bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc supplementation decreased ZIP4 mRNA abundance in peripheral blood mononuclear cells of rural women in south Ethiopia (1043.4)
Authors: Bogale Alemtsehay , Clarke Stephen , Stoecker Barbara
Published: June 2021

Understanding the control of zinc homeostasis as a result of chronic deficiency is hampered due to a lack of effective measures to assess Zn status. This randomized placebo‐controlled Zn supplementation trial in apparently healthy women evaluated effects of 20 mg Zn as Zn sulfate for 23 d on Zn transporter and metallothionein (MT) mRNA abundance and plasma Zn concentration. Non‐pregnant, non‐lactating women from rural southern Ethiopia with body mass index of 18.5‐24.9 were participants. PBMC collected from fasting blood at baseline and endpoint were stored in RNAlater. RNA was extracted from PBMC, reverse‐transcribed, and used for gene expression analyses. The mRNA abundance of Zn transporters (ZnT1/ZIP3/ZIP4) and MT1 was determined by qPCR. Plasma Zn was measured by inductively coupled plasma mass spectrometry. Abundance of ZIP4 decreased following Zn supplementation (p =0.04), though ZnT1, ZIP3 and MT1 abundance was unchanged. Despite these differences in expression, ZnT1 and ZIP4 abundance were correlated following Zn supplementation (r = 0. 934; p = 0.002). The mean (±SD) plasma Zn at baseline was 680±105 µg/L for the Zn group and 741±92 µg/L for the placebo group. At endpoint, plasma Zn in the Zn group increased to 735±79 µg/L (p<0.01) while the placebo group (717±103 µg/L) did not change from baseline (p = 0.31). Plasma Zn was not significantly related to abundance of mRNA for any Zn transporter or for MT. The observed change in ZIP4 mRNA abundance with Zn supplementation merits further investigation.

Grant Funding Source: Supported by Oklahoma Agricultural Experiment Station
Bioaccessibility of carotenoids and their distribution in lipoprotein fractions: comparison from two different sources in a randomized clinical trial (1044.21)
Authors: Ok Hyang , Jung Hye , Park Min , Do Gyeong‐Min , Kwon Oran
Published: June 2021

Both Chlorella and marigold are good sources of carotenoids. This study was performed to compare the bioaccessibility and lipoprotein distribution of carotenoids (lutein, zeaxanthin, α‐carotene, and β‐carotene) from Chlorella and marigold in human plasma and buccal mucosa cells (BMC). In a double‐blind, randomized, placebo‐controlled, and parallel design, twenty‐six healthy subjects were randomly assigned to receive placebo (n=6), Chlorella (n=10), or marigold (n=10) for 6 weeks. The doses of Chlorella and marigold were adjusted to provide the same amount of lutein at 10mg/d. At each week, fasting plasma levels of carotenoids were determined by HPLC, resulting in a significantly higher bioaccessibility in the Chlorella group compared to the marigold group. The levels of carotenoids in plasma and BMC were positively correlated at week 6, suggesting a potential use of BMC as a non‐invasive biomarker for bioaccessibility. Plasma levels of VLDL, HDL, and LDL were measured together with the levels of carotenoids in each lipoprotein fraction and then distribution of carotenoids in lipoprotein fractions was studied, showing a predominant localization of carotenoids in LDL fraction regardless of the sources. Future study is warranted for the potential role of carotenoids as preventive agents against LDL oxidation.

*This research was supported by MSIP/NRF Project (2012M3A9C4048761) and MEST/BK21 PLUS (22A20130012143).
The bioaccessibility of phenolic compounds from pineapple, mango and papaya are not compromised by their dietary fiber but to their amylaceous carbohydrates (1044.23)
Authors: Velderrain‐Rodriguez Gustavo , González‐Aguilar Gustavo , Ayala‐Zavala Jesus , Wall‐Medrano Abraham , Astiazarán‐García Humberto , Robles‐Sanchez Rosario , Sáyago‐Ayerdi Sonia
Published: June 2021

Physicochemical studies have shown that dietary fiber (DF) interacts with phenolic compounds (PC) present in many fruits. However PC release from DF in simulated gastrointestinal conditions has been scarcely reported. The aim of this study was to compare PC bio accessibility from freeze‐dried pulps of Mango (Mangifera indica cv. Ataulfo; MA), papaya (Carica papaya cv. Maradol; PM) and pineapple (Anana comosus cv. Esmeralda; PE) in an enzymatic digestion model (EDM). Soluble (SDF), insoluble (IDF) and total (TDF) DF and Klason lignin (KL) were assayed in 100g of dry weight (DW). MetOH : Water (80:20 v/v‐ Extractable (EPC) and total (TPC) PC, condensed (CT) and hydrolysable (HT) tannins, total antioxidant capacity (TAC) to reduce DPPH and ABTS radicals and fractions of PC associated (PCADF) to DF (SDF, IDF and TDF) were expressed in 100g fresh weight (FW). PC release from MA, PM and PE was also evaluated in EDM. PE had the highest (p<0.05) content of TDF (6.8g) and IDF (5.0g) but lesser SDF (1.8g) than MA & PM but KL was not detected in any fruit. MA showed a higher content (p蠄0.001) of EPC, HT and TPF (144.3, 130.0 y 274.3 mgEAG, respectively) than PM or PE. TAC of PE and PM was 73.4%/28.2% less than in MA (p蠄0.001) which directly correlate (rs蠅0.95) with EPC, HT and TPF content but inversely with TDF and IDF (rs蠅 0.87) of all three fruits. The lower the TDF and IDF (rs= ‐0.62) or higher TPC and EPC (rs=0.88) content the higher PCADF even when they represented 蠄6.1% TPC of all three fruits. In EDM 58, 47 and 56% of TFC present in MA, PM and PE were released with gastric pepsin and a further 33, 20 and 47% was liberated with α‐amylase which coincided with DPPH‐TAC at both enzymatic stages (rs=0.98 and 0.91, respectively). It is concluded that bio‐accessibility of phenolic compounds from Pineapple, Mango and Papaya are not compromised by their dietary fiber but for their amylaceous carbohydrates.

