bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory Effects of Silibinin on Inflammation via Attenuation of HO‐1 in Murine Macrophage Cell
Authors: Song Jihyeon
Published: November 2023

Silibinin is the major bioactive compound of silymarin which is the mixture of flavonoliigans extracted from milk thistle. Silibinin has been shown to possess anti‐inflammatory activity, in inflammation associated with the activation of inducible nitric oxide (iNOS), cyclooxygenase (COX2), and pro‐inflammatory cytokines. However, the underlying mechanisms still remain unclear.

The aims of this study were to determine the effect of silibinin on lipopolysaccharide (LPS)‐induced RAW264.7 murine macrophage cell.

In the present study, we confirmed that silibinin attenuated the production of inflammatory cytokines including interleukin (IL)‐1β, IL‐6, and tumor necrosis factor‐alpha(TNFα). This effect was associated with nitric oxide (NO) production and iNOS protein activity. Silibiinin also suppressed the procution of NO and the expression of iNOS level in LPS‐inducedf RAW264.7. Heme oxygenase‐1 (HO‐1) is know to be an inducible cytoprotective enzyme that has been suggested suppression of HO‐1 activity reduce airway inflammation in arsthma mouse model. We similarly found that HO‐1 was increased in LPS‐induced RAW264.7, while silibinin derepressed the abnormal expression of HO‐1 by LPS in RAW264.7. To demonstrate the anti‐inflammatory effect of silibiin through the regulation of HO‐1 in RAW264.7, we used short‐interfering RNA (siRNA) against to HO‐1. Our results showed that silibinin suppressed inflammatory response via HO‐1 and subsequent involvements with absence of cell cytotocixity in LPS‐induced RAW264.7.

Thus, we conclude that silibinin might be useful to prevent the exogenous pathogen‐mediated inflammation.
Transcription factor HMG box‐containing protein 1 (HBP1) modulates mitotic clonal expansion (MCE) during adipocyte differentiation
Authors: YU PING , CHAN CHIEN‐YI , CHANG FENG‐TZU , HUANG CHUN‐YIN
Published: November 2023

Adipocyte differentiation is delicately controlled by a series of transcription factors, including CCAAT/enhancer‐binding protein (C/EBP)β. Previously, we demonstrated that suppression of transcription factor HMG box‐containing protein 1 (HBP1) by either HBP1 siRNA or shRNA in 3T3‐L1 preadipocytes accelerated cell cycle progression in the mitotic clonal expansion (MCE) stage, but impaired adipogenesis, indicating that HBP1 might be involved in the regulation of adipocyte differentiation. To further gain insight into the role of HBP1 in the MCE stage, we examined the expression pattern of HBP1 upon hormonal stimulation in growth‐arrested 3T3‐L1 cells, and found that, immediately following stimulation, HBP1 expression was down‐regulated, whereas (C/EBP)β was up‐regulated. Further, ectopic expression of HBP1 dose‐dependently suppressed C/EBPβ expression. In addition, administration of methylisobutylxanthine (MIX), a C/EBPβ activator, inhibited both the expression of HBP1 and its promoter activity as determined by a 2 kb HBP1 promoter‐reporter gene assay. These data indicate that HBP1 functions as a negative regulator of MCE, in which down‐regulation of HBP1 ensures the expression of C/EBPβ and the reentry of growth‐arrested 3T3‐L1 into the proliferation mode. In contrast, HBP1 expression was gradually elevated, along with a concomitant induction of p27, at the end of the MCE. Overexpression of HBP1 activated a p27 promoter‐reporter system, suggesting that HBP1‐mediated p27 expression may be needed for the termination of cell cycle as well as the transition of growth arrest into terminal differentiation at the end of MCE, whereas HBP1 knockdown may delay the progression. Taken together, our findings add HBP1 as a key transcription factor in the complicated regulatory cascade during adipocyte differentiation.

