bluuebunny/crossref_metadata_2025 huggingface dataset:
CNS‐Permeant Tamoxifen Analog Modulates The Dopamine Transporter And Reduces Amphetamine Reinforcing Effects
Authors: Carpenter Colleen , Zestos Alexander , Altshuler Rachel , Sorenson Roderick , Guptaroy Bipasha , Jutkiewicz Emily , Kennedy Robert , Showalter Hollis , Gnegy Margaret
Published: November 2023

Amphetamine (AMPH) is the second most widely abused drug worldwide, yet an effective therapeutic for AMPH abuse and addiction remains elusive. The reinforcing properties of amphetamine are due to its ability to increase extracellular dopamine in the nucleus accumbens. This process is facilitated by protein kinase C (PKC) activation and blocking PKC reduces AMPH activities.

The selective estrogen receptor modulator tamoxifen remains the only available brain‐permeant PKC inhibitor. We have created a small library of novel tamoxifen analogs with increased selectivity for PKC inhibition and reduced affinity at the estrogen receptor. With an IC50 of 80 nM, our lead compound (6c) inhibits PKC 250 times more potently than tamoxifen and displays no binding at the estrogen receptor α. In determining the efficacy of 6c to reduce the neurochemical and behavioral effects of AMPH, we firstly investigated the ability of the compound to modulate the dopamine transporter in both cellular and rat striatal synaptosomal preparations.

6c showed negligible effects in blocking dopamine uptake or AMPH‐induced dopamine release in HEK cells stably expressing the dopamine transporter. However, 0.3–3 μM 6c dose dependently inhibits dopamine release stimulated by 10 μM AMPH in synaptosomes. 6c less potently blocks dopamine uptake and does not displace [3H]WIN 35428 binding at the dopamine transporter in synaptosomes, showing that it does not bind near the active site. 6 mg/kg 6c s.c. does not affect baseline dopamine levels or locomotion but significantly reduces both dopamine release and locomotion induced by 2 mg/kg i.p. AMPH as measured with microdialysis in freely‐moving rats. 6 mg/kg s.c. 6c reduces i.v. AMPH self‐administration (0.032 mg/kg AMPH per infusion) by ~40% but does not affect food self‐administration in rats.

6c effectively reduces the actions of AMPH both in vitro and in vivo. The fact that 6c modulates the dopamine transporter in synaptosomes but not in cells overexpressing DAT, suggests its action is via a mediator versus a direct action at the transporter. This study also highlights the importance of choosing appropriate models of the dopaminergic terminal in screening compounds for AMPH abuse.

Support or Funding Information

NIH grant 1R01 DA11697, HHMI International Student Research Fellowship
Regulation of Goal‐directed Action Selection by Cocaine, MDMA, and Orbitofrontal BDNF‐trkB
Authors: Pitts Elizabeth , Gourley Shannon
Published: November 2023

Cocaine dependence is characterized by compulsive drug use and maladaptive decision‐making. Adolescents are particularly vulnerable to the effects of cocaine; for example, cocaine exposure during adolescence increases the risk of developing lifelong addictions. Here we show that subchronic cocaine exposure during adolescence results in increased responding during acquisition of cocaine self‐administration and a bias towards stimulus‐driven habits in adulthood in mice. Changes in Brain‐derived Neurotrophic Factor (BDNF) in the orbitofrontal prefrontal cortex (oPFC) could underlie, in part, this habit bias, while strategies that stimulate cortical BDNF systems could be protective. We utilized viral‐mediated gene transfer to interfere with the activity of BDNF's high‐affinity receptor tyrosine kinase receptor B (trkB) selectively in the oPFC. This manipulation induced habit‐like behavior in an instrumental contingency degradation task. Next, we hypothesized that stimulating BDNF expression in the oPFC could block habits by enhancing response‐outcome learning and memory. We report that 3,4‐methylenedioxymethamphetamine (MDMA) increases BDNF levels in the oPFC, but not the amygdala or dorsal striatum, and also “breaks” habits resulting from adolescent cocaine exposure. Interfering with trkB activity in the oPFC blocks MDMA‐mediated rescue of goal‐directed decision making. MDMA was delivered immediately following instrumental contingency degradation, suggesting that it enhances the consolidation of new response‐outcome learning and memory. Finally, 7,8‐dihydroxyflavone (7,8‐DHF), a trkB agonist, also reverses cocaine‐induced habits, further suggesting that BDNF‐trkB systems are a point of intervention in combatting maladaptive decision making following repeated cocaine exposure.

Support or Funding Information

This work was supported by DA036737 and DA042358. The Yerkes National Primate Research Center is supported by the Office of Research Infrastructure Programs: OD P51OD011132
Dimethyl Fumarate Improves Depression and Anxiety by Reducing Brain Catalase Levels in Mice
Authors: Iniaghe Loretta Oghenekome , Ilondu Chinenye Amara , Eseka Ewere Ogechuwu , Gabriel Benjamin
Published: November 2023

Introduction

Depression and anxiety are psychiatric disorders which are leading causes of disability and are estimated to be major contributors to overall global burden of disease; both often accompany chronic diseases. This study investigated the effects of dimethyl fumarate (DMF) in animal models of depression and anxiety.

Methods

Mice (n=40) were treated with either the vehicle ‐dimethyl sulfoxide/distilled water (DMSO solution), 50 and 100 mg/kg DMF and Imipramine and subjected to either the forced swim test (FST), n=20 or the tail suspension test (TST) after they were euthanized, whole brains isolated, homogenized and assayed for brain catalase levels. Another set of mice (n=40) were treated daily with either the vehicle, DMF 50 and 100 mg/kg and Imipramine for two weeks and subjected to FST and TST on the fourteenth day. Thereafter, they were sacrificed, whole brains isolated, homogenized and assayed for brain catalase levels. Changes in body weight were also recorded daily.