Grant Funding Source: Supported by CONACyT (Investigación Científica Básica 2012, Proyecto 179574)
A single ascending dose, initial clinical pharmacokinetic and safety study of ursolic acid in healthy adult volunteers (1044.6)
Authors: Hirsh Steven , Huber Luke , Zhang Peter , Stein Richard , Joyal Steven
Published: June 2021

Objective: To assess the safety and pK parameters after administration of a single ascending oral dose of ursolic acid.

Methods: 14 subjects were administered a single oral ascending dose of ursolic acid 100mg, 500mg and 1000mg. pK assessements were conducted at pre‐dose, 0.5 hr, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr, 24 hr and 48 hr.

Safety assessments were also conducted.

Results: Statistical analysis was conducted for 11 subjects who had completed the trial with no serious adverse events reported. For the 100mg dose: 4 subjects‐ ursolic content was noted at 5 timepoints at 0.5hr‐8hr with 4 timepoints‐ levels were lower than LLOQ (lower limit of quantification‐10.5ng/ml) and in 7 subjects, ursolic acid content was not observed. For the 500mg dose: ursolic acid content was observed in 6 subjects at 12 timepoints at 1‐12hr with 10 timepoints‐the levels were lower than LLOQ and in 5 subjects, ursolic acid content was not observed. For the 1000mg dose: ursolic acid content was observed in 9 subjects at 21 timepoints at 1‐12 hr with 12 timepoints‐the levels were lower than LLOQ and in 2 subjects, ursolic acid content was not observed.

Conclusion: These results revealed low and variable bioavailability for oral ursolic acid in human subjects. Possible reasons could include: Poor water solubility with decreased absorption and rapid elimination, metabolism by the gut wall and liver with poor intestinal absorption. Metabolism of ursolic acid by the human gut microbiome may yield variability in the peak plasma levels. Human peak level results differ remarkably from the peak levels observed in the preclinical animal research.
Melibiose, a non‐digestible saccharide, promotes absorption of quercetin glycosides in rat small intestine with a novel mechanism (1044.9)
Authors: Tanaka Seiya , Shinoki Aki , Hara Hiroshi
Published: June 2021

We examined effects of melibiose a non‐digestible saccharide, on intestinal absorption of quercetin glycosides, which are low bioavailability but have antioxidant, anti‐inflammatory and anti‐carcinogenic properties. We used water‐soluble quercetin glycosides (a mixture of ~7 glucose adducts of quercetin‐3‐O‐glucoside, Q3GM). Melibiose (150 µmol) with or without Q3GM (15 or 60 µmol) was directly injected into a ligated segment of rat jejunum in anesthetized rats or into the duodenum in conscious rats. Before and after an injection, the concentrations of quercetin derivatives in the portal blood collected through a cannula were measured by LC‐MS. Melibiose enhanced absorption of quercetin aglycon and total quercetin derivative including conjugated and methylated forms in situ experiments. We found a similar tendency in in vivo experiment using portal cannulated rats. Additionaly, we found that Q3GM was very rapidly hydrolyzed to Quercetin‐3‐O‐glucoside (Q3G) by a homogenate of the small intestinal mucosa, but melibiose promoted further hydrolysis of Q3G to quercetin aglycone. In conclusion, non‐digestible saccharides, melibiose, enhances intestinal absorption of quercetin glycosides with increasing hydrolysis to quercetin aglycone in the small intestinal lumen. This study suggests that melibiose contribute health promotive effects of quercetin glycosides.
Degradation of chromatin modifiers, histone deacetylases, by Spirulina platensis extracts in macrophages (1045.11)
Authors: Pham Tho , Lee Ji‐Young
Published: June 2021

The roles of dietary components in the modulation of chromatin modifiers, such as histone deacetylases (HDACs), to control gene transcription are largely unknown. We previously demonstrated that the organic extract of Spirulina platensis (SPE), a blue‐green alga, potently inhibited the expression and secretion of pro‐inflammatory mediators in macrophages. Therefore, the objective of our study was to investigate whether SPE ameliorates inflammation through the modulation of HDACs. We found that SPE facilitated rapid degradation of HDAC2, 4 and 9 in a time and dose‐dependent manner in RAW 264.7 macrophages. More specifically, HDAC4 was more labile and sensitive to SPE when compared to HDAC2. The increased HDAC degradation by SPE was not unique to RAW 264.7 macrophages, but also occurred in bone marrow‐derived macrophages and other cell lines. Inhibition of proteasomes, lysosomal acidification, or calpain proteases with MG132 (10 μM), chloroquine (50 μM), or calpeptin (20 μg/mL), respectively, revealed that SPE facilitated the degradation of HDAC4 at least partly via lysosomes and calpain proteases, but not proteasomes. In contrast, the degradation of HDAC2 by SPE was not attenuated by the inhibitors. Our results demonstrate that SPE can regulate the protein levels of HDACs and therefore might potentially possess an epigenetic mode of action for the regulation of gene transcription.

