bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synergistic Impact of d‐δ‐Tocotrienol and Geranylgeraniol on the Growth and HMG CoA Reductase of Human DU145 Prostate Carcinoma Cells
Authors: Yeganehjoo Hoda , DeBose‐Boyd Russell , McFarlin Brian , Mo Huanbiao 
Published: November 2023

d‐δ‐Tocotrienol and geranylgeraniol suppress the growth of diverse types of tumors in vitro and in vivo. Their growth‐suppressive effect is at least partially attributed to their impact on 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, the rate‐limiting enzyme in the mevalonate pathway that provides essential intermediates for the posttranslational modification of growth‐related proteins including RAS and nuclear lamins. d‐δ‐Tocotrienol blocks the processing and nuclear localization of sterol regulatory element binding protein 2, the transcriptional factor for HMG CoA reductase, while d‐δ‐tocotrienol and geranylgeraniol collaboratively enhance the proteasome‐mediated degradation of HMG CoA reductase. We hypothesize that these agents have synergistic inhibitory impact on cell growth based on their complementary mechanisms of action with HMG CoA reductase. d‐δ‐Tocotrienol (0 – 40 μmol/L) and geranylgeraniol (0 – 100 μmol/L) each induced concentration‐dependent suppression of the growth of human DU145 prostate carcinoma cells as measured by CellTiter 96® AQueous One Solution. Blends of the two agents synergistically suppressed the growth of DU145 cells, with combination index values in the range of 0.67–0.75. While 7.5 μmol/L d‐δ‐tocotrienol and 30 μmol/L geranylgeraniol individually had no impact on cell cycle distribution in DU145 cells, a blend of the agents at these concentrations induced cell cycle arrest at the G1 phase. The synergistic downregulation of the expression of HMG CoA reductase by 7.5 μmol/L d‐δ‐tocotrienol and 30 μmol/L geranylgeraniol was accompanied by a reduction in membrane RAS protein induced by the blend. Our finding lends further support to the cancer chemopreventive action of plant‐based diets and their isoprenoid constituents. Properly formulated isoprenoid combinations and their derivatives with complementary mechanisms may provide novel approaches in prostate cancer therapy.Support or Funding InformationTDA FFRP, TWU REP, and American River Nutrition, Inc.
NRG1‐ERBB4‐PSEN1 Dysfunction in Human Myocarditis and Dilated Cardiomyopathy
Authors: Hanson Paul J , Jang Erika L , Rai Harpreet , Chang Angela , Mo Angela , McManus Bruce M , Seidman Michael A 
Published: November 2023

Myocarditis, inflammation of the muscular portion of the heart, can be caused by infection (most often viral), autoimmunity, or chemical exposure. Clinical symptoms are also variable, with some patients, regardless of etiology, experiencing life threatening acute illness, others suffering from chronic disease, and still others who never come to clinical attention at all. The risk factors for developing different patterns of disease are poorly elucidated, but preclinical and limited clinical studies have implicated a role for the signaling cascade involving neuregulin 1 (NRG1), epidermal growth factor related receptor ERBB4, and presenilin 1 (PSEN1). In this study we sought to characterize the same signaling pathway in archival human tissue specimens from patients with a diagnosis of myocarditis. It is hypothesized that variable expression and function of the NRG1‐ERBB4‐PSEN1 signaling axis is associated with different clinical manifestations of myocarditis. Twenty (20) cases of myocarditis and five (5) normal controls were identified from the Cardiovascular Tissue Registry in the Centre for Heart Lung Innovation. Using archival tissue from the left ventricle of each case, protein expression levels, cellular localization, and post‐translational modifications of NRG1, ERBB4, and PSEN1 were evaluated using immunohistochemistry, immunofluorescence, and western blotting. Data analysis was performed using ImagePro Plus for color segmentation and quantification and ImageJ software for densitometry. NRG1, ERBB4 and PSEN1 are expressed at higher levels, up to 4.18‐, 19.96‐ and 38.41‐fold, respectively, in the majority of myocarditis cases, increasing with severity and chronicity, as compared to control cases. All three proteins are seen in extracellular protein aggregates (plaques) in myocarditis cases. ERBB4 and PSEN1 accumulate in the nucleus of myocarditis hearts, and ERBB4 also accumulates at Z lines. NRG1 isoforms range from 50–75 kDa, ERBB4 shows 80 and 120 kDa cleavage products, and PSEN1 aggregates range from 50–250 kDa. Preliminary studies show a distinct difference in expression patterns of NRG1, ERBB4, and PSEN1 in cases of myocarditis as compared to normal controls. Further experiments are likely to establish patterns of expression that correlate with disease severity and temporality. These data are expected to eventually provide diagnostic and therapeutic guidance with respect to the different clinical manifestations of myocarditis.Support or Funding InformationThis research is funded by the Providence Health Care Research Institute and through donations made to the St. Paul's Hospital Foundation.
Cortical Neurons Exposed to HIV‐1 Nef Exhibit Increased MAP2 Levels
Authors: Vargas‐Zapata Valerie , Martínez‐Orengo Neysha , Noel Richard J. 
Published: November 2023