The same procedure was followed for determination of anxiety using the stair case and hole board tests instead of the FST and TST and Diazepam as the reference drug.

Results

In the test for depression, 100 mg/kg DMF significantly (p<0.05) reduced periods of immobility in both the FST and TST after acute and chronic drug administration. DMF also significantly (p<0.05) decreased brain catalase levels and improved body weights when compared to the control.

In the test for anxiolysis, 50 mg/kg DMF significantly (p<0.05) improved upward climbs and number of head dips in both the stair case and hole board tests respectively.

Conclusion

DMF exhibited anti‐depressant activity and anxiolytic properties at doses used in this study

Support or Funding Information

Personal Funds of the Authors
DSS‐induced intestinal damage exacerbates liver injury by acute alcohol exposure in mice: role of intestinal hypoxia‐inducible factor 1α
Authors: Shao Tuo , McClain Craig , Feng Wenke
Published: November 2023

Inflammatory bowel diseases (IBD) are characterized by repeated injury of the mucosa and loss of the intestinal epithelial barrier function, which has also been considered one of the major contributors to the development of alcoholic liver disease (ALD). Hypoxia induced factor 1α (HIF1α) plays a critical role in the intestinal barrier integrity by regulating several mucus and tight junction protecting genes. In this study, we hypothesize that intestinal HIF1α deletion increases intestinal permeability resulting in an elevation of endotoxemia in the dextran sulfate sodium (DSS)‐induced colitis murine model, and this exacerbates acute alcohol‐induced liver injury. To test this hypothesis, Wide type (WT) and intestinal epithelial‐specific HIF1α knockout (IEhif‐1α−/−) mice were fed 3% dextran sodium sulfate (DSS) in drinking water for 7 days and a bolus of alcohol at 6g/kg was gavaged 6 hours before sacrificing. DSS decreased body weight of the WT mice, and this decrease was more significant in the IEhif‐1α−/− mice. Intestinal inflammation and mucosal abnormality were increased in the IEhif‐1α−/− mice than that in the WT mice, along with an elevated serum LPS concentration, indicating an increase in intestinal barrier permeability. Binge alcohol, in addition to DSS, further damaged barrier function and increased serum LPS concentrations. Further analyses showed that DSS+alcohol induced a decrease in the expression of caludin‐1 and intestinal trefoil factor, which play an important role in intestinal barrier integrity. Intestine epithelial HIF1α deletion aggravated this effect. Unlike alcohol exposure, DSS treatment alone did not affect liver function in either WT or IEhif‐1α−/− mice, as determined by serum ALT and AST analysis. However, ALT and AST levels were significantly elevated in DSS + alcohol treatment groups, and these elevations were more pronounced in IEhif‐1α−/− mice. HIF‐1α deletion also enhanced alcohol‐induced hepatic triglyceride concentration in DSS‐treated mice. Hepatic histology analysis showed that IEhif‐1α−/− mice had an increased fat accumulation and inflammatory cell infiltration, confirming the hepatic steatosis and liver injury. In conclusion, our results demonstrated that DSS‐induced colitis increases intestinal permeability and endotoxemia, resulting in an worsened liver steatosis and injury due to binge alcohol exposure. Deleting intestinal HIF1α further exacerbates these deleterious effects. Targeting intestinal HIF1α might be an effective strategy for the treatment of colitis‐associated ALD.

Support or Funding Information

Supported by NIH and Veterans Administration.
Repairing Interstrand DNA Crosslinks (ICL): Characterization of an ICL Incision/Lesion Bypass Polymerase Complex regulated by the Fanconi anemia pathway
Authors: Jasti Vijay Prasad , Sharma Shilpy , Althaus Irene , Weinstein Abby , Ramani Shreyas , Canman Christine
Published: November 2023

The ‘REVersionless’ genes, REV1, REV3, and REV7, were discovered as being necessary for both spontaneous and DNA damage‐induced mutagenesis in yeast. REV3 is the catalytic subunit of DNA polymerase Zeta (ζ) and together with its accessory subunit REV7 extends nucleotides inserted opposite multiple types of DNA lesions. REV1 is a deoxycytidyl transferase and performs a scaffolding function in orchestrating the switch between insertion TLS polymerases directly opposite a DNA adduct and Polζ for the extension step. REV1 and Polζ are also involved in ICL repair and homologous recombination (HR) repair of DNA double stranded breaks (DSB). The phenotypes of cells deficient in expression of REV1, REV3, or REV7 are similar to cells derived from patients diagnosed with Fanconi Anemia (FA), a genetic disorder characterized by bone marrow failure, ICL hypersensitivity, and increased risk for cancer. Previous studies have demonstrated an epistatic relationship between REV1 or REV3 and genes encoding proteins comprising the FA core signaling complex in resistance to cisplatin. Additionally, REV1, Polζ, and the FANCD2/FANCI heterodimer have been shown individually to be involved in homologous recombination (HR) repair, which is crucial for resolution of DNA DSBs and ICL repair during S phase. We demonstrate that human REV1 and REV3 exhibit an epistatic relationship with FANCD2 and FANCI in cellular resistance and ICL repair following mitomycin C (MMC) treatment, as well as HR repair. MMC‐induced chromosomal anomalies are increased in REV1 or REV3‐depleted cells and FANCD2 depletion does not further exacerbate this phenotype. We further show that the FANCA core complex protein regulates MMC‐induced chromatin association of REV7 and by extension REV3 to sites of ICL repair. Co‐immunoprecipitation studies show that REV1, REV3 and REV7 interact with FANCD2 and FANCI independent of DNA damage or monoubiquitination of FANCD2 by the FA core complex. In addition, REV1, REV3, and REV7 co‐immunoprecipitate with the SLX4/FANCP scaffold protein, which regulates the unhooking step of an ICL DNA adduct by the ERCC1/XPF (FANCQ) endonuclease and HR repair of a DSB. We propose a model where REV1/Polζ‐dependent activities during ICL and HR repair are regulated by multiple FA proteins including FANCA, FANCD2 and SLX4 (FANCP). Together, these results suggest that REV1 and Polζ are part of a macromolecular complex involved in resistance to many anticancer agents and interactions within this complex may be viable targets for small molecule inhibitors to sensitize resistant tumors to chemotherapy.

