption of tea is of great interest due to its effect on weight control, inflammation and glucose metabolism. It is our hypothesis that alterations of the gut microbiota in part contributes to the effect of tea on weight control, inflammation and glucose metabolism. 48 C57BL/6 male mice were randomly assigned to one of four treatment groups: control high fat (HF)/high sucrose (HS)/Western diet (32% kcal from butter fat and corn oil, 25% kcal from sucrose), a HF/HS‐diet supplemented with either GT or BT extract providing 0.25% polyphenols or a low fat diet (10.6% kcal from fat) for 4 weeks. All observations are in comparison to the HF/HS‐diet fed control mice. GT and BT administration decreased body weight, epidydimal, mesenteric and subcutaneous fat as determined by weight. In the cecum both GT and BT treatment significantly increased cecum weight normalized to body weight and total cecum DNA content. As determined by qPCR and MiSeq in the cecum and duodenum GT and BT treatment increased the phyla Bacteroidetes (genera Parabacteroides ssp‐Prevotella ssp) significantly and decreased the phyla Firmicutes (genus Roseburia), and Firmicutes/ Bacteroidetes ratio. In addition the effect of tea consumption on Lactobacillus varied by gut location. GT treatment was associated with an increase in Lactobacillus spp in the duodenum, while Lactobacillus spp was decrease significantly by GT and BT in the cecum. The shift in the ratio of Firmicutes/Bacteroidetes may have contributed to weight loss and change in body composition.
DNA Damage Inhibitory Activities, Anti‐acetylcholinesterase, Anti‐procine α‐Amylase and Antioxidant Properties of Dolichos Lablab Bean
Authors: Theuri Serah , Habib Hosam , Mohamed Fedah
Published: June 2021

This underutilized variety of Dolichos lablab bean seed serves as a good source of natural antioxidants, which may be effective in reducing the risk of occurrence of certain diseases. This study was undertaken for the first time to address the limited knowledge regarding the, antioxidant activities, DNA damage inhibitory activity, bovine serum albumin (BSA) damage inhibitory activity, anti‐acetylcholinesterase, and anti‐porcine α–amylase in the lablab bean seed. The antioxidant capacity of lablab bean seeds were evaluated for their potential antioxidant activities using the ferric‐reducing/antioxidant power assay (FRAP), free radical‐scavenging activity (DPPH), nitric oxide (NO) radical‐scavenging assay, and 2,20‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS). The lablab bean extract exhibited good antioxidant activity by effectively scavenging FRAP, DPPH, NO, and ABTS radicals. Additionally, excellent protective effects against BSA damage, as well as natural α‐amylase inhibitors were also shown. The bean extract had protective effects against DNA damage. The lablab bean may have a future role as an ingredient in the development of functional foods
Functional Studies of CtBP in Breast Cancer Using the Lentiviral pINDUCER System
Authors: Yi Dae Ik , Park Sam , Liang Genqing , Yan Tingfen , Byun Jung , Gardner Kevin
Published: June 2021

As a transcriptional corepressor, CtBP plays a significant role in tumorigenesis and tumor progression through epigenetic regulation. Interacting with transcription factors and chromatin modifying enzymes, CtBP mediates repression of tumor suppressor genes that maintain genome stability, repress cellular proliferation and inhibit EMT. This contributes to the evolution of more aggressive forms of multiple epithelial malignancies, including breast cancer. CtBP is also considered a metabolic sensor due to its ability to bind to NADH, the high energy intermediate generated during carbohydrate metabolism. However, despite its potential role in tumor progression and its link to metabolic imbalance, a detailed mechanism explaining how CtBP promotes aggressive breast cancer behavior is unclear. Thus, we are employing an inducible gene expression approach that utilizes the lentiviral pINDUCER system to inducibly manipulate the levels of CtBPs using Doxycycline. The fidelity of this system allows us to characterize CtBP‐regulated pathways including EMT, invasion, metastasis, genome instability, and uncontrolled self‐renewal. To explore the role of CtBP in increasing cancer malignancy, we are implementing a broad array of biological functional assays including wound healing migration assays, Boyden chamber invasion assays, and 3D gland formation assays. We are also conducting clonogenic assays, comet assays, gamma‐H2AX foci assays to further understand how CtBP drives genome instability and activates self‐renewal pathways. Eventually, we will develop orthotopic xenograft mouse models to study breast cancers in vivo. Together, the functional assays manipulated by the pINDUCER system will shed light on the role of CtBP in breast tumorigenesis and progression.
Maresin 1: a Novel Macrophage Derived Endogenous Proresolving Inhibitor of Primary Tumor Growth and Metastasis
Authors: Lehner Kristen , Sulciner Megan , Ramon Sesquile , Mudge Dayna , Huang Sui , Serhan Charles , Panigrahy Dipak
Published: June 2021

Background

Chemotherapy and radiation reduce tumor burden by killing tumor cells, but as a consequence, create tumor cell debris. The debris of dead cancer cells acts as a source of tumor‐stimulation, via inflammation, on the few surviving cells contributing to tumor relapse. Thus chemotherapy may be a doubled‐edged sword. Maresin 1 (MaR1), a key mediator of inflammation resolution, is biosynthesized by human macrophages from endogenous docosahexaenoic acid. We hypothesize that maresins represent a novel modality in cancer treatment by inhibiting tumor growth and metastasis by enhancing endogenous clearance of tumor debris by macrophage phagocytosis.