Support or Funding Information

This work was supported by the grant to NSC 102‐2320‐B‐039‐036 C‐Y Huang.
Effect of Sex on the Selenotranscriptomic Changes by Long‐term Dietary Selenium Deprivation and Aging in Short Telomere Mice
Authors: Cao Lei , Wu Ryan T.Y. , Cheng Wen‐Hsing
Published: November 2023

Male Terc−/− short telomere mice on a diet deficient in selenium (Se) and during the aging process show selenotranscriptomic changes in a tissue‐ and selenoprotein‐specific manner. The objective of this study was to determine the effect of sex on selenotranscriptome in Se‐deficient and ‐adequate (0.15 ppm Se as sodium selenate) Terc−/− mice at 18 and 24 months of age (early and late stage aging). Various organs were collected and examined for mRNA expression of the 24 mouse selenoproteins by quantitative RT‐PCR analyses. In the heart, the mRNA expression of glutathione peroxidase‐1, selenoprotein H, selenoprotein W, and selenoprotein M were decreased by dietary Se deprivation in both male and female Terc−/− mice. In contrast, the age‐dependent selenotranscriptomic changes differed dramatically by sex. Compared to 18 months of age, the mRNA expression of selenoprotein T, selenophosphate synthetase‐2, thioredoxin glutathione reductase, and selenoprotein O were decreased and iodothyronine deiodinase‐2 were increased only in females, while selenoprotein H were decreased only in males at 24 months of age. The sex effect on the selenotranscriptomic changes in other organs will later be presented. In conclusion, there are sex differences in selenotranscriptomic changes under these dietary and aging conditions.
Despite the Introduction of Mandatory Folic Acid Fortification, Australian Women of Childbearing Age may not obtain the Estimated Average Requirements for Folate
Authors: Borradale David , Kimlin Michael
Published: November 2023

Background

Despite the importance of folate in reducing the risk of Neural Tube Defects (NTD's) and the widespread introduction of mandatory folic acid fortification, there remains a lack of data on the sources where child bearing age women are obtaining their folate requirements.

Objective

We aimed to assess the folate status and dietary intake of folate in a sample of Australian women of childbearing age and to estimate the major contributors of folate intake.

Methods

Across‐sectional study of forty‐five (N=45) women (aged 18–47) conducted in Brisbane, Australia. Dietary intake of folate, assessed as Dietary Folate Equivalents (DFE's) was measured via a validated food frequency questionnaire. Serum and red blood cell folate of participants were measured via commercial automated competitive binding assay.

Results

No participants were deficient in either serum or red blood cell folate, however, over one third of the group (n=16) did not consume an adequate intake of folate according to the Estimated Average requirements (EAR). Meeting the EAR of dietary intake for folate was significantly associated with increased odds of having higher red blood cell folate status (OR=3.83;p=<0.05). Proportionally, folic acid fortified bread contributed 15.4% of total dietary folate intake.

Conclusions

Mandatoryfolic acid fortification provided a substantial proportion of the dietaryfolate intake in this sample. However, despite the mandatory fortification of bread making flour with folic acid a considerable proportion (36%) did not meet the estimated average requirement of dietary folate. Future studies should include females of childbearing age belonging to vulnerable populations, to elucidate the adequacy of folate intake in these groups and assess the impact of low intakes in these populations on blood folate status

Support or Funding Information

David Borradale was supported via Queensland University of Technology's Postgraduate Research Award and project costs were supported via a Queensland University of Technology's small grant award
Soy Proteins Deficient in the α’ Subunit of β‐Conglycinin and Various Glycinin Subunits Maintain the Ability to Modulate Hepatic Lipid and Glucose Metabolism
Authors: Chatterjee Cynthia , Liu Jiajie , Wood Carla , Gagnon Christine , Cober Elroy R. , Fregeau‐Reid Judith A. , Gleddie Stephen , Xiao Chaowu
Published: November 2023