Support or Funding Information

R01 CA133046
Activation of the D2 dopamine receptor hampers the protective effect of the A2A adenosine receptor on TDP‐43 mislocalization
Authors: Chern Yijuang , Lai Cha‐You , Liu Yu‐Ju , Lai Hsing‐Lin
Published: November 2023

TAR DNA‐binding protein (TDP‐43) is an RNA‐binding protein that exists mostly in the nucleus and regulates transcription and RNA splicing. Mislocalization of TDP‐43 to the cytoplasm has been observed in several neurodegeneration diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), and is believed to initiate the pathogenesis of TDP‐43 proteinopathy. We have previously published that several adenosine analogues (T1‐11 and its analogues) prevented the reactive oxygen species (ROS)‐induced TDP‐43 mislocalization in a mouse motor neuron cell line (NSC34) via the A2A adenosine receptor (A2AR), and prevented the degenerated motor functions in a TDP‐43 transgenic ALS mouse model. In the striatum, previous studies suggest that A2AR and the D2 dopamine receptor (D2R) formed dimmer and negatively cross‐regulated each other's functions. In the present study, we first demonstrated that A2AR and D2R both exist in the motor neurons of the spinal cord of normal subjects and ALS patients by using immunofluorescence assay. Expression of both A2AR and D2R in NSC34 cells led to dimer formation as assessed using the proximity ligation assay (in situ PLA). No change in the binding affinity of T1‐11 was observed when A2AR formed dimmer with D2R. Nonetheless, activation of D2R reversed the A2AR‐mediated prevention of ROS‐induced AMPK activation and TDP‐43 mislocalization in NSC34 cells. Given that D2R agonists have been commonly used in clinical settings, the impact of D2R activation on the normalization of TDP‐43 mislocalization by A2AR in ALS warrants further investigation.
AKT signaling is essential for functional and structural integrity of the heart
Authors: Goedecke Stefanie , Heinen Andre , Moeller Finja , Deenen Rene , Koehrer Karl , Goedecke Axel
Published: November 2023

The mammalian heart expresses predominantly two isoforms of protein kinase B (AKT), namely AKT1 and AKT2. Knockout mice for AKT1 and AKT2 respectively, have demonstrated that AKT1 primarily appears to regulate growth whereas AKT2 is rather involved in regulating metabolism. Here we have analyzed common functions of both AKT isoforms in the heart by generating Tamoxifen(OHTX)‐inducible, cardiac myocyte specific AKT1 and AKT2 single and double KO mice (iCMAKT1/2KO).

Inactivation of AKT1 and AKT2 resulted in a maximal loss of both isoforms 5 days after start of OHTX‐treatment without a compensatory upregulation of AKT3. The basal level of Ser9 Phosphorylation of GSK3β was significantly reduced in hearts of double KO only, demonstrating that GSK3β is controlled by AKT1 and AKT2 isoforms in CM. Insulin did not stimulate GSK3β phosphorylation. Furthermore, insulin dependent phosphorylation of classical direct and indirect AKT downstream targets like mTor (Ser2448, Ser2841), p70S6 kinase (Thre 389) or S6 ribosomal protein, among others, was shown to be significantly downregulated in iCMAKT1/2KO heart.

Functional analysis by echocardiography revealed a progressive decline of pump function characterized by a drop of ejection fraction from initially 62 to 37 % and 31 % on day 10 and day 21, respectively resulting in cardiac failure on day 24. In contrast, the single iCMAKT1 and iCMAKT2 KO mice showed no cardiac depression. Interestingly, wall thickness declined from day 14 to day 21. Concomitantly, cardiac mass declined to 70 %. Gene expression analysis using Agilent 60K microarrays were performed using hearts from single and double knock out mice. On day 10 after KO induction 1095 genes were differentially expressed (P<0.05) and on day 21 this number increased to 7707 (P<0.01, moderated t‐test, Benjamini‐Hochberg correction). On day 10 upon Txf induction Gdf15 (4.9 × up), a typical stress marker for diseased hearts but not ANP was induced in double knock out hearts. On day 21 this expression increased to Gdf15 (58 × up) and also ANP was induced (17.8 × up). None of these expression alterations coud be detected in Akt1 or Akt2 single knock out heart. Ingenuity Pathway analysis identified particularly metabolic pathways as well as EIF2 signalling to be substantially affected.

Conclusion

Expression of either AKT1 or AKT2 in cardiac myocytes is sufficient to preserve cardiac function whereas loss of both isoforms results in progessive cardiac atrophy and failure.