Results

Flow cytometry confirmed chemotherapy and targeted therapy (tamoxifen)‐induced apoptotic tumor cell debris. MaR1 stimulated macrophage phagocytosis of chemotherapy‐induced cell debris (e.g. breast carcinoma and melanoma) by 123% to 206%. Systemic administration of MaR1 inhibits primary tumor growth by promoting the resolution of inflammation without toxicity.

Conclusions

We demonstrate for the first time that maresins inhibit primary tumor growth and metastasis via the stimulation of macrophage phagocytosis of chemotherapy‐induced tumor cell debris. Thus, the MaR1 pathway or the enhancement of endogenous resolution processes, offers an entirely novel approach for clearing tumor cells debris induced by conventional cancer therapy.
Comparative Studies on the Molecular and Functional Profile of Sheep Mucosal Derived Enoxaparin
Authors: Abro Schuharazad , Iqbal Omer , Kahn Daniel , Syed Daneyal , Hoppensteadt Debra , Yao Yiming , Jin Yong Sheng , Fareed Jawed
Published: June 2021

Enoxaparin is a Low Molecular weight heparin which is derived from Porcine mucosal tissues. Enoxaparins derived from Bovine and Sheep Mucosal heparin have become available. This study compared 3 sheep enoxaparin preparations with varying amounts of anhydromanno contents with branded enoxaparin in routinely carried out biological assays.

Enoxaparins of sheep origin were obtained from Ronssi Pharmaceuticals China, Branded enoxaparin was obtained commercially. Normal human plasma was supplemented with each of these agents individually to obtained final concentrations of 0‐10ug/mL to profile the anticoagulant and anti‐protease activities. Protamine neutralization studies were carried out. HIT antibody mediated platelet aggregation studies were also carried out.

The molecular profile of the individual sheep enoxaprins irrespective of the anhydromanno component was comparable to branded enoxaparins (4.1‐4.3kDA vs 4.2kDA). In the global tests such as the aPTT, Heptest and Thrombin time both the branded and sheep heparin provide comparable results. Similarly in the protamine neutralization comparable neutralization profiles were noted in different tests. In the Anti‐xa assay the relative potency for the sheep enoxaparin (98‐104U/mg) compare to branded (99U/mg). In the HIT studies all agents exhibited comparable responses.

These results indicate that the sheep mucosal heparin derived enoxaparin exhibit comparable molecular profile and biological activities in the laboratory assays. Based on these studies it is concluded the sheep mucosal heparin is comparable to porcine mucosal heparin derived enoxaparin.
Just Say Know to Drugs!: A Pilot Study for High School Student Science Enrichment
Authors: Akers James , Thompson Katie , Carrier Raeann
Published: June 2021

The purpose of the Just Say Know to Drugs! camp was twofold: our primary goal was to increase student understanding of basic science and pharmacology concepts and our secondary goal was to develop student self‐assurance in the sciences. The program introduced the fundamentals of pharmacology through the study of basic science. Different teaching modalities were utilized to keep learning fun and engaging. The camp included a pre‐test given on the first day and an identical post‐test administered on the last day. An interest survey was included in the post‐test. Students were given one hour to complete the tests which contained a series of basic science and pharmacology questions. The results of the tests and survey were analyzed using paired t‐tests to determine the impact of the camp on participant knowledge and performance. The post‐test revealed significantly higher scores in biology, chemistry, and pharmacology and the test overall (p < 0.05). Furthermore, survey data revealed that the camp increased student knowledge regarding pharmacology and drugs of abuse. Survey questions also highlighted student confidence in their current understanding of and progress through their high school science courses. The data collected are indicative of the success of this pilot program. Given these findings, we intend to continue educating students about pharmacology using this model. Additional studies will evaluate whether students take this educational success and interest further, choosing to direct their focus on pharmacology or other related fields. This project was funded by the WVSOM RHI and South East AHEC.
Carborane Derived Local Anesthetics Show Isomer‐Dependent Analgesia
Authors: Kracke George , VanGordon Monika , Kabytaev Kuanysh , Sevryugina Yulia , Kueffer Peter , Jalisatgi Satish , Hawthorne M.
Published: June 2021

Clinically there is a need for local anesthetics with a greater specificity of action and longer duration. We have synthesized a series of local anesthetic derivatives we call boronicaines in which the aromatic, phenyl ring of lidocaine was replaced with ortho‐, meta‐, dimethyl meta‐ and para‐carborane clusters. The boronicaine derivatives were tested for their analgesic activity and compared to lidocaine using standard procedures in mice following a plantar injection. The compounds differed in their analgesic activity in the following order: ortho‐carborane = dimethyl meta‐carborane > para‐carborane > lidocaine > meta‐carborane derivative. Both ortho‐boronicaine and dimethyl meta‐boronicaine had longer durations of analgesia than lidocaine. Differences in analgesic efficacies are rationalized by variations in chemical structure and protein binding characteristics. Financial support to G.R.K. from the University of Missouri Intellectual Property Fast Track Funding Program is acknowledged.
Effects of Isoflurane on Dopamine Synaptic Vesicle Exocytosis
Authors: Bonvicino Christina , Zhou Zhenyu , Hemmings Hugh
Published: June 2021