Soy consumption has been associated with improved blood lipid profiles and decreased incidence of metabolic diseases. However, the involved molecular mechanism(s) and the bioactive components in soy remain unclear. This study examined the effect of soy proteins with depletion of different subunits of the two major storage proteins, β‐conglycinin and glycinin, on hepatic lipids and proteins involved in lipid and glucose metabolism as well as inflammation in rats and cultured human hepatocytes. Sprague‐Dawley rats (8 males and 8 females/group) were fed diets containing either 20% casein or 20% conventional soy proteins or soy proteins depleted in different β‐conglycinin and glycinin subunits. After 8 weeks, the rats were necropsied for collection of blood and tissue samples. Human HepG2 liver cells were treated with casein or test soy protein hydrolysates in the media containing low (5 mM) or high (25 mM) levels of glucose with or without insulin and tumour necrosis factor alpha (TNF‐α). Hepatic lipids were extracted and measured. Various modulators of lipid and glucose metabolisms in the liver and cultured human hepatocytes were determined by Western blot. Serum inflammation markers were analysed by a magnetic beads‐based multiplexing immunoassay. The results showed that the soy diets lacking glycinins reduced hepatic lipid droplet formation, total cholesterol, triglycerides, and non‐esterified fatty acids content in the male rats compared to rats on the casein control diets, and a similar trend was observed in the females. The soy diets lacking glycinins significantly attenuated serum inflammation markers and the abundance of mature hepatic sterol regulatory element binding protein ‐1 (SREBP‐1) and fatty acid synthase (FAS) in both genders and decreased hydroxymethylglutaryl‐CoA reductase (HMGCR) and low‐density lipoprotein receptor (LDL‐R) in female livers. Soy protein hydrolysates devoid of glycinins significantly increased Akt phosphorylation in high glucose concentrations. These results suggest that the remaining soy protein components other than α’ subunit of β‐conglycinin and glycinin may be associated with the health benefits of soy intake such as hypolipidemic function, improved insulin signalling and suppression of inflammation.

Support or Funding Information

This research was supported by Health Canada, Grain Farmers of Ontario Research Fund and the partnership of Agriculture and Agri‐Food Canada/Canadian Field Crops Research Alliance DIAP Fund. C.C. is supported by the National Science and Engineering Research Council (NSERC) Visiting Fellowship in Canadian Government Laboratories.
Effect of Enzyme‐Treated Asparagus Extract on brain function
Authors: Takahashi Makiko , Sakurai Takuya , Hisajima Tatsuya
Published: November 2023

Enzyme‐Treated Asparagus Extract (ETAS) is an anti‐stress functional material which is made from asparagus native to Hokkaido, Japan. It shows a variety of anti‐stress effects including heat shock protein 70 (HSP70)‐inducing activity. Several investigations were conducted to evaluate the anti‐stress effects of ETAS in in vitro and in vivo studies and in a clinical trial.

In the in vitro study, when NG108‐15 neuronal cells were treated with ETAS, it upregulated the expressions of cytoprotective and anti‐apoptotic mRNAs such as HSP70, heme oxygenase‐1, growth differentiation factor 15, and neuronal apoptosis inhibitor protein 2. In the in vivo experiment, a senescence‐accelerated prone mouse (SAMP8) which shows early deficits of learning and memory, was supplemented with ETAS. When a contextual fear conditioning test was conducted, the ratio of freezing responses of SAMP8 mice treated with ETAS increased significantly compared to that of SAMP8 mice without ETAS.

A randomized, double‐blind, placebo‐controlled, cross‐over study was also carried out. Healthy volunteers (n=25) received ETAS (150 mg/day) or placebo, each of 28 days, with a 14‐day wash‐out. The subjects took the mental arithmetic (the Uchida‐Kraepelin test) for psychological test. After the mental arithmetic test, stress markers were measured using blood and saliva collected, and profile of mood states (POMS) was performed. The intake of ETAS inhibited the elevation of noradrenaline in blood and enhanced s‐IgA level in saliva. Furthermore, the number of answers and correct answers in the mental arithmetic test increased significantly.

In conclusion, it was demonstrated that ETAS has neuroprotective effects and ability to maintain homeostasis against psychological stress, suggesting that ETAS may have beneficial effects in brain function.
Preliminary analysis of bioactive components in aqueous garlic extract
Authors: Thomson Martha , Al‐Qattan Khaled K , Divya JS , Ali Muslim
Published: November 2023