Grant Funding Source: Funded by NIH R21AT005152
Sulforaphane influences histone methylation in advanced prostate cancer cells (1045.15)
Authors: Watson G , Wickramasekara S , Palomera‐Sanchez Z , Black C , Maier C , Williams D , Dashwood R , Ho E
Published: June 2021

Sulforaphane (SFN) supplementation is well tolerated and exhibits cancer‐selective cytotoxicity in preclinical models. SFN decreases global DNA‐methyltransferase (DNMT) and histone deacetylase (HDAC) activity in prostate cancer (PCa) cells, two classes of chromatin modifying enzymes (CME) involved in heterochromatin (HC) establishment and stability. HC marks co‐occur due to coordinated CME activity; therefore, we hypothesized that deficiencies in DNMT and HDAC activity would lead to alterations in histone methyltransferase (HMT) activity. SFN (15 µM) led to a global decrease in histone H3 lysine 9 trimethylation (H3K9me3) in PC3 PCa cells. Investigation into the underlying mechanism suggests SFN influences the H3K9 HMT SUV39H1. SFN leads to SUV39H1 ubiquitination and acetylation, posttranslational marks that are associated with SUV39H1 instability and reduced enzymatic activity. SUV39H1 modification coincided with decreased HC stability. Inhibition of SUV39H1 activity by small molecule inhibitor is cytotoxic to PCa cells, suggesting SUV39H1 inactivation contributes to the cancer‐suppressive activity associated with SFN. Our results suggest that destabilization of HC plays an active role in mediating cytotoxicity in response to SFN and supports further development of SFN as a therapeutic option and SUV39H1 as a therapeutic target for PCa.

Grant Funding Source: Supported by NIH P01 CA090890
Priming of the antiviral immune transcriptome by a mixed flavonoid‐fish oil supplement in adult overweight women (1045.23)
Authors: Cialdella‐Kam Lynn , Nieman David , Knab Amy , Shanely R. Andrew , Meaney Mary Pat , Jin Fuxia , Sha Wei , Ghosh Sujoy
Published: June 2021

Flavonoids and fish oils have anti‐inflammatory and immune modulating influences. The purpose of this study was to determine if a mixed flavonoid‐nutrient‐fish oil supplement (Q‐Mix; 1000 mg quercetin, 400 mg isoquercetin, 120 mg EGCG, 220 mg EPA, 180 mg DHA) would alter genomic profiles in overweight, middle‐aged women. In this double‐blinded placebo‐controlled trial, women (n=48; age=56.1±8.3y; BMI=33.2±0.8kg/m2) were randomly assigned to either Q‐Mix supplement or placebo for 10 weeks. Whole blood derived mRNA, collected at 0‐ and 10‐wk, was analyzed using Affymetrix U133 Plus 2.0 microarrays. Plasma levels increased with Q‐mix supplementation by 388% for quercetin, 95% for EPA, 18% DHA, and 20% DPA. Gene Set Enrichment Analysis revealed upregulation of three pathways in Q‐Mix vs. placebo related to interferon‐induced antiviral mechanism. Consistent with this finding, over‐representation analysis also disclosed that infection, immune cell trafficking, and inflammatory pathways were inhibited in Q‐mix compared to placebo. Thus, relative to the placebo, a 10‐wk Q‐mix supplementation elicited a significant rise in plasma quercetin, EPA, DHA, and DPA and stimulated an antiviral whole‐blood transcriptomic response in adult overweight women.
Milk lactoferrin supplementation enhances early brain development and cognition in postnatal piglets (1045.27)
Authors: Wang Bing , Chen Yue , Zheng Zhi , Zhu Xi , Shi Yu , Tian Dan , Troy Frederic
Published: June 2021

Background: Lactoferrin (Lf) is an iron‐binding, sialylated glycoprotein prevalent in milk, and known to have multifunctional health benefits. Its potential role in neurodevelopment and cognition remains unknown.

Objective: To determine the functional effect and underlying molecular biochemical mechanism of milk lactoferrin on early brain development and cognitive function in postnatal piglets.

Method: 3‐day‐old male piglets (n=33) were randomly allocated 2 groups with supplemented Lf at a dose level of 155 mg/kg/d for 38 days. Global gene expression profile and the selected target gene and protein expression were determined. Learning and memory were assessed using an eight‐arm radial maze.

Results: Dietary Lf enhanced the piglet learning speed and long‐term memory. Several gene expression pathways associated with promoting neurodevelopment and cognitive functions in Lf treatment group were up‐regulated in hippocampus. At least ten genes involved in the brain‐derived neurotrophic factor (BDNF) signaling pathway in the hippocampus were affected.

Conclusion: Dietary promotes early brain development and cognitive functions by up‐regulating the BDNF signaling pathway in postnatal piglets.

Grant Funding Source: Supported by Xiamen University and Nestle Research Centre Beijing
Astragalin inhibition of oxidative stress‐induced airway epithelial fibrosis by blocking autophagy and mitochondrial dysfunction (1045.30)
Authors: Cho In‐Hee , Gong Ju‐Hyun , Kang Young‐Hee
Published: June 2021

Chronic asthma is characterized by ongoing airway inflammation with involvement of reactive oxygen species (ROS) in the cellular signaling cascades, leading to subepithelial fibrosis. This study examined that 72 h‐exposure of 20 μM H2O2 promoted airway fibrosis with concurrent loss of E‐cadherin and induction of collagen type 1 in airway epithelial BEAS‐2B cells, which was reversed by astragalin non‐toxic at 1‐20 μM. This study further investigated that the regulation of autophagy was responsible for inducing airway fibrosis. Growing evidence has revealed the crucial involvement of autophagy in the ROS‐associated pathogenic processes underlying pulmonary diseases. H2O2 highly enhanced the induction of the autophagy‐related beclin‐1 expression within 48 h. Astragalin blocked the beclin‐1 induction and mTOR activation by H2O2, indicating that this compound may inhibit ROS‐promoted the initiation of the formation of the autophagosome in autophagy. In addition, astragalin deterred the expression induction of the mitophagy marker LC3A/B in 48 h‐H2O2‐exposed BEAS‐2B cells. These results demonstrate that dietary astragalin can be effective in blocking ROS‐promoted bronchial epithelial fibrosis by inhibiting autophagosome initiation through mTOR activation and by ameliorating mitochondrial dysfunction.