Although volatile anesthetics are extensively used in clinical medicine and animal research, their exact mechanism of action is not completely understood. Previous results from our laboratory show that isoflurane, a commonly used volatile anesthetic, attenuates synaptic transmission by differentially inhibiting synaptic vesicle exocytosis. Most research has been focused on glutamatergic and GABAergic neurons, while little is known of other types of neurons. Dopaminergic neurons play important roles in cognition, attention, reward, and movement, and have been implicated in emergence from anesthesia. Here we test the hypothesis that isoflurane inhibits synaptic dopamine release. Live‐cell imaging of cultured rat dopaminergic neurons were used to study action potential‐evoked exocytosis and Ca2+ influx measured by using optical methods. Neurons were transfected with a pH‐sensitive variant of GFP (pHluorin) fused to the luminal domain of the vesicular monoamine transporter (vMAT) to monitor exocytosis, or were treated with the cell permeant calcium indicator Fluo‐5F. The roles of specific Na+ and Ca2+ channel subtypes mediating the inhibition of Ca2+ influx and exocytosis are assessed using both pharmacological and genetic approaches for comparison to the effects on other synaptic vesicle phenotypes. Preliminary evidence indicates that clinical concentrations of isoflurane reduce Ca2+ influx and synaptic vesicle exocytosis. The goal of this work is to discern the molecular targets for the inhibitory effects of isoflurane on synaptic vesicle exocytosis and to elucidate additional presynaptic mechanisms of anesthetic action.

Research supported by grant GM 58055
Ferulic Acid Alleviates Neuropathic Pain: Involvement of Descending Monoaminergic System and Opioid Receptors
Authors: Xu Ying , Yu Yingcong , Wang Weijie , Cao Liang , Zhang Hanting , O'Donnell James
Published: June 2021

Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress‐related depression and respond to similar treatment. FA is a major active component of angelica sinensis and has been reported to exert antidepressant‐like effects; however, it remains unknown whether resveratrol ameliorate CCI‐induced neuropathic pain. Chronic treatment with FA (10, 20, 40 and 80 mg/kg) twice daily for 3 weeks ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal monoamine levels. Pharmacological depletion of spinal noradrenaline (NA) or serotonin (5‐HT) by 6‐hydroxydopamine (6‐OHDA) or p‐chlorophenylalanine (PCPA) completely blocked FA's effects on mechanical allodynia or thermal hyperalgesia, respectively. The anti‐allodynic action of FA on mechanical stimuli was prevented by co‐treatment with beta2‐adrenoceptor antagonist ICI 118,551, or by co‐treatment with delta‐opioid receptor antagonist naltrindole. Moreover, the anti‐hyperalgesia on thermal stimuli induced by FA was blocked by co‐treatment with 5‐HT1A receptor antagonist WAY‐100635, or with the irreversible mu‐opioid receptor antagonist beta‐funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via modulation of descending monoaminergic system that coupled with spinal b2‐ and 5‐HT1A receptors and the downstream opioid receptors.
Comparison of Opioid Antagonist Modulation of Buprenorphine‐Induced Neurochemical Changes
Authors: Eyerman David , Li Wen‐I , Cunningham Jacobi , Dean Reginald , Deaver Daniel
Published: June 2021

Objective

Evaluate differences in onset and duration of action of µ‐opioid antagonists on buprenorphine (BUP)‐induced elevations of nucleus accumbens shell (NAc‐sh) neurotransmitters.

Methods: Studies utilized male Wistar rats (275 ‐ 375g) with microdialysis probes inserted into the NAc‐sh (A/P +1.70; M/L ±0.8; D/V ‐7.8 from bregma). Bioanalytical analyses of dopamine (DA) and its metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were performed using HPLC‐EC. Data were converted to percent change from baseline and analyzed using a repeated measures two‐way ANOVA and mechanistic pharmacodynamic modeling.

Results: BUP (0.1 mg/kg) elevated extracellular concentrations of DA, DOPAC and HVA in the NAc‐sh for approximately 20 hours after subcutaneous administration. Co‐administration of either samidorphan (SAM) or naltrexone (NTX) (0.3 ‐ 3.0 mg/kg) dose dependently attenuated BUP‐induced dopaminergic activity. However, the temporal pattern of this attenuation was different for NTX and SAM. NTX displayed a rapid Kon and Koff resulting in an immediate but short‐lived (~4 hours) attenuation of BUP's effects. SAM displayed a longer Kon and Koff resulting in a protracted but longer‐lasting (~8‐10 hours) attenuation of BUP's effects.

Conclusions

Differences observed between SAM and NTX in onset and duration of action were not predicted by in‐vitro pharmacology or pharmacokinetic profiles. Importantly, the duration of action reported previously in humans for both antagonists is much longer than in rats. These data illustrate the importance of understanding the duration of action when assessing the activity of different opioid antagonists in nonclinical models.
Characterization of the behavioral effects of lorcaserin in rats
Authors: Serafine Katherine , France Charles
Published: June 2021