Support or Funding Information

This work was funded by DFG Grant IRTG1902.
Analysis of Cell Spreading as a Function of Integrin Inhibition
Authors: Soneral Paula , Zipoy Hannah , Lunacek Kaitlyn
Published: November 2023

Glioblastoma multiforme is an aggressive form of glioma due to its high metastasis rates and unresponsiveness to curative treatments such as surgical resection and existing chemotherapies. The purpose of this research was to find potential antibody inhibitors to block dimerization of alpha and beta integrin receptors and stop cell migration. A set of inhibitory antibodies were screened for their ability to inhibit adhesion in a cell‐drop spreading assay using the U251 human glioblastoma astrocytoma cell line. Cells pretreated with inhibitory concentrations of anti‐beta 1, anti‐alphaV, and anti‐alpha 2 and integrin antibodies were seeded on synthetic collagen surfaces and recorded using time‐lapse phase‐contrast microscopy to monitor cell attachment and spreading. Under each of these conditions, the frequency of rounded vs. spread cells were scored and analyzed. Cells inhibited with the anti beta‐1 antibody were completely rounded (half‐time = 0) compared to untreated cells, which exhibited an exponential decay function with half‐time of 19 minutes, indicating full inhibition of cell spreading. In contrast, both anti alpha integrin antibodies showed a delay in cell spreading with a characteristic half‐time of 25 minutes, and a logistic spread curve. ELISA binding assays revealed that a large number of alpha subunits (alpha V, alpha 2, others) partner with the beta 1 subunit dimerization. Furthermore removal of extra‐cellular matrix binding proteins destroys all cell‐substratum interactions and cell morphology. Taken together, these data suggest that beta‐integrin is a key cell adhesion receptor in glioma cells, and is partially modulated by alphaV and alpha 2 receptors during dimerization. Adhesion may be blocked to different extents by antibody inhibitors, and anti‐beta 1 antibody in particular may serve as potent future chemotherapy for glioma patients.
Antioxidant Activity and Protective Effect on PC12 against H2O2 of Epimedium koreanum
Authors: You SoHyeon , Jang Miran , Kim Gun‐Hee
Published: November 2023

It is well known that Epimedium is bioactive genus of Asian pharmacopoeia. Recent studies have attributed most of these functional properties to its flavonoid glycosides, especially icariin which is the major constituent. In this study, we investigated flavonoids glycosides in Epimedium koreanum and icariin, icaritin, icariside□, icariside□, epimedin A‐C have been identified using HPLC‐DAD. After screening the seven identified flavonoid glycosides by 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (DPPH) assay, icariin and icariside□ presented significant effect on radical scavenging activity. We examined the antioxidant effect of Epimedium koreanum extract, icariin and icariside□ using DPPH, 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and ferric reducing ability power (FRAP) assay. The results showed Epimedium koreanum extract, icariin and icariside□ have effect on antioxidant peroperty in dose dependant manner. Neuroprotective effect against H2O2–induced cell viability in PC12 cells was analyzed using MTT assay. It revealed that Epimedium koreanum extract, icariin and icariside□ showed cell protective effect dose dependantly. From these outcomes, we could identified that icariside□ has potency of functional effect as natural source for human health and further research needs to be carried on.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.2016R1A2B4014740)
First‐in‐class Naturally ‐ occurring Bioenergetic Peptide from Hibiscus sabdariffa
Authors: Kam Antony , Loo Shining , Tam James P
Published: November 2023

Mitochondria produce more than 90% of cellular energy and defects in energy metabolism are a common cause of age‐related diseases1. These diseases include cardiovascular disease, diabetes, neurodegeneration and fatigue2. In a program to identify potential therapeutics against age‐related diseases, we have identified cysteine‐rich peptides, designated as roseltides, from the medicinal plant Hibiscus sabdariffa. Hibiscus sabdariffa of the Malvaceae family, commonly known as roselle, is a shrub cultivated in South‐east Asian countries. The dark red fruit is used to make drinks, syrups and jams. As a folk medicine, it has been used for treating diabetes, cardiovascular and neurodegeneration‐related diseases3. Here, we report the discovery of roseltide rT1, a bioenergetic‐mitochondria‐targeting peptide from Hibiscus sabdariffa that improves bioenergy traits. Roseltide rT1 was isolated and purified through repeated rounds of liquid chromatography. Using a combination of transriptomic and proteomic methods, roseltide rT1 is identified as a 27‐residue peptide with high metabolic stability4. Roseltide rT1 is a cationic and amphipathic peptide which is favorable for cell‐penetration. Using flow cytometry and live‐cell confocal microscopy, we demonstrated that roseltide rT1 is internalized into living cells via endocytosis. Upon cell entry, roseltide rT1 escapes the endosome and localizes to the mitochondria. Functionally, roseltide rT1 increases cellular ATP level. Taken together, this study highlights the discovery of roseltide rT1, as the first naturally‐occurring mitochondria‐targeting peptide with enhancing properties of ATP production. The features of roseltide rT1 display potential as a new mitochondrial‐targeting agent with therapeutic potentials for mitochondrial dysfunctions.

Support or Funding Information

This project was supported in part by the National Research Foundation (NRF‐CRP8‐2011‐05) of the Prime Minister's Office of Singapore.
Cellulose degradation in anaerobic condition by filament fungi
Authors: Mercado‐Garcia Benjamin
Published: November 2023

Cellulases are powerful enzymes with many potential applications, including agriculture, textiles and biofuels production. Many Fungi are known to be cellulose degraders, secreting powerful cellulases in aerobic environments. But can fungi degrade cellulose in anaerobic conditions? Rose Bengal Agar enriched with sodium nitrate and antibiotics was used to isolate fungi in wood incubated in anaerobic conditions using anaerobic jars together with Gas Pak systems. Isolated fungi were transferred to PDA medium until purified colonies were obtained. Purified colonies were identified using PCR techniques with ITS‐1 F and ITS‐4 R primers. All isolated fungi were grown on PDA and PDA enriched with sodium nitrate in anaerobic conditions to evaluate growth abundance and colony pigmentation. Also, growth was tested for all fungi in liquid media using cellulose strips as the only carbon source. Eight genera were identified using primers ITS‐1 and ITS‐4 including: Aspergillus, Mucor, Phlebia, Fusarium, Botryosphaeria, Neofussicoccum, Schizophyllum and Penicillium. Twenty two of the forty two fungi tested showed good growth in PDA and PDA + nitrate in anaerobic conditions. Secretome was collected from fungi growing in aerobic and anaerobic conditions in liquid media with cellulose strips and proteins were precipitated with acetone:water 4:1 and stored at −20°C, for specific activities assays. SDS PAGE was used to separate cellulases secreted and proteins extracted from SDS PAGE were analyzed by mass spectroscopy. This project will help to identify possible new cellulases produced in anaerobic conditions by fungi traditionally classified as aerobic cellulose degraders.