Grant Funding Source: Supported by Brain Korea 21 plus
Resveratrol suppresses TH2 cytokine production linked to MAPK activation in IgE‐antigen complex‐exposed basophilic mast cells (1045.31)
Authors: Han Seon‐Young , Kang Young‐Hee
Published: June 2021

Mast cells contribute to the development and progression of TH2 cytokine‐mediated allergic inflammation and cooperate with other immune cells to promote optimal TH2 cytokine responses in vivo. play important roles in allergic reaction. Our current study examined whether resveratrol suppressed the production of inflammatory TH2 cytokines in the allergic model using cultured rat basophilic leukemia RBL‐2H3 cells. Basophilic mast cells pre‐treated with resveratrol non‐toxic at 1‐25 μM were sensitized with anti‐dinitrophenyl‐immunoglobulin E (anti‐DNP‐IgE), and subsequently stimulated by DNP‐human serum albumin antigen. Resveratrol dose‐dependently diminished the secretion of interleukin (IL)‐3, IL‐4, IL‐13 and tumor necrosis factor‐α from anti‐DNP‐IgE‐sensitized cells, evidenced by ELISA. In addition, Western blot analysis showed that 1‐25 μM resveratrol dose‐dependently mitigated the phosphorylation of p38 MAPK, ERK and JNK elevated in mast cells exposed to IgE‐antigen complex. Taken together, these results showed that resveratrol inhibited TH2 cytokine responses linked to MAPK signaling. Therefore, resveratrol may possess the therapeutic potential of targeting basophilic mast cells in preventing or alleviating the development and progression of allergic inflammation.

Grant Funding Source: Supported by Brain Korea 21 plus
Effects of cinnamon metabolites on glucose utilization and glycolysis (1045.43)
Authors: Jiang T. Alan , Moss‐Pierce Tijuana , Tu Zheng
Published: June 2021

Studies have shown that proanthocyanidins (oligomers/polymers of flavan‐3‐ols) in cinnamon may play a role in hypoglycemic activities. However, bioavailability of the oligomers and polymers is low, and they need to be biotransformed by the colonic microbiota into smaller metabolites in order to be absorbed. Therefore, phenolic metabolites, rather than the original high molecular weight compounds may be responsible for the biological activity derived from cinnamon consumption. We examined the effect of the following metabolites on glucose metabolism in vitro: 3 Hydroxyphenylacetic acid (3‐HPAA), 3,4‐dihydroxyphenylacetic acid (3,4‐DHPAA), 3‐(4‐hydroxyphenyl)propionic acid (3‐(4‐H)PA), 4‐hydroxyphenylacetic acid (4‐HPAA), α‐hydroxyhippuric acid, ferulic acid, and 4‐hydroxybenzoic acid. All were purchased from Sigma‐Aldrich. Glucose consumption in serum starved C2C12 myocytes culture medium was measured using a glucose assay kit (Sigma‐Aldrich). Glycolysis was measured in C2C12 myocytes by the extracellular acidification rate (ECAR) using a Seahorse XF96 analyzer with 10mM glucose. 3‐HPAA, 3,4 DHPAA, and ferulic acid increased glucose utilization by 60%, 118%, and 247%, respectively. In addition, 3,4‐DHPAA and ferulic acid increased glycolysis by 57%, and 62%, respectively. It therefore appears that certain proanthocyanidin metabolites exert biological activity in vitro.

Grant Funding Source: This research was supported by McCormick and Company.
Modulation of estrogen metabolism by broccoli: mechanistic investigation of cancer prevention (1045.45)
Authors: Gaikwad Nilesh , Kucera Heidi , Olivera Deborah , Shibamoto Takayuki
Published: June 2021

Modulation of Estrogen Metabolism by Broccoli: Mechanistic Investigation of Cancer Prevention Nilesh W. Gaikwad, Heidi R. Kucera, Deborah, Olivera, Taka Shibamoto Departments of Nutrition and Environmental Toxicology, University of California, Davis, CA 95616, USA Broccoli is commonly used as a popular vegetable in the human diet. Recently, broccoli sprouts have also received much attention because of their different bioactive constituents from mature broccoli. They are reported to be more effective in the prevention of carcinogenesis by reactive oxygen species than broccoli. However, very little information on their biological activities such as, antioxidant, anti‐inflammatory, anticancer, of broccoli sprouts, are available. Moreover, there are no reports on the antioxidant activity of the volatile compounds from broccoli sprouts. We hypothesize that bioactive compound(s) from broccoli sprouts could modulate estrogen metabolism towards non carcinogenic pathways resulting in a significant preventive effect. Present evidence shows the involvement of estrogens in carcinogenic processes within estrogen‐responsive tissues including the breast, endometrium and ovaries. Epidemiologic studies have indicated that breast cancer risk is higher in women with early menarche and late menopause, who have longer lifetime exposure to estrogens. Obese postmenopausal women have higher serum concentrations of free estrogen and are at greater risk of breast cancer. Gathering data suggests metabolism of estrogens to methides as well as catechol‐o‐quinones play a major role in estrogen carcinogenesis. To test our hypothesis, we have treated the MCF 10A cells with estrogens and broccoli sprout extract and using recently developed mass spectrometry based metabolomic platform we investigated the molecular mechanisms which leads to alteration of estrogen metabolic pathways.