Lorcaserin was recently approved by the FDA for the treatment of obesity, and its therapeutic effects are thought to be due to agonist activity at serotonin (5‐HT)2C receptors. Lorcaserin also has affinity for other 5‐HT receptor subtypes, although its activity at those subtypes is not fully described. The current study examined the behavioral effects of lorcaserin (0.0032 – 32.0 mg/kg) in comparison to those of mCPP (0.032 – 1.0 mg/kg; 5‐HT2C receptor agonist) and DOM (0.1 – 3.2 mg/kg; 5‐HT2A receptor agonist) in 8 male Sprague Dawley rats. The behavioral profile of lorcaserin was similar to that of mCPP, in that both drugs dose‐dependently induced yawning and penile grooming. However, at some doses, lorcaserin (but not mCPP) also induced forepaw treading, an effect typically produced by 5‐HT1A receptor agonists. In contrast, DOM dose‐dependently induced head twitching, back contractions, and hyperthermia. Pretreatment with lorcaserin (3.2 mg/kg), mCPP (3.2 mg/kg), or the 5‐HT2A receptor selective antagonist MDL 100907 (0.01 mg/kg) attenuated DOM‐induced head twitching. Only lorcaserin significantly attenuated DOM‐induced hyperthermia. While the behavioral effects of lorcaserin are consistent with it having agonist activity at 5‐HT2C receptors, these data suggest that it likely also has actions at 5‐HT2A (e.g., antagonism) and 5‐HT1A (e.g., agonism) receptors. A better understanding of the activity of lorcaserin at these receptor subtypes might facilitate its use for treating other disorders (e.g., drug abuse).
Differential effects of the dopamine D3 receptor antagonist PG01037 on cocaine and methamphetamine self‐administration in rhesus monkeys
Authors: John William , Newman Amy , Nader Michael
Published: June 2021

Within the dopaminergic system, the DA D3 receptor (D3R) has been shown to mediate many of the behavioral effects of psychostimulants associated with high abuse potential. The present study sought to extend the assessment of the highly selective D3R antagonist PG01037 on cocaine and methamphetamine (MA) self‐administration to include a food‐drug choice procedure. Complete cocaine and MA self‐administration dose‐response curves were determined daily in each session for eight male rhesus monkeys (n=4/group). When choice was stable, monkeys received acute and five‐day treatment of PG01037 (1.0‐10 mg/kg, i.v.). Acute administration of PG01037 significantly reallocated choice behavior from cocaine to food and decreased cocaine intake, however, tolerance developed by day 5 of treatment. Up to doses that disrupted responding, MA choice and intake were not decreased following acute or 5‐day PG01037 treatment but the behavioral potency was significantly greater on MA choice compared to cocaine choice. Furthermore, using an unconditioned behavior, yawning elicited by the D3/D2R agonist quinpirole (0.003‐0.3 mg/kg) in these same monkeys, the acute efficacy of PG01037 was negatively correlated with the sensitivity to the behavioral effects of quinpirole. These data suggest (1) that efficacy of D3R compounds to decrease drug choice is greater in subjects with lower D3R, perhaps suggesting that it is percent occupancy that is the critical variable in determining efficacy and (2) differences in D3R activity in chronic cocaine vs. MA users. Supported by DA012460, AA007565, and NIDA‐IRP.
Effects of Intranasal Neuropeptide Y and HS014 in Preventing Anxiety and Depressive‐like Behavior Elicited by Single Prolonged Stress Animal Model of PTSD: Comparative Study
Authors: Sabban Esther , Serova Lidia , Alaluf Lishay , Laukova Marcela , Peddu Chandan
Published: June 2021

Stress triggered neuropsychiatric disorders are a serious societal problem. Prophylactic treatment or early intervention has great potential in increasing resilience to traumatic stress and reducing its harmful impact. Previously, we demonstrated proof of concept that intranasal administration of NPY or the MC4 receptor antagonist, HS014, prior to single prolonged stress (SPS) rodent PTSD model, can prevent or attenuate many PTSD associated impairments. Here, we compare effects of NPY and HS014 given 30 min before or immediately after SPS stressors on development of anxiety, depressive‐like behavior and associated biochemical abnormalities. SPS triggered anxiety on EPM was reduced by intranasal pre‐administration of 100 µg NPY and to even greater extent HS014 (3.5 ng or 100 µg). The SPS‐elicited depressive‐like behavior on FST was prevented with 100 µg NPY or the high dose HS014. Combined administration of low HS014 and NPY, ineffective by themselves, prevented development of depressive‐like behavior. Reductions in stress triggered activation of LC/noradrenergic system and HPA axis were observed with both HS014 and NPY. In contrast to NPY, early intervention with HS014 immediately after SPS did not prevent the development of anxiogenic behavior on EPM. However, HS014 given after SPS stressors, even at very low dose, prevented development of depressive‐like behavior. Thus, both MC4R antagonist and NPY, alone or combined, have potential for prophylactic treatment against traumatic stress triggered anxiety or depressive‐like behaviors, while NPY has more widespread potential for early intervention. (Supported by US Amy grant DM102281)
Behavioral, Cognitive and Biochemical Consequences of Witnessing Maternal Abuse: Insights from an Animal Model
Authors: Liu Hesong , Patki Gaurav , Salvi Ankita , Kelly Matthew , Salim Samina
Published: June 2021

Recent studies from our lab have shown that post‐traumatic stress disorder (PTSD)‐like behaviors are triggered not only in rats that physically experienced traumatic events of social defeat, but also in rats that witnessed their cage mates undergo traumatic exposure. Extending this line of research we have attempted to establish a rat model to study the consequences of early‐life exposure of witnessing maternal abuse (MAW). In this MAW model we introduced a female Spraugue‐Dawley rat (mother) into the cage of a male Long Evans (LE) rat. The female rat undergoes defeat from the LE rat. Natural litters at postnatal day 21 were placed in separate chambers surrounding the cage to witness 3 attacks per day, for 7 consecutive days. One month later, when the pups became 60 day old (considered adults), cognitive and behavioral outcomes of witnessing maternal abuse during early life were examined. MAW rats exhibited depression‐like behavior (examined via forced‐swim test) when compared to age matched controls. No changes were noted in anxiety‐like behavior (examined via open‐field and elevated plus maze tests) or learning and memory function (Radial arm water maze test) in MAW rats. Elevated levels of oxidative stress correlate with depressive phenotype of MAW rats. Studies to investigate the underlying neurobiology of MAW phenotype are currently ongoing.
Sigma‐1 receptor agonist and fluvoxamine rescue depressive behaviors in CaMKIV null mice
Authors: Fukunaga Kohji , Sakagami Hiroyuki , Moriguchi Shigeki
Published: June 2021