Support or Funding Information

University of Puerto Rico

RISE Program
Integrase Move‐In Day: Mechanisms of HIV DNA Integration
Authors: Windgassen Tricia A , Fahlberg Sarah , Hanson Bradley , Klauser Katherine , Semia Sanaa , Keck James L
Published: November 2023

More than 1.2 million people in the United States are living with the HIV virus, with about 50,000 new cases each year. All viruses, including HIV, need the viral protein Integrase, which integrates the viral genome into the host genome for replication. The integrated DNA is then accessible to make more viruses or is able to stay dormant until the best opportunity to activate, generating viruses. Currently, the reaction mechanism of HIV DNA insertion into host DNA is poorly understood, and exactly how this works is key to understanding how the whole of a virus, like HIV, functions. The Monona Grove High School SMART (Students Modeling a Research Topic) Team is modeling Integrase found in a prototype foamy virus to better understand how the mechanisms of strand transfer are carried out and how this could relate to the HIV virus found in humans. By studying the post‐catalytic strand transfer structure of Integrase1,PDB 3OS0, we will be able to better understand reaction intermediates and host DNA sequence preference of the HIV DNA integration process. It is important to study viral DNA integration because it will aid in understanding HIV, as well as other viruses, and potentially help design more, novel drugs that block this integration and even cure retroviral infections like HIV. This infection is a global problem from a virus that is constantly evolving, and studying integrase is just one step to further solving this epidemic.
TolC: An important component of bacterial efflux pumps and potential target for ameliorating antibiotic resistance
Authors: Kessler Eric , Whalen Joe , Bradford Hanna , Chavali Vamsi , Engle Davis , Jeng Joshua , Meier Tristin , Merchant Grace , Narayanan Srivats , Song Anna , Sundar Archana , Van Lieshout Emma
Published: November 2023

Antibiotics are a hallmark of modern medicine, having saved countless lives since the discovery of penicillin in 1928. Bacteria resistant to antibiotics emerged shortly after introduction, and is an increasing problem as bacteria evolve multidrug resistance. Membrane efflux pumps, including AcrAB‐TolC, in gram‐negative bacteria export a variety of substances and provide an intrinsic means for the development of antibiotic resistance. The Blue Valley CAPS 2016–17 SMART (Students Modeling a Research Topic) Team modeled the TolC component of a bacterial efflux pump using JMol and 3D printing technology, and is working to increase antibiotic susceptibility of E. coli using a CRISPR/dCas9 system to inhibit expression of TolC. Efflux pumps, located in the plasma membrane, consist of three proteins; TolC serves as a channel through the periplasmic and outer membrane spaces, while AcrA and AcrB, located in the inner membrane, act as translocases. TolC is a trimeric protein forming a right‐twisted 12‐stranded antiparallel β‐barrel domain, anchoring the protein in the outer membrane, and a left‐twisted 12‐stranded antiparallel α‐helical coiled‐coil domain, extending across the periplasmic space. When translocases AcrA‐B bind a suitable substrate and are energized, TolC is recruited and forms a transient complex. Conformational changes within the coiled‐coils of the α‐helical domain transforms TolC into an open state, allowing entry and movement through its tunnel. Neutralizing the TolC component of efflux pumps using CRISPR/dCas9 may help to prevent bacteria from exporting antibiotics, making them more susceptible to these important drugs.

Support or Funding Information

Supported by NIH‐SEPA and ASBMB HOPES grants.
Effects of Tart Cherry Juice on Brachial and Aortic Hemodynamics, Arterial Stiffness, and Blood Biomarkers of Cardiovascular Health in Adults with Metabolic Syndrome
Authors: Johnson Sarah A. , Navaei Negin , Pourafshar Shirin , Jaime Salvador J. , Akhavan Neda S. , Alvarez‐Alvarado Stacey , Litwin Nicole S. , Elam Marcus L. , Payton Mark E. , Arjmandi Bahram H. , Figueroa Arturo
Published: November 2023

Metabolic syndrome affects greater than one‐third of adults in the United States and is highly associated with the development of cardiovascular diseases. In fact, premature vascular dysfunction in metabolic syndrome may lead to accelerated atherogenesis and arterial stiffening, in large part due to oxidative stress and inflammation in the vasculature. Montmorency tart cherries (Prunus cerasus L.) are rich in bioactive compounds, particularly anthocyanins, known to exert cardiovascular‐protective effects. Previous research suggests that tart cherry juice consumption may improve physiological processes associated with cardiovascular and metabolic health. We conducted a randomized, parallel‐arm, single‐blind, placebo‐controlled pilot clinical trial to investigate the extent to which daily consumption of tart cherry juice would improve parameters associated with cardiovascular and metabolic health including brachial and aortic hemodynamic parameters such as blood pressures and wave reflection, arterial stiffness as assessed by pulse wave analysis and pulse wave velocity, and blood biomarkers of cardiovascular and metabolic health, inflammation, and oxidative stress in men and women between the ages of 20 and 60 years with metabolic syndrome. Nineteen adults with metabolic syndrome consumed 480 mL of tart cherry juice (Tart Cherry; n = 5 males, 4 females) or an isocaloric placebo‐control drink (Control; n = 5 males, 5 females) daily for 12 weeks. Measurements were taken and blood was collected at baseline, 6 and 12 weeks. Oxidized low‐density lipoprotein and soluble vascular cell adhesion molecule‐1 were significantly lower (P = 0.047 and P = 0.036, respectively) in Tart Cherry than Control at 12 weeks. There was a trend for total cholesterol to be lower (P = 0.08) in Tart Cherry than Control at 12 weeks. These results suggest that daily tart cherry consumption may attenuate processes involved in accelerated atherogenesis without improvements on brachial blood pressure, aortic hemodynamics, and arterial stiffness in this population; however, further research is needed to evaluate this.