Grant Funding Source: NA
Versican is a critical extracellular matrix protein in lung and brain during embryonic development in the mouse (1046.9)
Authors: Snyder Jessica , Washington Ida , Chang Mary , Frevert Charles
Published: June 2021

Versican, a chondroitin sulfate proteoglycan and a component of the extracellular matrix, is associated with fatal cardiac malformations if absent during embryonic development and is increased with injury in adult tissues. Changes in versican gene expression and accumulation have not been examined comprehensively in the lung and brain during mid to late gestation in the mouse. The objective of this work was to determine if versican accumulation and degradation change during lung and brain embryogenesis. Quantitative PCR and immunohistochemistry were used to measure versican expression, accumulation and degradation in the lung, brain, heart and liver of C57Bl/6 mice for five consecutive days during embryonic development. Consistent with previous reports, we found that total versican gene expression was higher in embryonic tissues when compared to adults and the highest total versican gene expression in embryos occurred at embryonic day 13.5 (E13.5). At E15.5, versican gene expression was lowest in the liver, highest in the brain, and intermediate in the heart and lung. There was differential expression of versican isoforms, with V0, V1, and V2 isoforms increased in the brain but only V0 and V1 isoforms increased in the lung. Versican expression in the lung peaked at E13.5, at which time there was strong versican immunoreactivity in the interstitium and airway basement membrane but little accumulation in airway epithelial cells. In the brain, immunoreactivity was strongest in the roof of the hind brain and neopallial cortex and in the deeper white matter tracts, and at later gestational ages there was little accumulation in the developing choroid plexus and periventricular gray matter. During embryonic development of the lung and brain, we found areas of versican expression that colocalized with the expression of DPEAAE, a versican degradation product. These results suggest that versican plays an important role in embryogenesis of the lung and brain.

Grant Funding Source: Supported by HL098067, RR030249
Dissecting the role of IKKβ in tumor‐specific cell death (1048.1)
Authors: Fields Peter , Beattie Amy , Limoge Michelle , Bakin Andrei
Published: June 2021

The TAK1 cell signaling pathway has been found to be very important for tumor invasion, metastasis, and angiogenesis in breast cancer. Disruption of TAK1 signaling in breast and prostate carcinoma cells reduces invasive and angiogenic capacities of tumor cells. In addition, disruption of TAK1 reduces the ability of tumor cells to metastasize to the lungs and bones. Recent studies in our lab indicate that TAK1 is also involved in tumor specific cell‐death. Inhibition of TAK1 sensitizes tumor cells to TNFα and cytotoxic drugs such as doxorubicin. TAK1 signaling is mediated via Mitogen‐activated protein kinases (MAPKs) and the heterotrimeric IKKα/IKKβ/NEMO complex, activating NFkB transcription factor. Given that NFkB is required for pro‐invasive and angiogenic activities of TAK1, we hypothesized that the IKKα/IKKß/NEMO complex may play a significant role in TAK1‐mediated survival of tumor cells. In the IKK complex, TAK1 can activate IKKβ or IKKα that phosphorylates IκB (an inhibitor of the transcription factor NFκB), triggering the K48‐linked polyubiquitination and degradation of IκB. As a result, NFκB is free to translocate to the nucleus and regulate the transcription of target genes such as MMP9 and CIAP2. The former contributes to tumor invasion and angiogenesis, while CIAP2 may protect cells from death. To address the role of IKKβ, which regulates canonical NFκB factors, we downregulated IKKβ using an RNA interference approach. In addition, we assessed the balance between the non‐canonical and canonical pathways by comparing levels of NFκB subunits such as RelA/p65 and p100/p52 in the nucleus and in the cytosol. It was found that a knockdown of IKKβ did not sensitize cells to TNFα. It was also found that a disruption of TAK1 increased non‐canonical NFκB activity in tumor cells. Further studies will be needed to clarify the role of TAK1 in tumor cell signaling.
Immunoexpression of the human kallikrein‐10 in surgically removed human pituitary tumors (1048.12)
Authors: Rotondo Fabio , Di Ieva Antonio , Kovacs Kalman , Syro Luis , Cusimano Michael , Yousef George
Published: June 2021

Human kallikrein 10 (hK10), a serine protease, plays an important role in the regulation of cell proliferation and tumor growth. Its presence was demonstrated in lactotrophs and corticotrophs of the nontumorous human pituitary. In this work, we investigated hK10 immunoexpression in 59 surgically removed human pituitary tumors. Tissues were fixed in formalin, embedded in paraffin. Immunostaining was performed by the streptavidin‐biotin‐peroxidase complex protocol using the LSAB+ Kit (DAKO, Carpenteria, CA) and an hK10‐specific rabbit polyclonal antibody (1:150).

Results showed that lactotroph adenomas removed from patients receiving no dopamine agonist medication were conclusively immunopositive for hK10. Immunopositivity was localized in the cytoplasm and was clearly visible in many adenoma cells. In the various tumor types (silent corticotroph subtype 1 & 2, oncocytic and gonadotrophic adenomas, somatotroph adenomas, and carcinomas), immunopositivity was very mild, seen only in few unevenly distributed tumor cells. Previous studies showed that hK10 possesses tumor suppressing properties and is decreased in rapidly growing tumor cells. Future studies should focus on hK10 immunoexpression in lactotroph adenomas exposed to dopamine agonist therapy which inhibits tumor growth.

This study was supported by the Jarislowsky and Lloyd Carr‐Harris Foundations.
Ameloblastoma driver mutations revealed by next‐generation sequencing of formalin‐fixed paraffin‐embedded specimens (1048.18)
Authors: Pollack Jonathan , McClary Andrew , Sweeney Robert , Biscocho Jewison , Myers Benjamin , Neahring Lila , Kwei Kevin , Qu Kunbin , Gong Xu , Ng Tony , Jones Carol , Varma Sushama , Odegaard Justin , Rubin Brian , Troxell Megan , Pelham Robert , Zehnder James , Beachy Philip , West Robert
Published: June 2021

Rare cancer types are not only understudied, but are typically represented by formalin‐fixed paraffin‐embedded (FFPE) (rather than freshly‐frozen) specimens that are suboptimal for genomic analysis. Ameloblastoma is one such rare tumor type, thought to arise from ameloblasts, the cells that deposit enamel during tooth development. Though typically benign, ameloblastomas are locally destructive to the jaw and face, and new non‐surgical interventions are needed. To discover novel driver mutations and therapeutic targets, we optimized methods and performed whole‐transcriptome sequencing and/or targeted exon sequencing (TruSeq Cancer Panel) of 8 FFPE cases. Identified mutations were verified, and then evaluated on a larger, independent set of 22 FFPE cases by PCR and Sanger sequencing. From the analysis, we identified recurrent somatic mutations in three key developmental or signaling pathways, including Hedgehog, fibroblast growth factor, and MAP kinase pathways. Functional interrogation of a novel Hedgehog pathway mutation confirmed increased basal pathway activity, and defined the response profile to various pharmacologic Hedgehog inhibitors. Together, our results define new ameloblastoma drivers and nominate new molecularly‐directed therapies for this rare but disfiguring disease. More generally, our findings validate a robust approach for discovering driver mutations in rare cancers.