Sigma‐1 receptor (Sig‐1R) is a molecular chaperon regulating calcium transport from the neuronal endoplasmic reticulum to mitochondria (Shioda and Fukunaga, JBC 2012;287:23318). Recently Song et al (Int J Neuropsychopharmacol 2012;16:803) reported that CaMKIV null mice exhibit depressive behaviors and impaired neurogenesis as assessed by bromodeoxyuridine (BrdU) incorporation into newborn cells of the hippocampal dentate gyrus (DG). Moreover, the typical SSRI fluoxetine fails to induced DG neurogenesis and lacks of its anti‐depressive effects in CaMKIV null mice. Here, we addressed whether Sig‐1R agonist (SA4503) and SSRI (fluvoxamine) with affinity for Sig‐1R rescue the depressive behaviors in CaMKIV null mice. Treatment with SA4503 or fluvoxamine for 14 days completely rescued the depressive behaviors in CaMKIV null mice. By contrast, treatment with paroxetine, which lacks affinity for Sig‐1R, did not alter these behaviors. Reduced numbers of BrdU‐positive cells and decreased BDNF mRNA expression seen in the DG of CaMKIV null mice were significantly restored by chronic Sig‐1R stimulation with SA4503 or fluvoxamine. We previously reported that Sig‐1R stimulation with SA4503 and fluvoxamine promotes ATP production through increased mitochondrial calcium transport (Tagashira et al., BBA2013;1830:3082). As expected, the reduced ATP production in the DG of CaMKIV null mice was improved by chronic Sig‐1R stimulation. Taken together, Sig‐1R stimulation rescues the SSRI‐resistant depressive behaviors observed in CaMKIV null mice, suggesting attractive therapeutics for SSRI‐resistant depressive patients.
Differential Activity of Decynium‐22 Analogs: Novel Targets for Probing Low‐Affinity/High‐Capacity Biogenic Amine Transporters
Authors: Fraser Rheaclare , Owens W Anthony , Wyatt Naomi , Krause‐Heuer Anwen , Greguric Ivan , Callaghan Paul , Fraser Benjamin , Daws Lynette
Published: June 2021

We study neurotransmitter clearance by low‐affinity, high‐capacity uptake‐2 transporters. This family includes plasma membrane monoamine transporter (PMAT) and three organic cation transporter isoforms (OCT1‐3). We have shown uptake‐2 transporters limit the effectiveness of the selective serotonin reuptake inhibitor (SSRI) fluvoxamine. Discerning the transporter type(s) involved is restricted by the lack of highly selective ligands. This project examines the pharmacological characteristics of novel uptake‐2 compounds.

Activity of ANSTO analogs, structurally based on the non‐selective inhibitor decynium 22 (D22), was tested in human OCT3‐HEK cells. Ligand competitions of [3H]MPP+ uptake were measured in whole, attached cells. Compared to D22, dose‐responses of ANSTO compounds shifted 1‐ or 2 log‐rightward, indicating reduced potency to inhibit OCT3 mediated [3H]MPP+ uptake. ANSTO analogs displayed similar potencies to corticosterone and may have higher selectivity at alternate uptake 2 subtypes.

[3H]MPP+ Uptake Competition in hOCT3‐HEK Cells

COMPOUNDS
IC50 (µM)

ANSTO 301
3.9

ANSTO 302
2.5

ANSTO 303
0.76

ANSTO 304
0.71

ANSTO 305
0.41

ANSTO 306
0.71

ANSTO 307
1.2

Corticosterone
1.3

Decynium 22
0.072

Analyses in PMAT over‐expressing cells are ongoing. Analogs and SSRI competitions will be measured in brain preparations from OCT3 knockout & PMAT knockout mice for physiological comparisons to our findings in cells. These studies will reveal more about the pharmacological profile of these novel compounds for their potential therapeutic application to treat disorders with neurotransmitter dysregulation, such as depression and drug abuse.

Supported by NIH MH093320 and NRSA T32DA031115.
Characterization of Real Time Adenosine Effluxes in the Brain of Anesthetized Rats using Fast‐Scan Cyclic Voltammetry
Authors: Adamah‐Biassi Ekue , Weiner Jeff , Bonin Keith , Budygin Evgeny
Published: June 2021