Limited analysis of bioactive components of garlic (Allium sativum) extracts by GC‐MS as well as analysis of amino acid and fatty acid composition and trace metal analysis were carried out. The total phenol content of garlic was 0.374 mg/g dry weight. GC‐MS determination of sulfur compounds in fresh garlic extract and fresh chopped garlic revealed similar compositions with vinyl dithins being the major component in both (40% and 46% respectively), with lesser amounts of allylmethyl trisulfide, diallyl disulfide and S‐allyl cysteine. Freezing and storage of garlic extract resulted in increased amounts of S‐allylcysteine and diallyldisulide, and decreased levels of allylmethyl trisulfide. Vinyldithins remained at approximately 40% throughout the storage period. Boiling of garlic extract vastly changed the composition with allylmethyl disulfide, allyl alcohol and dimethyl disulfide being the major components. Amino acid and metal analysis of garlic extract revealed the presence of 17 amino acids and potassium as the major metal. Fourteen fatty acids were identified in garlic extract with the major fatty acids identified as palmitic, linoleic and stearic acids;

A major bioactive component from garlic extract, S‐allyl cysteine (SAC), was purified by preparative HPLC and assessed for biological activity in streptozotocin (STZ) ‐ induced diabetic rats during a 4 week daily treatment experiment. SAC (5mg/kg daily intraperitoneally) was shown to be effective in limiting weight loss and lowering blood glucose levels of diabetic rats. However, SAC was effective in decreasing polyphagia and polydipsia typical of diabetic rats.

Support or Funding Information

The study was supported by KU grant #SL 07/14.
The Effect of Moringa Oleífera Leaves in Anthropometric and Biochemical Parameters in Obese Type 2 Diabetes Mellitus Subjects
Authors: Dominguez‐Rodriguez Maribel , Valenzuela‐Rubio Nancy Guadalupe , Ochoa‐Acosta Dora Alicia , Fierros‐Valdez Jesus Antonio , Castro‐Sanchez Francisco Humberto , Vergara‐Jimenez Marcela
Published: November 2023

Obesity is associated with the development of diabetes. Diabetes is one of the mayor public health problems worldwide. Several studies have demonstrated the hypoglycemic and hypolipidemic effects of Moringa oleífera leaves. The objective of this study was to evaluate the effect of Moringa oleífera leaves on anthropometric and biochemical parameters in obese‐type 2 diabetic subjects from Mexico. A crossover double‐blind design was carried out through 22w (10w for Stage 1, 2w for a washout period and 10w for Stage 2). We recruited 24 obese type 2 diabetics using metformin (17 woman and 7 men, 20–60y). All subjects were randomly assigned into 2 groups (AB or BA). Letter A represents placebo and B Moringa Oleifera leaves. During the study all subjects received moderate carbohydrate diet (Carbohydrates 40%, Protein 30% and fat 30%) and they were advised to maintain their usual level of physical activity. 4 blood draw points were collected (at the beginning and ending of each stage). Only 3 subjects finished the whole crossover study.

Body weight and Insulin were reduced in A (p<0.05). BMI was significantly reduced by the end of the study. Although the differences between biochemical parameters were not statistically significant, c‐LDL and HbA1c showed a trend to be reduced in B. blood pressure was lower in B. c‐HDL increased in both groups. Fasting glucose concentrations were reduced in A and B. Our results suggest that Moringa oleifera leaves improves parameters associated with obese‐type 2 Diabetics and therefore it could be used as an alternative treatment to control the diabetic conditions of obese adults.
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 Information

TDA FFRP, TWU REP, and American River Nutrition, Inc.
Genetic Modifiers of Cardiac Development 22q11DS Mouse Models
Authors: Arroyo Kevin Cintron
Published: November 2023