Grant Funding Source: Supported by Stanford University Department of Pathology
The effect of CBD, CBG and a combination of CBD plus CBG on Haccat human skin carcinoma cells (1048.19)
Authors: Javid Farideh , Afshinjavid Saeed
Published: June 2021

The effect of CBD, CBG and a combination of CBD plus CBG on Haccat human skin carcinoma cells Cannabinoid pharmacology has received attention in cancer research for their potential in combating cancer (Alexander et al., 2009). The present study investigated the potential anti‐tumour activity of cannabidiol and cannabigerol on skin tumour cells.

Haccat cells (American Type Culture Collection) were grown and maintained in DBEM medium plus10% fetal bovine serum at 37oC, 5% CO2 .The cells were plated in 96‐well culture plates at a density of 1x104 cells/well and allowed to adhere at 37oC for 24 hours. The following day, various doses of the compounds or were added to the cells and further incubated for 24, 48, 72 and 96 hours. Then the supernatant was removed and MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide) was added for 4 hours. The ability of cells to form formazan crystals by active mitochondrial respiration was determined after dissolving the crystals in DMSO. Cytotoxicity was expressed as a relative percentage of the absorbance measured at 540 nm.

CBD induced dose‐dependent cytotoxic effect (IC50 of 4.83+1.4, 4.75+0.06; 5.22+0.3 and 5.19+1.27 micromolar for 24, 48, 72 and 96 h contact time respectively). Addition of CBG also induced cell toxicity with IC50 of 1.96+0.3, 5.5+0.34, 8.32+0.19 and 7.85+1.47 for 24, 48, 72 and 96 h respectively. A combination of CBD plus CBG induced cell toxicity with an IC50 of 2.4 + 0.05 over 96 h contact time.

The data suggest a significant (p<0.001) cytotoxicity when compounds are applied in combination.

We thank GW Pharmaceuticals for providing cannabinoid extracts.

Grant Funding Source: University of Huddersfield
An upregulated long noncoding RNA RP3‐337D23.3 in lung adenocarcinoma in never‐smokers promotes metastasis (1049.1)
Authors: Yin Rong , Qiu Mantang , Jiang Feng , Xu Lin
Published: June 2021

Objective: The present study was designed to screen and validate lung adenocarcinoma in never smokers (LACINS) specific long noncoding RNAs (lncRNAs).

Methods: A custom‐designed human lncRNA expression microarray was utilized to determine lncRNA expression profiles of LACINS and indentify LACINS specific lncRNA. Five LACINS and paired adjacent normal lung tissues were used for microarray analysis and another 10 LACINS were used for further searching of LACINS specific lncRNAs by quantitative real‐time polymerase chain reaction. Function of RP3‐337D23.3 was investigated by cell proliferation, transwell, and wound healing assay in vitro.

Results: A total of 3353 lncRNAs were differentially expressed in LACINS (fold change >2), 1509 upregulated and 1849 downregulated. Eleven candidate LACINS specific lncRNAs were identified and further validated in 10 pairs of LACINS and corresponding lung tissues. We identified RP3‐337D23.3 as a LACINS specific lncRNAs which is significantly up‐regulated in tumors as compared with adjacent normal tissues. Additionally, RP3‐337D23.3 is significantly overexpressed in LACINS with metastasis compared with those without metastasis (p=0.0006). In vitro siRNA mediated silencing of RP3‐337D23.3 led to remarkable decreased invasion, migration, and proliferation abilities of H1975 cell line.

Conclusion: We identified the first LACINS specific lncRNA, RP3‐337D23.3, and provided preliminary evidence that RP3‐337D23.3 could promote LACINS metastasis.

Grant Funding Source: National Natural Science Foundation of China (81372321)
Glypican‐5 regulates lung cancer cell metastasis through Wnt signaling pathway (1049.3)
Authors: Xu Lin , Yin Rong , Jiang Feng , Yang Xin
Published: June 2021

Glypican‐5 (GPC5) may be a potential tumor suppressor gene in non‐small cell lung cancer (NSCLC). Previously, we determined that GPC5 over‐expression in lung cancer cells could significantly suppress their migration, invasion, and proliferation activities and also induced G1/S phase arrest of the cells in vitro. However, the signaling mechanism of GPC5 is unclear. We reported here that GPC5 inhibited the metastasis of lung cancer cells in vitro and in vivo by blocking canonical WNT signals. Wnt signaling in vitro and in vivo was assessed in A549 cells transfected with GPC5 recombinant lentivirus. The interaction between GPC5, E‐Cadherin andβ‐Catenin was assessed by coimmunoprecipitation. β‐catenin‐dependent transcriptional activity was assessed by the TOPFLASH luciferase assay. Expression of GPC5, Wnt signaling molecules and target genes was analyzed by real‐time PCR and Western blot. We found that GPC5 is able to inhibit canonical Wnt signals involved in cell migration, invasion, and proliferation. Our results are consistent with the potential role of GPC5 as a metastasis suppressor in lung cancer.