The present study was designed to characterize adenosine effluxes in the brain using fast scan cyclic voltammetry and explore how spontaneous adenosine release may be affected by a tail pinch. Long Evans rats were anesthetized and a carbon fiber electrode was positioned in the motor cortex or dorsal striatum. Voltammetric recordings were made every 100 ms by applying a triangular waveform from ‐0.4 to +1.5 V and back versus an Ag/AgCl reference electrode at a rate of 400 V/s. Frequency of adenosine spikes detected was relatively stable in both tested regions and the time intervals between spikes were regular and lasted from 1 to 6 seconds. The number of spikes per 10 seconds ranged from 7.5 ± 0.56 to 9.5 ± 0.62 in the motor cortex and 8.0 ± 1.24 to 9.8 ± 1.54 in the dorsal striatum. The average amplitude fluctuated from 75.0 ± 10.18 to 94.3 ± 18.12 nM in the motor cortex and from 122.7 ± 29.17 to 150.3 ± 43.20 nM in the dorsal striatum. Statistical analysis did not reveal any significant difference in the frequency or the amplitude between the two regions (n=6 rats, p>0.05). The striatal and cortical clearance rate of adenosine was equivalent (n=7 rats, 137.4 ± 23.23 and 195.9 ± 44.14 nM/s for the cortex and striatum, respectively). Furthermore, a 3 s tail pinch transiently increased adenosine signaling in both brain areas. The increase was significantly higher in the dorsal striatum than the motor cortex (n=6, p<0.05). Our study confirmed that adenosine signaling can operate on a faster time scale to modulate brain functions. This study was supported by NIAAA R01AA022449.
The Marine Pharmacology and Pharmaceuticals Pipeline in 2014
Authors: Mayer Alejandro , Nguyen Dom , Glaser Keith
Published: June 2021

In October 2014 there were six FDA‐approved marine‐derived drugs: for cancer, cytarabine (Cytosar‐U®, Depocyt®, FDA‐approved 1969), eribulin mesylate (Halaven®, FDA‐approved 2010), trabectedin (Yondelis®, FDA‐orphan drug approval 2005) being EU‐registered, and brentuximab vedotin (Adcetris®, FDA‐approved 2011); for pain, ziconotide (Prialt®, FDA‐approved 2004); for hypertriglyceridemia omega‐3‐acid ethyl esters (Lovaza®, FDAapproved2004). The clinical marine pharmaceutical pipeline (Mayer et al. TIPS 31:255‐265, 2010) consisted of 10 marine‐derived compounds in clinical development in 2010, including three monoclonal antibodies conjugated to monomethyl auristatin E, a synthetic analog of the marine compound dolastatin, in either Phase I, Phase II or Phase III clinical trials. The current clinical marine pharmaceutical pipeline consists of 9 marine‐derived compounds, and greater than 14 other auristatin‐containing antibody drug conjugates in clinical development. Updated information on the clinical marine pharmaceutical pipeline is available at http://marinepharmacology.midwestern.edu/clinPipeline.htm. Finally, the global preclinical marine pharmacology pipeline was reviewed in Mayer et al. MARINE DRUGS 11:2510‐2573, 2013). In late 2014, both the marine pharmacology preclinical and clinical pharmaceutical pipelines remained very active. Supported by Midwestern University.
Idiosyncratic Drug‐Induced Liver Injury Potential of Zileuton is Detected in Diversity Outbred Mice
Authors: Harrill Alison , Lyn‐cook Lascelles , Gatti Daniel , Luo Shaoke , Churchill Gary
Published: June 2021

Zileuton is an orally active inhibitor of 5‐lipoxygenase used clinically for maintenance treatment of asthma. While also a promising candidate for other inflammatory diseases involving leukotrienes, such as rheumatoid arthritis and inflammatory bowel disease, enthusiasm for expanding its indications is tempered by rare instances of associated drug‐induced liver injury (DILI). Mechanistic understanding of zileuton DILI is hampered by the rarity of the cases and lack of an animal model. A promising model for rare liver injury is the Diversity Outbred (DO) mouse population, which contains genetic variation that is similar to that found in humans. We hypothesized that zileuton DILI could be mediated by rare genetic alleles that are harbored by sensitive patients and thus used the DO mice as a surrogate model for clinical populations. Female DO mice (N=50/group) were orally administered zileuton (300 mg/kg) or vehicle for seven days. Significant elevations in alanine aminotransferase (ALT) were elicited by zileuton (P<0.05), but not the vehicle. Sensitive and resistant individuals were observed in the zileuton group with ALT elevations ranging from 0.2‐13.6 fold over baseline. Group mean±SEM for the zileuton and vehicle groups were 123.8±10.0 U/L and 32.39±5.4 U/L, respectively. MiR‐122 elevations in plasma confirmed the liver origin of the ALT release (P<0.05). The data provide an important first step to identifying the DO as an animal model for idiosyncratic DILI due to zileuton.

This work is supported by the Burroughs Wellcome Fund, Arkansas Biosciences Institute, NIH [TRI UL1TR000039 and T32 GM106999].
Characterization of Mice with Liver‐Specific Knockout of Histone Methyltransferase G9a
Authors: Lu Hong , Lei Xiaohong , Zhang Qinghao
Published: June 2021