BackgroundApproximately 50% of HIV‐1 patients suffer from neuropathologies despite the success of anti‐retroviral therapy. The HIV viral protein Nef has been found in post mortem brain tissues of HIV‐1 patients with neurocognitive disorders. Studies have demonstrated that Nef is present in the brain even in the absence of viral replication raising the possibility for ongoing neurotoxicity at undetectable viral loads. Our work showed that rats suffer from learning impairments and neuronal loss when infused with astrocytes expressing Nef. This also correlates with an increased expression of inflammatory cytokines that promote neuroinflammation, but this mechanism is not well understood. We hypothesize that neurons undergo morphological changes in response to Nef‐induced inflammation. We tested the effects of Nef on neuron/dendritic marker MAP2 and the synaptic marker synaptophysin in cortical neurons derived from rats.MethodsCortical neurons were co‐cultured with astrocytes expressing Nef or GFP for 48 and 72 hours. Astrocyte lysates were collected for protein quantification and the supernatants for testing pro‐inflammatory markers. Neurons were stained for MAP2 and synaptophysin using immunoflourescense microscopy.ResultsQualitative comparison of cortical neurons exposed to Nef show higher expression of MAP2 when compared to GFP control, most noticeable at 72 hours. Synaptophysin expression was stronger at 72 hours for both Nef and GFP exposed cells.ConclusionsThis preliminary data suggests that Nef increases MAP2 levels in cortical neurons in a time dependent manner. It is of our interest to investigate if the increments we are observing in MAP2 and synaptophysin expression are related to neurogenesis in response to inflammation or a mechanism of Nef leading to cell proliferation.Future directionsFurther studies will let us better understand the co‐localization and functional effects of MAP2 and synaptophysin in neurons exposed to Nef. Inflammatory markers will be used to study the correlation between Nef, inflammation, and neuronal morphologic changes. Experiments will also be performed in human cell lines and eventually in our in vivo model.Support or Funding InformationMBRS‐RISE Program (R25GM082406), Dr. Noel's Lab (GM106970), PHSU‐PRI MAGIC Laboratory (MD007579)
CRISPR‐Cas9: Power And Challenges
Authors: Charpentier Emmanuelle 
Published: November 2023