Over 70% of patients with 22q11.2 Deletion Syndrome (22q11DS) have congenital heart defects. The prevalence and severity of these defects make it critical for us to strive to understand the underlying mechanism of the syndrome by elucidating the gene pathways that are involved in heart development, as their disruption may lead to congenital heart diseases (CHDs). TBX1 and CRKL, the main genes that have implicated in 22q11DS as responsible for patient CHDs, are required for normal development of the heart. It's been suggested that some genes located outside of the 22q11 deleted region can act as genetic modifiers of CHDs in the syndrome and may also be required for normal development of the heart. Three of these genes are NTRK3, WNT5A and ROR2. Mutations in WNT5A and ROR2 were identified in patients with CHDs and 22q11DS, while mutations in NTRK3 were identified in non‐syndromic patients with CHDs. One of the overarching aims of this study is to test whether genetic modifiers of heart defects associated with the 22q11.2 deletion are the same genes as the genes that alter risk for heart defects in non‐syndromic CHD patients. First, we must test where these three genes are expressed in normal developing embryonic hearts and we have decided to use the mouse as a model to do this. Digoxigenin‐labeled RNA probes for Ntrk3, Wnt5a and Ror2 were designed and used for in situ hybridization in order to observe the expression of these genes in mouse embryos specifically in the heart and regions that contribute to the formation of the heart. Ntrk3, Ror2 and Wnt5a were expressed in the pharyngeal arches and heart region. The expression of the genes Ntrk3, Ror2 and Wnt5a were not only restricted to the heart regions but were also seen in the neural tube area. We also examined the expression of Ror2 in Crkl knock‐out embryos, but observed no change. The results may support our hypothesis that NTRK3, WNT5A, and ROR2 may be causative of CHDs, since they were found to be expressed in the heart and regions required for heart development, however, more work is needed to prove this. More work is also needed to show whether any of these genes act in the same pathway as genes in the 22q11 deleted region to function in normal heart development.
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 Information

This 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

Background

Approximately 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.

Methods

Cortical 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.

Results

Qualitative 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.

Conclusions

This 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 directions

Further 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 Information

MBRS‐RISE Program (R25GM082406), Dr. Noel's Lab (GM106970), PHSU‐PRI MAGIC Laboratory (MD007579)
R‐Ras Subfamily Proteins Elicit Distinct Physiologic Effects and Phosphoproteome Alterations in Neurofibromin‐Null MPNST Cells
Authors: Prechtl Amanda , Brossier Nicole , Barnes Stephen , Wilson Landon , Brosius Stephanie , Byer Stephanie , Carroll Steven
Published: November 2023

Loss of the Ras GTPase‐activating protein neurofibromin promotes the development of multiple nervous system tumors in patients with the genetic disorder neurofibromatosis type 1 (NF1). Neurofibromin loss potentially results in the activation of both classic Ras (H‐Ras, N‐Ras, K‐Ras) and R‐Ras (R‐Ras, R‐Ras2/TC21, R‐Ras3/M‐Ras) subfamily proteins. Previously, we have shown that the classic Ras family inhibit neurofibromin‐null malignant peripheral nerve sheath tumor (MPNST) cell mitogenesis. In order to determine whether the R‐Ras proteins differentially contribute to the pathogenesis of neurofibromin‐null MPNST cells, we inhibited the R‐Ras family proteins and assessed the impact of R‐Ras inhibition on mitogenesis, survival, migration and the phosphoproteome. We show that R‐Ras and R‐Ras2 are expressed and can be activated in neurofibromin‐null MPNST cells. Additionally, like DN H‐Ras, DN R‐Ras inhibits MPNST mitogenesis, however only DN R‐Ras inhibits migration. These findings were further validated by individual shRNA knockdown of the R‐Ras proteins. Using mass spectrometry‐based phosphoproteomics, we found that DN R‐Ras regulated thirteen largely distinct networks, one of which regulates cellular movement via effects on microtubules. We conclude that like classic Ras proteins, R‐Ras proteins can promote tumorigenesis, albeit via different effects that involve the activation of distinct signaling pathways.
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 Information

P01 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 Information

This 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 Information

American Heart Association

National Ataxia Foundation
Activation of Human Aryl Hydrocarbon Receptor in Promoting Hepatocellular Carcinoma
Authors: Lu Peipei , Xie Wen
Published: November 2023

Background & Aims

TCDD, 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.

Methods

Humanized 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.

Results

We 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.