Grant Funding Source: National Natural Science Foundation of China (81201830)
Evaluation of tissue section cryostorage on immunohistochemistry (1050.1)
Authors: Estrada Larissa , Amaral Ana , Giannaris Eustathia , Heyworth Nadine , Mortazavi Farzad , Ngwenya Laura , Roberts Debra , Robinson Amy , Killiany Ronald , Rosene Douglas
Published: June 2021

Storage of tissue sections for significant periods is often required to allow tissue from multiple samples, acquired over months to years, to be processed together, in the same reagents, for quantitative studies using immunohistochemistry. Several protocols for storage in sucrose with different additives have been reported that assert there is no effect on immunogenicity. However, there have been no quantitative assessments of how long‐term frozen storage in any cryoprotectant affects immunogenicity. The present study analyzed existing datasets, derived from monkey brain sections stored at ‐80°C in 15% phosphate buffered glycerol to determine whether time frozen in this cryoprotectant affected stereology counts of cells immunostained for NeuN, Parvalbumin, Orexin‐A, Doublecortin, BrdU or pro‐BDNF. Multiple regression analyses controlling for age and sex of the subjects in the different data sets showed that time in 15% glycerol did not affect stereological counts for any marker. Experiments are underway to compare our 15% glycerol cryoprotectant to four 30% sucrose based cryoprotectants for preservation of immunogenicity after frozen storage.

Grant Funding Source: Supported by NIH grants R01‐AG043640 and R01‐AG042512
Laminitis‐specific microRNAs in equine plasma (1050.6)
Authors: Kim Myung‐Chul , Lee Seung‐Woo , Cui Feng‐Ji , Ryu Doug‐Young , Kim Yongbaek
Published: June 2021

Purpose: To identify laminitis‐specific plasma microRNAs in a horse Method and materials: In order to induce laminitis experimentally, oligofructose was administered to a horse through nasogastric tube. Peripheral blood was collected from the jugular vein at the time of pre/post induction of laminitis. After euthanasia, various tissues including laminae were collected from the horse. Total RNA was extracted and subjected to the reverse transcription to cDNA. The cDNA products of the small RNA fragments ranging from 40bp to 60bp were sequenced directly using Illumina HiSeq 2000 sequencer.

Results: A total of 288 miRNAs were identified in laminar tissues and 262 miRNAs were identified in the plasma of horse with laminitis. Fifteen miRNAs were identified as laminitis‐specific plasma miRNAs, based on the following criteria: laminar tissue‐specific expression compared to other tissues and significant increase of the plasma miRNA level after the induction of laminitis.

Conclusions: The present study revealed a subset of plasma miRNAs specific to horse laminitis, suggesting the potential value of the miRNAs as a biomarker for the diagnosis of equine laminitis.

Grant Funding Source: Supported by Bio‐industry Technology Development Program of iPET, Ministry of Agriculture, Korea
Low molecular weight fucoidan against diabetic nephropathy (1051.17)
Authors: Zhou Hong
Published: June 2021

The objective of the present study was to investigate the therapeutic effect and underlying mechanisms of LMWF on diabetic nephropathy.LMWF was administered to the diabetic rats at a daily dose of 100 mg/kg body weight by intragastric administration. Following 12 weeks treatment, we measured body and kidney weight, blood glucose, blood pressure, creatinine and urea concentration in serum and urine, urine output, urinary osmolality, urinary protein excretion. α‐SMA, E‐cadherin, fibronectin, TGF‐β1, CTGF were measured by Western blot to evaluate the level of epithelial‐to‐mesenchymal transition (EMT). In addition, the phosphorylation of Akt, Smad3, p38 and ERK in renal tissue were evaluated. In virio, HK2 renal tubular epithelial cells pretreated with LMWF were stimulated by the recombinant human TGF‐β1 (10 ng/ml) for 48 h. α‐SMA, vimentin, E‐cadherin, fibronectin, CTGF were measured by Western blot to evaluate the level of EMT. In addition, activity of Akt, Smad and MAPK signal pathway in HK2 cell were evaluated.Renal expression of TGF‐β1, CTGF, fibronectin and α‐SMA protein were increased significantly in diabetic rats compared to those in the controls. E‐cadherin protein expression in diabetic kidneys decreased significantly. LMWF effectively reduced hypertension, proteinuria, renal TGF‐β1, CTGF, fibronectin and α‐SMA up‐regulation, as well as renal E‐cadherin down‐regulation in diabetic rats without a significant effect on blood glucose. LMWF also inhibited the activation of Smad, Akt, MAPK pathways. In vitro, LMWF alleviated TGF‐β1 induced EMT in HK2 cells via inhibition of Smad, Akt, MAPK pathways in a dose‐dependent manner.

Our in vivo and in vitro studies show that LMWF ameliorates diabetic nephropathy, possibly through inhibiting the expression of TGF‐β1 and Smad, Akt, MAPK signaling pathways downstream, then subsequent suppressing EMT. The data provide evidence that LMWF may serve as a potential therapeutic agent for diabetic nephropathy.
microRNA‐34a mediates impaired angiogenesis in diabetes: role of SIRT1 and metformin (1051.2)
Authors: Arunachalam Gnanapragasam , Samuel Samson , Ghosh Suparna , Triggle Christopher , Ding Hong
Published: June 2021

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with diabetes mellitus. In particularly, hyperglycemia‐associated impaired angiogenesis and vascular dysfunction critically contributes to the pathogenesis of diabtes‐associated CVD. Recent findings demonstrated the importance of microRNAs (miRNAs) in the vasculature and emerged as important regulators of angiogenesis. Our data with microvascular endothelial cells (MMECs) cultured in high glucose (HG, 40 mM) indicate a significant increase in miR‐34a expression and this induction paralleled and correlated with altered expression of SIRT1, eNOS (phospho/acetylation) and makers of angiogenesis (VEGF, angiopoietins and thrombospondins) as well as a significant impairment of tube‐forming activity (angiogenesis) when compared with normal glucose (NG, 11mM). Interestingly, inhibition of miR‐34a (anti‐miR) increased SIRT1 expression and attenuated changes in downstream signaling and impaired angiogenesis in HG‐exposed MMECs. Conversely, treatment with metformin (50μM) also inhibited miR‐34a expression and attenuated HG‐induced impaired angiogenesis in MMECs. miR‐34a, via the regulation of SIRT1 expression, has an anti‐angiogenic action in microvascular endothelial cells that is attenuated by metformin. miR‐34a may represent a new therapeutic target for the prevention/treatment of diabetic vascular disease.