The RNA‐programmable CRISPR‐Cas9 system has recently emerged as a transformative technology in biological sciences, allowing rapid and efficient targeted genome editing, chromosomal marking and gene regulation in a large variety of cells and organisms. In this system, the endonuclease Cas9 or catalytically inactive Cas9 variants are programmed with single guide RNAs (sgRNAs) to target site‐specifically any DNA sequence of interest given the presence of a short sequence (Protospacer Adjacent Motif, PAM) juxtaposed to the complementary region between the sgRNA and target DNA. The system is efficient, versatile and easily programmable.Originally, CRISPR‐Cas is an RNA‐mediated adaptive immune system that protects bacteria and archaea from invading mobile genetic elements (phages, plasmids). Short crRNA (CRISPR RNA) molecules containing unique genome‐targeting spacers commonly guide Cas protein(s) to invading cognate nucleic acids to affect their maintenance. CRISPR‐Cas has been classified into three main types and further subtypes. CRISPR‐Cas9 originates from the type II CRISPR‐Cas system that has evolved unique molecular mechanisms for maturation of crRNAs and targeting of invading DNA, which my laboratory has identified in the human pathogen Streptococcus pyogenes. During the step of crRNA biogenesis, a unique CRISPR‐associated RNA, tracrRNA, base pairs with the repeats of precursor‐crRNA to form anti‐repeat‐repeat dual‐RNAs that are cleaved by RNase III in the presence of Cas9 (formerly Csn1), generating mature tracrRNA and intermediate forms of crRNAs. Following a second maturation event, the mature dual‐tracrRNA‐crRNAs guide the endonuclease Cas9 to cleave cognate target DNA and thereby affect the maintenance of invading genomes. We have shown that the endonuclease Cas9 can be programmed with sgRNAs mimicking the natural dual‐tracrRNA‐crRNAs to target site‐specifically any DNA sequence of interest. I will discuss the biological roles of CRISPR‐Cas9, the mechanisms involved, the evolution of type II CRISPR‐Cas components in bacteria and the applications of CRISPR‐Cas9 as a novel genome engineering technology.
Global Analysis of LncRNAs in Gliomas and Glioblastomas
Authors: Reon Brian , Sinegra Andrew , Dutta Anindya 
Published: November 2023

Each year, over 15,000 patients will die from brain cancer. Long noncoding RNAs (lncRNAs) are a new regulatory entity that has large effects on cell physiology and have recently been recognized for their functional role in cancer. To better understand the role of lncRNAs in brain cancer, we identified and characterized all of the long non‐coding RNAs (lncRNAs) in both grade II and III gliomas as wells as grade IV glioblastomas (GBM). We determined the expression of all lncRNAs in over 750 brain cancer and normal brain tissue RNA‐seq datasets from the Cancer Genome Atlas (TCGA) and other publicly available datasets. Over a thousand lncRNAs were induced or repressed in gliomas and GBMs relative to normal brains. Interestingly, lncRNA expression profiles distinguished the three glioma subtypes (IDH1/2 wt, IDH1/2 mutant and IDH1/2 mutant with 1p19q deletion) and the four subtypes of GBM (Classical, Mesenchymal, Neural and Proneural). Using a multivariate Cox regression survival model, we identified several hundred lncRNAs that predict patient survival independent of grade, patient age or IDH1/2 mutational status. Compared to two published studies of lncRNAs in cancer (Yan et al. Cancer Cell 28:529–540 and Iyer et al. Nat Genetics 47:199–208), this is the first comprehensive profiling of lncRNAs in gliomas and GBMs correlating expression to tumor subtype and prognosis.We next tested whether prognostically predictive lncRNAs affect the oncogenic phenotypes of brain tumor cell lines. Ten prognostically significant lncRNAs were selected that were highly expressed in both A172 and U87 GBM cells lines. 8 of the lncRNAs were mainly located in the cytoplasm while 2 were predominantly found in the nucleus. We are currently measuring the effects of lncRNA knockdown using cell proliferation, scratch wound‐healing migration and transwell invasion assays. Although more work is needed to determine the functions of the prognostic lncRNAs, this work provides a critical resource for future studies examining the role of lncRNAs in brain cancers.Support or Funding InformationP01 CA104106
Homer2: A Negative Regulator of Methamphetamine Reward Sensitivity
Authors: Brown Chelsea N. , Fultz Elissa K. , Quadir Sema G. , Flaherty Daniel M. , Szumlinski Karen K. 
Published: November 2023