Support or Funding Information

Cherry Research Committee of the Cherry Marketing Institute
Soy protein nanoemulsions improve stability and bioavailability of vitamin D
Authors: Alruwaili Nawaf Wanas , Lopez Emely Cristina , Gaur Shashank , Feng H. , Engeseth N.J. , Andrade J.E.
Published: November 2023

BACKGROUND

Deficiency and insufficiency of vitamin D (VD) afflicts populations worldwide. Fortification of foods with VD has been present for decades; however, its stability in foods is often compromised by its rapid degradation, low absorption and poor water solubility. In this study, soy protein isolate was conditioned using pH shifting and ultrasound treatment to create soy protein‐based nanoemulsions (SPNEs). The objective was to evaluate the stability, bioaccessibility and bioavailability of VD dispersed in SPNEs.

METHODS

SPNEs were prepared by mixing canola oil (1%, w/w) and soluble SPI (30 mg/mL) containing VD (100 μg/mL; cholecalciferol) and sonicating (20 kHz) 5 min. VD‐containing SPNEs were compared to controls including VD dispersed in water after sonication and in lecithin with canola oil after sonication. VD‐containing SPNEs were freeze dried and evaluated after resuspension. Particle size was measured using dynamic light scattering spectroscopy. VD stability was evaluated after UVB light exposure for 0, 30, 60 and 120 min followed by quantification using and reversed phase HPLC‐UV. Bioaccessibility was evaluated in a two‐compartment in vitro digestion model. Bioavailability was evaluated in adult Long‐Evans hooded rats. Rats were conditioned for a week under a AIN‐93M diet before receiving a gavage dose of: 1) VD‐containing SPNEs (2 mL, 150 μg/mL), 2) Oil with VD (0.5 mL, 300 μg/mL), and 3) Oil without VD (0.6 mL canola oil). After gavage, specific groups of animals (n=3–5) within each treatment were euthanized at 1, 9, 24, and 48 hours after oral dose. Serum 25(OH) VD levels in rats were measured post‐mortem using a colorimetric (450 nm) sandwich ELISA.

RESULTS

Soy nanoparticles (15–30 nm) were larger (p<0.05) than VD‐containing SPNEs (10–20 nm). VD‐containing SPNEs had higher particle size after freeze drying (50–70 nm; p<0.05). Recovery of VD after 60 min UV exposure was 52.3%, 12.8%, and 9.8% in SPNEs, emulsified in lecithin, or without emulsifier in water, respectively. VD bioaccessibility was higher (p<0.05) in SPNEs (96%) than emulsified in lecithin (69%), or without emulsifier in water (63%). Pharmacokinetic studies in rats showed that oral delivery of VD in SPNEs resulted in 8.1‐fold increase in the AUC (p<0.05) and reaching a Cmax at 24 hours compared to the Oil with VD control.

These results revealed that dispersion of VD in SPNEs protects VD from UV exposure and increases its bioaccessibility and oral bioavailability. Future studies will evaluate the sensory properties of SPNEs in food products.

Support or Funding Information

University of Illinois at Urbana‐Champaign, King Saud University
Effects of Physical Exercise on Adult Hippocampal Neurogenesis: Changes in DNA Methylation and a Focus on Smad2 Gene
Authors: Trejo Jose Luis , Herrera Antonio , Fontán‐Lozano Ángela , Pons Sebastián , Gradari Simona
Published: November 2023

Physical activity is an important factor capable of inducing changes in the brain. This study aims to investigate if the expression of certain genes is regulated by changes in the DNA methylation in the hippocampus of the adult mouse, in response to exercise. In particular, the research has focused on the Smad2 gene, manipulating its expression in the dentate gyrus with lentiviral vectors, and studying its role during the process of neurogenesis.

First of all, the hippocampus regions from sedentary and runner animals were analyzed using a microarray detecting the DNA methylation status of 24 transcription factors, and then gene expression was assessed by means of a qPCR. Lentiviral vectors were therefore created to over‐express Smad2, or to silence it by means of a shRNA. Afterwards, mice were subjected to bilateral stereotactic injections into the dentate gyrus to inoculate the different kind of lentivirus. Half of the animals from each group remained in their cage for the duration of the experiment, while the other half undertook physical exercise for two weeks. Finally, animals performed behavioural tests to assess activity, state of anxiety and ability in spatial learning and memory. Neurogenesis and modifications in synaptic plasticity were analyzed in the granule neurons with immunohistochemistry techniques.

Results show that exercise induces changes in the DNA methylation and it regulates the expression of many transcription factors, such as Smad2. The silencing and over‐expression studies elucidate a role for this gene in the late‐stages of adult hippocampal neurogenesis: Smad2 does not promote cell proliferation and survival but it induces neuron's maturation and differentiation. Moreover, Smad2 silencing compromises the spatial learning abilities in sedentary mice, while exercise is able to reverse the impairments due to the genetic manipulations. This study confirms that exercise alters DNA methylation, it has a role in neural plasticity and, moreover, that Smad2 is involved in the late‐stages of hippocampal neurogenesis.