Conclusions

Human 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 Information

This 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 Information

This 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 Information

This work was supported by the National Institutes of Health (EY10291, MH101679, and T32).
Structural and Functional Characterization of the Metastatic RhoGEF P‐Rex1 and its Regulation by PtdIns(3,4,5)P3: Towards Inhibitory Small Molecule Development
Authors: Cash Jennifer N. , Davis Ellen M. , Tesmer John J.G.
Published: November 2023

Phosphatidylinositol 3,4,5‐trisphosphate (PIP3)‐dependent Rac exchanger 1 (P‐Rex1) is a Rho guanine‐nucleotide exchange factor (RhoGEF) that regulates cell motility and is strongly associated with cancer metastasis. P‐Rex1 is synergistically recruited to the cell membrane and activated by PIP3 and heterotrimeric G protein βγ (Gβγ) subunits, positioning the enzyme downstream of multiple classes of cell surface receptors that control processes such as cytoskeleton rearrangement and cell migration. P‐Rex1 has thus become an attractive therapeutic target for the suppression of cancer metastasis. However, development of inhibitors against P‐Rex1 is hindered by the fact that its regulatory mechanisms are poorly understood. My primary goal is to define the molecular basis for regulation of P‐Rex1 by PIP3 and Gβγ using X‐ray crystallography and biochemical and cell‐based assays, with the aim of identifying the surfaces of P‐Rex1 that are important for interaction with these molecules as well as their mechanisms of activation. Thus far, I have determined crystal structures of the tandem Dbl homology (DH)/pleckstrin homology (PH) domain catalytic core of P‐Rex1 in complexes with its substrate small GTPases Cdc42 and Rac1, as well as of the independent PH domain in complex with Ins(1,3,4,5)P4, a soluble analog of PIP3. Using site‐directed mutagenesis, we have shown that the PH domain is necessary and sufficient for PIP3‐dependent activation. Interestingly, mutation of residues within the PIP3 binding site does not abrogate membrane binding, suggesting that PIP3 activates P‐Rex1 through an allosteric mechanism as opposed to simple recruitment to the cell membrane. These studies have also opened the door to medium‐throughput screens anticipated to identify small molecule probes that compete with physiological regulators of P‐Rex1. These compounds could potentially be developed into molecules that block PIP3 binding, thereby inhibiting P‐Rex1 activation in cells and, ultimately, blocking cancer metastasis.

Support or Funding Information

This work was supported by an American Cancer Society – Michigan Cancer Research Fund Postdoctoral Fellowship (PF‐14‐224‐01‐DMC) to J.C. and an American Heart Association Undergraduate Student Research Program Award (14UFEL20510027) to E.D.
Comparative Analysis of Organophosphate Developmental Neurotoxicity in a Freshwater Planarian
Authors: Hagstrom Danielle , Zhang Siqi , Hirokawa Hideto , Taylor Palmer , Collins Eva‐Maria S.
Published: November 2023

Organophosphates (OPs) are among the most commonly used pesticides in the U.S. and act by irreversibly inhibiting acetylcholinesterase (AChE), leading to cholinergic overstimulation due to increased synaptic levels of acetylcholine, paralysis and death. Their environmental abundance, although below acutely toxic levels to humans, has been suggested to be at least partially responsible for the observed increase in childhood neurodevelopmental disorders. However, it is unknown through which mechanisms chronic, low‐dose prenatal and infant OP exposure causes neurotoxicity in the developing brain. In this study, we analyzed the toxicological profiles of five common OPs (chlorpyrifos, dichlorvos, diazinon, malathion and parathion) using a multi‐endpoint planarian screening platform. The freshwater planarian, Dugesia japonica, is an excellent in vivo model for developmental neurotoxicology studies because its remarkable regenerative capabilities allows development to be induced at will by amputation. Moreover, because of their short development time (12 days) and similar size, regenerating and adult animals can be tested simultaneously using the same assays to identify development‐specific toxicity. Importantly, despite its simplicity, features of the planarian brain are conserved in the mammalian brain on the molecular and structural levels making mechanistic analysis of neurotoxicity in this animal directly relevant to human health. With this platform, we characterized effects on lethality, regeneration and a range of stimulated and unstimulated behaviors. We found that the different OPs varied widely in toxic dose, affected endpoints and developmental sensitivity. Lastly, through biochemical analysis of AChE activity in OP‐treated animals, we found that, depending on the OP, prolonged inhibition of AChE was not necessary for or necessarily indicative of toxic effects. This indicates that toxicity may be induced by alternative mechanisms, necessitating further in depth mechanistic studies.

Support or Funding Information

DH supported by the NIH Cellular and Molecular Genetics Training Grant. EMSC supported by the Hellman foundation, the Burroughs Wellcome Fund CASI award and the Alfred P. Sloan Fellowship