Homer2 is a post‐synaptic scaffolding protein involved in regulating glutamate receptor function that has been highly implicated in drug‐induced neural plasticity. Withdrawal from repeated methamphetamine injections increases Homer2 protein expression within the nucleus accumbens (NAC). Moreover, both idiopathic & genetic vulnerability to high MA reward is associated with elevated Homer2 expression within NAC subregions. The present series of studies were designed to examine the functional relevance of NAC Homer2 expression for methamphetamine reward and reinforcement. Under place‐conditioning procedures [4 pairings of 0.5–2 mg/kg], the dose‐response function for a methamphetamine‐conditioned place‐preference was shifted upwards and to the right in Homer2 KO mice, relative to their wild‐type counterparts. A follow‐up study using shRNA‐mediated knock‐down of Homer2b within the NAC core of C57BL/6J mice recapitulated the KO effect upon the expression of a methamphetamine‐conditioned place‐preference (2 mg/kg) and shifted the dose‐response function for methamphetamine reinforcement upwards of controls. Together, these results provide novel evidence that Homer2 expression, particularly that within the NAC core, normally functions to dampen the reinforcing/rewarding properties of methamphetamine, posing an inhibitory role for Homer2‐dependent regulation of glutamate transmission within the NAC as a neurobiological substrate in the etiology of MA addiction.Support or Funding InformationThis work was funded by NIDA grant R01DA039168. The authors thank Drs. Matthias Klugmann and von Jonquires for constructing the AAVs.
The interactions between riluzole and SK2 channel
Authors: Zhang Miao , Nam Young Woo , Ali Sara , Alexander Tia , Chiang Stephen 
Published: November 2023

Riluzole, the only FDA‐approved drug for Amyotrophic Lateral Sclerosis (ALS), works by modulating multiple drug targets including small conductance Ca2+‐activated potassium (SK) channels. However the functional binding site of riluzole in any of its drug targets is still unclear. Here we determine the binding pocket of riluzole in SK2 channels through crystallography. This study investigates whether this binding pocket of riluzole in SK2 channels is the functional binding pocket through which the drug exerts its effects. With combined techniques including site‐directed mutagenesis and electrophysiology, we proved that the binding site identified by crystallography is indeed the functional binding pocket. Mutations A477V/L480M, that mimic the corresponding residues in SK4 channels, were introduced into SK2 channels to test whether these mutations increases the channel's sensitivity to riluzole. The mutant channel was then co‐expressed with calmodulin (CaM). Inside out macro‐patch recordings were performed to measure the SK2 current as a function of riluzole concentration (μM). Dose response curves for the potentiation of SK2 channel activity were constructed. The half maximal effective concentrations (EC50), were significantly different between the mutant and wild type channels. As a result, the binding site identified by crystallography is indeed the functional binding site through which riluzole exerts positive modulation of SK2 channels.Support or Funding InformationAmerican Heart AssociationNational Ataxia Foundation
Activation of Human Aryl Hydrocarbon Receptor in Promoting Hepatocellular Carcinoma
Authors: Lu Peipei , Xie Wen 
Published: November 2023

Background & AimsTCDD, or dioxin, is a potent liver tumor promoter in rodents through sustained activation of the aryl hydrocarbon receptor (AHR). In addition to the pharmacological model, constitutive activation of AHR by genetic approach has an equal potential in promotion of hepatocellular carcinoma (HCC). However, humans appear to be resistant to most of the adverse health effects of dioxin, and only chloracne is related to serum TCDD levels. This inter‐species difference is largely due to distinct ligand affinity and various gene transactivation selectivity and potential. Here we investigate the liver tumor promoting effect of human AHR and explore the mechanisms underlying its promoting effect.MethodsHumanized AHR transgenic mice expressing a constitutively‐activated human AHR (CA‐hAHR) were generated to investigate the direct involvement of human AHR in HCC. A two‐stage initiation‐promotion model of liver tumor was utilized in which a single dose of tumor initiator N‐Nitrosodiethylamine (DEN) was applied at 6 weeks old, followed by AHR activation for 9 months.ResultsWe found that strikingly, constitutively activated human AHR promoted both liver tumor incidence and multiplicity in mice, which was comparable to that in mouse Ahr transgenic mice. This liver tumor promoting effect was associated with increased inflammatory cytokine expression, induced hepatocyte proliferation, as well as enhanced hepatocyte apoptosis.ConclusionsHuman AHR works at least as efficiently in developing HCC when compared to that in mouse Ahr transgenic mice, suggesting the oncogenic potential of human AHR.Support or Funding InformationThis work was supported in part by the National Institutes of Health grants DK083952 and HD073070 to Wen Xie.
Deubiquitinating Enzymes for P450 NO Synthase
Authors: Bolles Amanda , Morishima Yoshihiro , Lau Miranda , Miller Siennah , Osawa Yoichi 
Published: November 2023