G9a is an epigenetic enzyme essential for a key gene silencing mark, histone H3 dimethylation at lysine‐9 (H3K9me2). G9a is important for embryonic development and differentiation of multiple tissues. However, little is known about the role of G9a in liver. To understand the role of G9a in regulating hepatic gene expression and pathophysiology, we generated mice with liver‐specific knockout (Liv‐KO) of G9a by crossing G9a floxed mice with Alb‐cre mice. G9a Liv‐KO mice had marked loss of G9a and H3K9me2 proteins in liver. Unstressed adult male G9a Liv‐KO mice had no overt liver injury or infiltration of inflammatory cells. However, G9a Liv‐KO mice had higher blood levels of total cholesterol; results of hepatic gene expression suggest that the underlying mechanism may be increased biosynthesis but decreased catabolism of cholesterol. Consistent with a key role of G9a in gene silencing, G9a Liv‐KO mice had dramatic ectopic induction of certain genes normally expressed in neural and immune systems. Additionally, G9a Liv‐KO mice had hepatic down‐regulation of many cytoprotective genes and certain important drug‐processing genes, elevated endogenous reactive oxygen species, but lower glutathione and nuclear Nrf2, a master regulator of cytoprotection. Sixteen hours after LPS treatment, G9a Liv‐KO mice had aggravated lipid peroxidation and proinflammatory response, manifested by much higher hepatic induction of IP‐10, Mcp‐1, Icam‐1, and iNos, as well as proinflammatory transcription factors c‐Jun, Stat1, Irf‐1, and Atf4. Thus, G9a plays a key role in regulating hepatic gene expression by silencing neural and proinflammatory genes but maintaining/activating cytoprotective genes, by which G9a may protect the liver from injuries induced by various environmental stressors.
Drugs associated with idiosyncratic hepatotoxicity synergize with cytokines to kill hepatocytes in vitro
Authors: Maiuri Ashley , Breier Anna , Turkus Jonathan , Lansdell Theresa , Kaur Gurpreet , Parkins Robert , Ganey Patricia , Roth Robert
Published: June 2021

Idiosyncratic drug‐induced liver injury (IDILI) occurs in a small fraction of patients and often results in removal of otherwise efficacious drugs from the market. The mechanisms of IDILI are unknown; however, in studies using animal models the inflammatory mediators tumor necrosis factor‐alpha (TNFα) and interferon‐gamma (IFNγ) are essential to the pathogenesis of liver injury. The purpose of this study was to test the hypothesis that a drug's ability to synergize with inflammatory cytokines to kill hepatocytes in vitro can classify dugs according to their potential to cause idiosyncratic hepatotoxicity in humans. Human hepatoma (HepG2) cells were treated with drugs associated with IDILI or with drugs lacking IDILI liability and cotreated with TNFα and/or IFNγ.Of 13 drugs associated with IDILI, 12 synergized with TNFα to kill HepG2 cells.IFNγ enhanced the toxicity mediated by 8 IDILI‐positive drugs in the presence of TNFα. Of 10 drugs with little/no IDILI potential, none synergized with inflammatory cytokines to kill HepG2 cells. These findings indicate that drugs associated with IDILI in humans synergize with TNFα and IFNγ in vitro to cause cell death and raise the possibility that drug‐cytokine synergy underlies hepatocellular killing in some IDILI reactions. These results suggest an in vitro approach that could be used during preclinical safety evaluation to identify drug candidates with the potential to cause IDILI.
Urinary KIM‐1 is a Novel Biomarker for Cisplatin‐Induced Subclinical Nephrotoxicity in Oncology Patients
Authors: George Blessy , Wen Xia , Ellison Lucas , Joy Melanie , Aleksunes Lauren
Published: June 2021

Acute kidney injury can be observed in 8 to 40% of oncology patients treated with cisplatin using traditional clinical markers, including serum creatinine and urinary albumin excretion. However, these indicators of nephrotoxicity are considered insensitive since a significant degree of tubular damage is needed in order to increase their levels. Preclinical studies support detection of kidney injury molecule‐1 (KIM‐1) in urine as an early and sensitive biomarker of cisplatin‐induced injury. Therefore, we sought to characterize urinary levels of KIM‐1 protein in patients prescribed cisplatin‐containing chemotherapy regimens. Thirty‐nine patients scheduled for outpatient chemotherapy for solid tumors with IV cisplatin 蠅 30 mg/m2 were recruited. Patients participated in the study during two cycles of cisplatin therapy. Urine was obtained at baseline as well as 3 and 10 days post infusion and analyzed for KIM‐1 protein concentrations using an ELISA assay. During the first cycle of cisplatin therapy, urinary KIM‐1 levels were increased 2‐ and 4‐fold at 3 and 10 days post infusion, respectively. A similar time‐dependent trend was observed with subsequent cisplatin cycles. Additionally, the baseline KIM‐1 levels were increased ~2‐fold in patients that had previously received cisplatin chemotherapy, although still within normal ranges. These data suggest KIM‐1 is a novel biomarker for detection of subclinical nephrotoxicity in patients treated with cisplatin. Supported by an AFPE Fellowship, DK080744, DK093903, and CA072720.
Oligomerization of Equilibrative Nucleoside Transporter 1
Authors: Cravetchi Xenia , Vilas Gonzalo , Hammond James
Published: June 2021

Equilibrative nucleoside transporter 1 (ENT1) is a ubiquitously expressed membrane transporter in mammalian cells responsible for the transmembrane flux of endogenous nucleosides such as adenosine, as well as, chemotherapeutic, anti‐vial, and anti‐parasitic nucleoside analogues. The present study was designed to assess whether ENT1, like many other solute transporters, functions as a dimer or higher order oligomer.

Previous functional studies from our laboratory suggested the presence of two populations of ENT1 with differential sensitivities to sulfhydryl reagents at the plasma membrane. Studies of ENT1 photoaffinity labelled membranes revealed multiple molecular mass bands on denaturing gels. Non‐denaturing Blue Native PAGE resulted in a molecular mass of ENT1 of 137 kDa (versus 55kDa monomer) suggesting a higher order oligomer or association with other proteins. Co‐immunoprecipitation studies on HEK293 cells expressing MYC‐ and HA‐hENT1 indicated that hENT1 can oligomerize. This was confirmed by immunofluorescence experiments, which showed a 1.5‐fold increase in colocalization of MYC‐hENT1 and HA‐hENT1 signal when the amount of transfected HA‐hENT1 cDNA was gradually increased while MYC‐hENT1 was kept constant in HEK293 cells. Finally, proximity ligation assays, performed on WT‐hENT1‐PK15 cells transiently transfected with MYC‐hENT1, showed interaction between the two pools of hENT1.