Support or Funding Information

Grant BFU2013‐48907‐R from Ministerio de Economía y Competitividad, Spain
Relationship between Muscularity Concerns and Body Checking Behaviors in Turkish Bodybuilders
Authors: Devrim Asli , Bilgic Pelin , Aslantas Burcu , Hongu Nobuko
Published: November 2023

Objective

Muscle dysmorphia is a common body dysmorphic disorder among male bodybuilders. The major problem is difficulty in diagnosing muscle dysmorphia and often underreported. Muscularity concern is one of the underlying problems in the process of body image disturbance. Researchers have defined muscularity concern as repetitive body checking behaviors more than once in the same day. We aim to investigate the association between muscularity concerns and body checking behaviors among Turkish bodybuilders.

Methods

Participants were 120 male bodybuilders, (mean age 31.05±10.60) recruited from bodybuilding gyms in this study. Their height and weight were measured and Fat Free Mass Index (FFMI) score were calculated. Body checking behaviors (numbers of checking their muscles in a mirror and mirror‐like surfaces) were self‐reported by the bodybuilders. Muscle Dysmorphia Disorder Inventory (MDDI) was used to determine muscularity concerns. The data were analyzed using the IBM SPSS Statistics 23.0. Correlation analyses were used to determine the relationship between the measures of MDDI and the numbers of body checking behaviors.

Results

In this study it is determined that participants check their muscles in the mirror mean average 9.2 (2–13) times a day, and it was higher in the high‐level MDDI scores compared with the low‐level MDDI scores (relatively 77.6% and 22.4% of the study population) The FFMI scores showed positive correlation with the MDDI scores and body checking behaviors (respectively; r=0,614 and r= 0,390, p=0,001).

Conclusions

It was found that bodybuilders with muscle dysmorphia concerned about their muscularity (higher MDDI score) and more frequent repeated body checking behaviors. Therefore, body checking behaviors and FFMI scores appeared to be a good diagnostic tool for health professionals to use when detecting muscle dysmorphia in bodybuilders.

Support or Funding Information

There is no funding support
Enhanced Lipoxygenase/LTD4 Activity Accounts For The Exaggerated Hypertensive And Nephrotoxic Effects Of Cyclosporine Plus Indomethacin In Rats
Authors: Helmy Maged W. , Helmy Mai M. , El‐Mas Mahmoud M.
Published: November 2023

Cyclosporine (CSA) and indomethacin are usually co‐prescribed for some arthritic conditions. This combined regimen is often associated with rises in systolic blood pressure (SBP) and exaggerated nephrotoxicity. Here, we examined whether the 5‐lipoxygenase (LOX) pathway and its products, LTD4 and/or LTB4, modulate the hemodynamic and renal interactions of this regimen in rats. The oral 10‐day treatment with CSA (20 mg/kg/day) or indomethacin (5 mg/kg/day) (i) elevated serum creatinine and blood urea nitrogen, (ii) caused renal tubular atrophy and interstitial fibrosis, (iii) increased renal LTD4, LTB4, TNF‐α, TGF‐β1, caspase 3, and (iv) reduced renal PGE2 and total antioxidant capacity (TAC). Non‐invasive blood pressure measurements by tail‐cuff plethysmography revealed significant increases in SBP and heart rate in rats treated with CSA but not indomethacin. These structural, hemodynamic, and biochemical derangements were mostly intensified in rats treated simultaneously with CSA plus indomethacin. We also report that the concurrent administration of montelukast (cysteinyl LT receptor blocker), but not ONO‐4057 (LTB4 receptor blocker), ameliorated the CSA/indomethacin‐evoked hypertension and renal structural and biochemical indices. Compared with ONO‐4057, montelukast exhibited a greater capacity in reversing the inflammatory, oxidative, apoptotic, and fibrotic abnormalities induced by CSA/indomethacin. Moreover, montelukast abolished the increases in renal LTD4 in contrast to no effect for ONO‐4057. Both montelukast and ONO‐4057 reversed the CSA‐evoked increases in renal LTB4 but had no effect on the concomitant decreases in PGE2. Together, the inflammatory and oxidative effects that follow renal LOX upregulation and rise in cysteinyl LTD4 level might account for the aggravated hypertension and nephrotoxicity caused by the CSA/indomethacin regimen. The data uphold the therapeutic potential of cysteinyl LT receptors antagonism.
6N‐Cyclohexyladenosine Suppresses Shivering in Anesthetized Swine
Authors: Drew Kelly L , Tøien Ø , Kumar Matthew M
Published: November 2023

Shivering compromises post‐operative recovery and patient comfort. Shivering is also an impediment to targeted temperature management (TTM), now standard of care for cardiac arrest. Without an effective drug to suppress shivering (referred to here as a thermolytic) high cost and difficulty of temperature management is restricting access to TTM outside of large hospitals and for conditions such as stroke. A more efficacious thermolytic is needed for post‐operative control of shivering and to improve patient comfort during TTM. Our prior work in rats, based on strong scientific premise from the study of hibernating animals, suggests that stimulating CNS A1 adenosine receptors (A1AR) by combining the A1AR agonist 6N cyclohexyladenosine (CHA) with the peripherally acting adenosine receptor antagonist 8‐(p‐sulphophenyl)theophylline (8‐SPT) will provide an effective means to control shivering. This approach works in rodents however, thermolytic efficacy has not been demonstrated in a larger mammal that, like humans, depends primarily on shivering thermogenesis to maintain body temperature. To assess the thermolytic efficacy of CHA in 50–55 kg swine, a species that lacks brown adipose tissue, three pigs were anesthetized with Telazol/Xylazine, propofol, or isoflurane and cooled to a target body temperature of 32 to 34°C with a water blanket set to 4°C. Probes measured core body temperature at the tympanum, lower esophagus, rectum and bladder. Electrocardiography, pulse oximetery and arterial blood pressure were monitored. Shivering was measured by respirometry and by a qualitative visual scale. When shivering was noted, CHA was administered (bolus, 4.2mg; infusion,113μg/kg/h). 8‐SPT (bolus, 186mg), atropine (bolus, 0.5mg) or epinephrine (bolus, 100μg) was then given IV to reverse bradycardia and hypotension. At completion, animals were euthanized and tissues collected for histopathology. Results show that CHA inhibits shivering and that 8‐SPT restores blood pressure and heart rate without reversing the thermolytic effect of CHA. No histopathology was noted. In conclusion, CHA is an effective thermolytic in swine.