Nitric Oxide Synthase (NOS) is a P450‐like enzyme that catalyzes the production of nitric oxide (NO) from L‐arginine, O2, and NADPH. The three isoforms of NOS, endothelial (eNOS), neuronal (nNOS), and inducible (iNOS), are essential for the regulation of blood pressure, synaptic response, and immune defense, respectively. It is known that NOS undergoes C‐terminus of Hsp70‐interacting protein (CHIP)‐dependent ubiquitination and proteasomal degradation. The steady state levels of ubiquitinated NOS are likely regulated by both ubiquitination and deubiquitination. We utilized a Ubiquigent screen of 50 common deubiquitinating enzymes (DUBs) to identify DUBs capable of cleaving CHIP‐dependent nNOS‐ and eNOS‐ubiquitin conjugates in a purified in vitro system. Numerous DUBs were identified that remove ubiquitin from both nNOS and eNOS, including USP2, USP6, USP7, and USP20. Other DUBs, including USP4 and UCHL3, displayed isoform selectivity towards eNOS‐ubiquitin conjugates. Consistent with the results on nNOS, treatment of HEK293 cells with the deubiquitinating enzyme inhibitor WP1130 increased nNOS ubiquitination. These results suggest that deubiquitinating enzymes play a role in the isoform selective regulation of steady state levels of ubiquitinated NOS.Support or Funding InformationThis work was supported, in whole or in part, by National Institutes of Health Grant GMO77430 and The University of Michigan Medical School's Protein Folding Diseases Initiative. AB is a trainee of the University of Michigan, Pharmacological Sciences Training Program T32‐GM007767.
The specificity of Gβγ subunits regulating exocytosis through the Gi/o‐coupled α2a adrenergic receptor
Authors: Yim Yun Young , Betke Katherine , McDonald Hayes , Gilsbach Ralf , Chen Yunjia , Hyde Karren , Wang Qin , Hein Lutz , Hamm Heidi 
Published: November 2023

Modulation of neurotransmitter exocytosis by activated Gi/o‐type G‐protein coupled receptors (GPCRs), such as the α2a adrenergic receptor (α2a‐ARs), is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. Gβγ and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) interaction is known as one of mechanisms that inhibit neurotransmitter release. There are 5 Gβ and 12 Gγ subunits, but protein level of each Gβs and Gγs in brain is not known. It is also not known whether specific Gβγs are activated by a given GPCR in vivo. However, presynaptic α2a‐ARs in both adrenergic (auto α2a‐ARs) and non‐adrenergic neurons (hetero α2a‐ARs) work in a similar manner to inhibit neurotransmitter release and have various physiological functions such as anesthetic sparing and working memory enhancement. In this project, we examine the protein level of each Gβ and Gγ in whole brain synaptosomes and in fractions of synaptosomes. In whole brain synaptosome, we have identified Gβ1 and Gγ2 as dominant subunits present. Although all Gβs and Gγs are highly present in cytosolic fractions, Gγs show interesting changes in protein expression patterns by fractions. We also investigate whether autoreceptors in sympathetic neurons use the same Gβγ subunits as heteroreceptors in other neuronal types. Using several mice models including transgenic Flag‐α2a‐ARs, knock‐in HA‐α2a‐ARs, and wild‐type mice, co‐immunoprecipitation, mass spectrometry analysis, we test our hypothesize that specific Gβγ subunits interact with activated auto‐ and hetero α2a‐ARs and inhibit exocytosis by interacting with SNARE. We have identified Gβ1, Gβ2, Gβ4, Gβ5, Gγ2, Gγ3, Gγ4, Gγ7, Gγ12, and Gγ13 which interact with α2a‐ARs and SNARE. Out of these G proteins, we found Gβ2 and Gγ13 preferentially interacting with activated auto α2a‐ARs. However, we found no difference in Gβγ specificity to SNARE following α2a‐ARs activation. Further understanding Gβγ specificity may offer new insights into the normal functioning of the brain, as well as better understanding of disease progression. The Gβγ‐SNARE interaction may be a new therapeutic target to modulate exocytosis in neural disorders in combination with drugs targeted to Gi/o‐type GPCRs.Support or Funding InformationThis work was supported by the National Institutes of Health (EY10291, MH101679, and T32).