These data support the hypothesis that ENT1 exists in the plasma membrane as a dimer, and possibly higher level protein complexes. Further work regarding the identity of the ENT1 complex components as well as the impact of these interactions on the function and inhibitor/substrate association with ENT1 remains to be determined.

Supported by the Natural Science and Engineering Research Council of Canada.
Rapid production of novel pre‐microRNA agent hsa‐mir‐27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells
Authors: Li Meimei , Wang Weipeng , Wu Wenjuan , Huang Min , Yu Aiming
Published: June 2021

MicroRNAs (miRNAs or miRs) are a large family of noncoding RNAs that have been identified as critical epigenetic factors in the regulation of various cellular processes including drug metabolism and disposition. However, the study on miRNA functions is limited to the use of synthetic RNA and recombinant DNA agents. Herein, we show that novel pre‐miRNA‐27b (mir‐27b) agents could be biosynthesized in Escherichia coli using tRNA‐based recombinant RNA technology, and recombinant tRNA/mir‐27b chimera was readily purified to a high degree of homogeneity (> 95%) using anion‐exchange fast protein liquid chromatography. The tRNA‐fusion mir‐27b was revealed to be processed to mature miRNA miR‐27b in human carcinoma LS‐180 cells in a dose and time dependent manner. Moreover, recombinant tRNA/mir‐27b agents were biologically active in reducing the mRNA and protein expression levels of cytochrome P450 3A4 (CYP3A4) which consequently led to a lower midazolam 1'‐hydroxylase activity. These findings demonstrate that human pre‐miRNAs may be produced in Escherichia coli by recombinant RNA technology as novel ncRNA agents for functional studies in drug metabolism.
Therapeutic Anti‐TNF alpha Antibodies : Structure‐Activity Investigations
Authors: Pierri Ciro Leonardo , De Grassi Anna , Cetrone Michela , Punzi Giuseppe , Tricarico Domenico
Published: June 2021

Therapeutic mAbs have high efficacy in treating TNFalpha‐related immunological diseases such as ankylosing spondylitis, psoriatic‐rheumatoid arthritis, and skin diseases. Three mAbs are available:Etanercept(Enbrel®), Infliximab(Remicade®), and Adalimumab(Humira®). Biosimilars(Inflectra® and Remsima®) of Infliximab have been recently approved. These IgG1 antibodies contain a single asn 297 glycosylation site that is essential for the Fc effector functions. Glycosylated antibodies lacking fucosylation show an increase in the affinity for FcγRIIIa, with an improved ADCC. However, no structural data on the Infliximab‐FcγRIII complex are available. For investigating the role of Fc fucosylation in the response to Infliximab we built a 3D mAb model that included the FAB portions of Infliximab complexed with TNFalpha(coordinates from 4G3Y.pdb), the Fc region of IGHG1 fucosylated(3SGJ) and afucosylated(3SGK) with FcγRIIIa. A few hundred steps of energy minimization were performed on the resulting 3D mAb‐model and on the minimized model we quantified interactions occurring between FcγRIII and the fucosylated/afucosylated Fc. While fucosylation does not affect FAB‐TNFalpha interactions, the superimposition of the 3D‐mAb models revealed a change in the number/quality of interactions at the carbo.‐carbo. interface between FcγRIIIa glycans and the fucosylated/afucosylated Fc. In the fucosylated Fc‐FcγRIIIa, these interactions are weakened/nonexistent supporting the reduced Fc‐FcγRIIIa affinity and ADCC activity also described in Infliximab biosimilars but not in the originator showing low fucosylation level of the Fc‐FcγRIIIa.
High Fructose Consumption Exacerbates Metabolic Alterations Compared to Glucose in Female Rats.
Authors: Sangüesa Gemma , Shaligram Sonali , Akther Farjana , Rahimian Roshanak , Alegret Marta , Laguna Juan
Published: June 2021

High fructose consumption, especially in soft drinks, has been associated with the development of obesity, diabetes and hypertension. The aim of this study was to investigate the effect of liquid fructose and glucose intake on metabolic parameters related to these pathologies in female rats.

Sprague‐Dawley female rats were supplemented with 20% w/v glucose (G) or fructose (F) in drinking water for 8 weeks. Control (C) rats received no sugar. Body weight, food and drink intake and blood pressure (BP) were monitored throughout the experiment, and after the sacrifice, organ weight and plasma analytes for metabolic parameters were measured.

Total caloric intake was significantly increased 1.8‐fold and 1.6‐fold in G and F groups vs C, respectively. Moreover, calorie consumption was significantly higher in G vs F group (p<0.01). Systolic BP was significantly increased in both G and F groups. Adiposity and plasma leptin levels were also significantly higher in treated groups vs C. However, total body and liver weights, as well as triglyceride (TG) and insulin levels were significantly increased only in F group. Interestingly, plasma adiponectin levels were increased by 3‐fold only in G group (p<0.001 vs F and C).

In conclusion, despite the higher total caloric consumption in G group, F but not G intake increased insulin and TG levels suggesting insulin resistance. The paradoxical increase in adiponectin levels only in G group might be a mechanism of protection from metabolic alterations.

Supported by NIDCR and F. Pedro i Pons.