Grant Funding Source: This project was funded by grant # UREP 13‐116‐3‐024, NPRP 5‐149‐3‐040
Sodium 4‐phenylbutyrate reverses glucose deprivation‐induced endoplasmic reticulum stress and death signal in cultured mouse microvascular endothelial cells (1051.3)
Authors: Samuel Samson , Arunachalam Gnanapragasam , Ghosh Suparna , Emara Mohamed , Ding Hong , Triggle Chris
Published: June 2021

In diabetes mellitus, despite higher than normal levels of blood glucose, deficiencies in signaling mechanisms results in a failure to utilize glucose, creating a scenario of variable glucose deprivation (GD). Reports suggest that stringent control of blood glucose in diabetic subjects invariably increases the risk of hypoglycemia, the severity of which is associated with increased cardiovascular risk. However, in such conditions the possible role of endoplasmic reticulum (ER) stress associated cell death during GD in microvascular endothelial cells has not been studied. Mouse Microvascular Endothelial cells (MMECs) subjected to GD for 24h showed significant increase in the levels of GRP78, p‐eIF2α, ATF4 & pro‐apoptotic CHOP, while levels of total PERK decreased & total‐ eIF2α did not change. Autophagic markers showed significant up‐regulation upon GD. 10mM of 4PBA, a chemical chaperone, significantly reversed the GD induced changes in ER stress & autophagic markers. Immunocytochemical analysis revealed that stress granules (SGs) are assembled at early hours (1h‐8h) of GD while very few SGs are observed at 16h‐24h. Our data suggests that GD induced ER stress & associated activation of death signal could play a major role in GD associated endothelial cell loss, which could be reversed by treatment with suitable chaperones.

Grant Funding Source: Supported by: QNRF grants # NPRP 4‐910‐3‐244 & JSREP 3‐016‐3‐009
Sexual dimorphism in aortic endothelial function of Zucker diabetic fatty rats: possible involvement of superoxide production (1051.8)
Authors: Han Xiaoyuan , Shaligram Sonali , Sangüesa Gemma , Anderson Leigh , Rahimian Roshanak
Published: June 2021

Little is known about the interaction between diabetes and sex in vasculature. This study was designed to investigate whether there were sex differences in rat aortic endothelium‐dependent vasodilation (EDV) in Zucker diabetic fatty (ZDF) rats, and the potential role of superoxide. EDV to acetylcholine (ACh) was measured in aortic rings pre‐contracted with phenylephrine before and after pretreatment with apocynin (100 μM), a NADPH oxidase (Nox) inhibitor. In addition, the level of Nox (a potent source of superoxide) and PKCβ mRNA expression were determined using real‐time RT‐PCR. ACh‐induced relaxations were significantly greater in female lean rats compared with male lean rats. Accordingly, male lean rats had higher PKCβI expression level than female lean rats. Diabetes significantly impaired EDV in aortic rings from female ZDF rats, however, potentiated the relaxation in males. Pre‐incubation of aortic rings with apocynin increased EDV only in diabetic female group, suggesting impairment of EDV in female ZDF aorta was partly due to an increase in the activity of superoxide. Accordingly, the levels of Nox1, Nox4, and PKCβ mRNA expression were substantially enhanced in aorta of female ZDF rats compared to those in lean animals. These data suggest that an elevation of superoxide may partially contribute to the predisposition of the female aorta to injury in type 2 diabetes.

Grant Funding Source: NIH/NIDCR
Evidence that chronic administration of 17β‐estradiol decreased the vasopressor responses in streptozotocin‐diabetic female rats (1051.9)
Authors: Centurion David , Acosta‐Cota Selene , Sanchez‐Lopez Araceli
Published: June 2021

In vitro studies have shown that 17β‐estradiol has beneficial effects in the cardiovascular system by activation of nitric oxide pathway. However, these effects have not been demonstrated in streptozotocin‐induced diabetic rats in the systemic vasculature in vivo. Thus, this study determined the effect of 17β‐estradiol on the vasopressor responses induced by sympathetic stimulation or i.v. injections of noradrenaline, methoxamine and B HT 933 in sham‐operated or ovariectomized, diabetic or non‐diabetic female rats. For this purpose, rats were ovariectomized or sham‐operated. One week later the animals were treated with streptozotocin (60 mg/kg, i.p.) or its vehicle. Two weeks later these rats were treated with 17β‐estradiol (10 μg/kg, s.c.) or its vehicle daily during five weeks. Then, under anaesthesia with ketamine and xylazine, the animals were pithed and prepared to measure blood pressure and heart rate. 17β‐estradiol failed to modify the vasopressor responses to: (i) sympathetic stimulation, noradrenaline, methoxamine or B HT 933 in sham‐operated non‐diabetic rats; (ii) sympathetic stimulation or B‐HT 933 in sham‐ operated diabetic rats; (iii) noradrenaline or methoxamine in ovariectomized non‐diabetic rats. In contrast, 17β‐estradiol significantly decreased the vasopressor responses to: (i) noradrenaline and methoxamine in sham‐operated diabetic rats; (ii) sympathetic stimulation or B‐HT 933 in ovariectomized non‐diabetic rats; and (iii) sympathetic stimulation, noradrenaline, methoxamine or B HT 933 in ovariectomized diabetic rats. These results, taken together, suggest that chronic administration of 17β‐estradiol produced a protective effect in the systemic vasculature in streptozotocin‐induced diabetic rats. This is the first in vivo study showing this protective effect of 17β‐estradiol during diabetes.

This project was financially supported by Conacyt (Grant number 152534).

Grant Funding Source: Supported by Conacyt 152534