Support or Funding Information

This work was supported by Be Cool Pharmaceutics LLC and the Mayo Clinic
Decreased Expression of ETS‐2 in EPCs as an Early Marker of Cardiovascular Instability in CABG Patients, Beneficial Effects of Sitagliptin
Authors: Tejerina Teresa , Medina Úrsula , Carnero Manuel , Navarro‐Dorado Jorge , García‐Bouza Mónica , Reguillo Fernando
Published: November 2023

Aim

In patients with cardiovascular disease (CVD), the endothelial progenitor cells (EPCs) play a key role in endothelial repair processes. It has been hypothesized that Ets2 transcription factor could be involved in the instability of CVD/hyperlipidaemia. Recent studies have shown that the dipeptidyl peptidase‐4 (DPP‐4) is expressed on endothelial cells and appears to modulate their kinetics. Our main objective was to determine the degree of expression of transcription factor Ets2 and the endothelial repair factors Endoglin and CXCR4 in peripheral blood mononuclear cells from patients undergoing coronary artery‐bypass grafting (CABG), and to prove a possible beneficial effect of Sitagliptin.

Methods

seventy patients undergoing CABG from the cardiac surgery service were selected for the study and categorized into five CVD stages. The Expression of Ets‐2, CXCR4, and Endoglin were measured by Western blot. Sitagliptin effect on EPCs and on the expression of different factors was studied by Western blot, ELISA and immunofluorescence. This study was conducted according to the World Medical Association Declaration of Helsinki and informed consent was obtained from all subjects before sampling.

Results

an increase in the expression of the transcription factor Ets‐2 in patients, without CV predictor risk factor associated with early stages of CV instability was observed (GI: 2.16 ± 1.4). Ets‐2 expression decreased in patients with longer evolution of CVD (GII: 1.16±0.8; GIII1.06±0.5; GIV: 0.91±0.3; GV: 0.56±0.3). A direct association of expression of Ets‐2 with age (p=0.04) and Endoglin expression (p=0.008), and indirectly with the evolution of CVD (p=0.008) and hyperlipidaemia (p=0.03) was found. A direct effect of Sitagliptin 1μM on the colony formation of EPCs at 6 and 12 hours (p=0.0077) and on expression of CXCR at 24 hours (p=0.0201) and SDF1 at 6 and 24 hours (p=0.0085) was observed.

Conclusions

The transcription factor Ets‐2 could be an early marker of cardiovascular instability associated with states of hyperlipidaemia. Our data suggest that a poor functionality of circulating EPCs could be associated with decreased expression of the transcription factor Ets‐2 in advanced stages of cardiovascular disease. Sitagliptin treatment of cultured EPCs, could help to revert EPCs deficient functionality. Therefore, the activatory response might depend on the SDF1‐CXCR4 axis.

Support or Funding Information

FIS PI12/00590. Convocatoria CONACYT‐Gobierno del Estado de San Luis Potosí‐México 2012.
Amelioration of galactosamine‐induced acute hepatic injury by betaine in rats
Authors: Choi Yeo Jin , Na Jong Deok , Kim Young Chul
Published: November 2023

Galactosamine (GalN) is a potent hepatotoxicant that inhibits RNA and protein synthesis via depletion of uridine nucleotide in the liver. Earlier studies suggested that some endogenous sulfur‐containing substances, particularly glutathione (GSH) and/or S‐adenosylmethionine (SAM), might antagonize the induction of liver injury by GalN. In this study, we determined the effect of betaine, a methyl donor used for remethylation of homocysteine to methionine, against GalN‐induced acute liver injury in association with alterations in the metabolomics of sulfur‐containing amino acids and related substances in the liver. Male Sprague‐Dawley rats were supplemented with betaine (1 % in drinking water) for 2 weeks prior to a GalN challenge (400 mg/kg, ip). Rats were sacrificed 24 h later for the assay. GalN treatment elevated serum alanine transaminase (ALT), aspartate transaminase (AST), and sorbitol dehydrogenase (SDH) activities significantly, and these changes were prevented by betaine supplementation. Histopathological examination revealed 30 % of multifocal necrosis with macrophage infiltration throughout the liver of GalN‐treated rats, which was also inhibited by betaine supplementation. GalN treatment increased methionine, but not SAM levels in the liver. Betaine supplementation further increased the hepatic levels of methionine, SAM, and the activity of methionine adenosyltransferase (MAT), an enzyme mediating the biosynthesis of SAM from methionine. However, hepatic GSH contents were not affected by betaine or GalN. GalN treatment elevated ornithine and spermidine levels significantly, but decreased putrescine levels in the liver, suggesting that the metabolic conversion of putrescine from ornithine was impaired. Betaine supplementation increased hepatic putrescine and spermidine levels significantly, but ornithine levels were unaltered. It is suggested that the hepatoprotective effect of betaine against GalN‐induced liver injury may be associated with an enhancement of polyamine biosynthesis via induction of MAT activity and SAM availability in the liver.

Support or Funding Information

Supported by National Research Foundation (NRF) grants (No. 2014‐R1A2A1A11052967 and No. 2009‐0083533) funded by the Ministry of Education, Science and Technology (MEST), Korea.