bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of TGFβ1 and HTRA1 in the pathogenesis of TMJ osteoarthritis (922.8)
Authors: Wong Phillip , Reece Daniel , Hepworth Jared , Wilhelm Spencer , Mecham David , Kooyman David , Seegmiller Robert
Published: June 2021

The objective of this study is to understand the pathogenesis of osteoarthritis (OA) in the temporomandibular joint (TMJ) by testing for the presence of established biomarkers present in OA of the knee.

Six wild‐type mice (M. musculus) and six mice heterozygous for the chondrodysplasia gene mutation (cho/+) were used in this study. The mice were euthanized at three months of age, and their TMJs were isolated, fixed, decalcified, embedded in paraffin, and sectioned at 6µ thickness. Fifteen sections of each mouse were stained with Safranin O and Fast Green to determine the extent of OA. Fifteen additional sections of each mouse were immunohistochemically stained for the presence of TGFß1 and HTRA1.

Using the Modified Mankin and OARSI scoring systems, it was determined that OA is present in the TMJ of cho/+ mice but not in the wild type. Also, staining for TGFß1 and HTRA1 was positive in cho/+ TMJs and absent in the wild type.

We have established that at three months of age, i.e., when early signs of OA histopathology are detectable, TGFß1 and HTRA1 are upregulated in the TMJ articular cartilage of cho/+ mice. We propose that TGFß1 is a biomarker for OA and can be used as an early diagnostic tool of TMJ OA. This finding is of clinical significance in light of the current practice to use TGFß1 as a therapeutic agent of TMJ OA. Despite the lack of type II collagen in TMJ articular cartilage relative to that of the knee joint, the presence of HTRA1 here suggests that the degenerative changes involve the same pathogenetic mechanism as in the knee joint.

Grant Funding Source: NIH grant 1‐R15‐AR056861‐01A1
Real‐time in vitro monitoring of impedance in rat bone‐like cells (UMR 106‐01 BSP) (923.10)
Authors: Joseph Eugene , Joshi Neil , Kim Kevin
Published: June 2021

Cultured mammalian cells have been exhaustively studied by biological and biochemical means. Such studies have been effective, but limited in providing real‐time cellular data in vitro. The purpose of this study was to validate electric cell‐substrate impedance sensing (ECIS) to assess and monitor spreading and attachment of bone‐like cells (UMR cells) under varying initial cell plating concentrations and under treatment of the cells with camptothecin, an inducer of oxidative stress. UMR cells were routinely passaged, counted, then plated at 250 X 10³, 125 X 10³ and 62.5 X 10³ cells/mL in eight‐well ECIS array culture plates equipped with gold electrodes. Designated cells were treated with 6.0 μM camptothecin. The array culture plates were placed on the ECIS array holder of the ECIS instrument, then incubated at 37 degrees Celsius under 5% carbon dioxide infusion. Impedance measurements of the cells were monitored for 48 hours. The results showed that the total impedance, attachment and rate of spreading were affected in a cell concentration‐dependent manner. The higher the concentration of cells, the higher the impedance measurements as well as the rate of spreading. Cells treated with 6.0 μM camptothecin exhibited significantly lower impedance values and a slower rate of spreading as compared to controls. F test ANOVA was used to compare variances and all values in the results were expressed as means ±SD. The results validate the use of ECIS technology as a reliable approach for real‐time monitoring of UMR cells in culture. Moreover, ECIS provides data regarding the physical properties of cells that can be used to corroborate the outcomes of biological and biochemical studies of UMR cells.

Grant Funding Source: Supported by La Sierra University, CAS Dean
Color‐coded chemotherapy: S/G2‐phase‐trapping by methioninase pre‐treatment, indicated by FUCCI imaging, enables highly effective cancer chemotherapy (923.11)
Authors: Yano Shuya , Tome Yasunori , Digman Michelle , Momiyama Masashi , Suetsugu Atsushi , Gratton Enrico , Hoffman Robert
Published: June 2021

Methionine deprivation by methionine α,γ lyase (methioninase or METase) selectively arrests cancer cells during late S/G2‐phase of the cell cycle, where the cancer cells are highly sensitive to DNA‐damaging chemotherapy. Fluorescent ubiquitination‐based cell cycle indicator (FUCCI) (Cell 132, 487‐498, 2008), was used to monitor the onset of the S/G2‐phase block due to methionine deprivation effected by METase. The S/G2‐phase‐blocked cancer cells fluoresced yellow or green, in contrast to cancer cells in G1/G0 which fluoresced red due to FUCCI. Cancer cells, synchronously blocked in S/G2‐phase by METase fluorescing yellow‐green, were treated with doxorubicin, cisplatin, or 5‐fluorouracil. As a control, cancer cells treated with drugs only without rMETAse, were resistant to the drugs. rMETase treatment, followed by chemotherapy, when FUCCI indicated the onset of S/G2 block, was highly effective. Color‐coded chemotherapy, whereby the cell cycle of cancer cells is blocked in S/G2‐phase, as identified by fluorescent reporters, may be a general approach to effective cancer treatment.
Protein acetylation contributes to higher genome fidelity during replication and repair (925.5)
Authors: Balakrishnan Lata , Bambara Robert
Published: June 2021

In eukaryotic nuclear DNA replication, one strand of DNA is synthesized continuously, but the other must be made as short Okazaki fragments that are later joined. The discontinuous process is inherently more complex and the steps involving fragmented intermediates create risks for disruptions of genomic integrity. Our recent observations suggest that the cell uses specific lysine (K) acetylation to alter the enzymatic properties of DNA replication and LP‐BER proteins. Adding to the knowledge that acetylation partially inhibits human FEN1, we found that it also stimulates human Dna2 nuclease/helicase activities and promotes strand displacement synthesis by human pol δ and human pol β. These observations led us to hypothesize that acetylation increases the extent of RNA‐DNA primer removal during Okazaki fragment synthesis. Acetylation would also increase the patch size made by pol β during BER. The likely reason for regulation by acetylation is to fully excise the error‐prone primer region made by pol α and the damaged region needing repair. Involvement of p300, a histone acetyltransferase an epigenetic regulator, suggests that there are times when it is advantageous to the cell to switch between efficient and high fidelity DNA synthesis. Differential regulation of the activities of replication/repair by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as insuring correct processing. Such intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the re‐synthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. Thus we propose acetylation to be a regulatory mechanism that promotes higher fidelity of DNA transactions, ensuring genome stability. Possibly this depends on development, nutritional state, or the region of the chromatin that is being duplicated.

Grant Funding Source: Supported by National Institutes of Health grants GM098328 (to L.B.) and GM24441 (to R.A.B.)
Mutation in the PPARG ligand binding domain impairs PPARG‐mediated turnover of the p65 subunit of NF‐κB (930.2)
Authors: Mukoda Masashi , Stump Madeliene , Ketsawatsomkron Pimonrat , Quelle Federick , Sigmund Curt
Published: June 2021

PPARG is a ligand activated transcriptional factor known to regulate metabolism. A recent study suggests that PPARG may elicit its anti‐inflammatory activity by promoting the degradation of the p65 subunit of nuclear factor kappa B (NF‐kB). We previously demonstrated that transgenic mice expressing dominant negative PPARG in vascular muscle (S‐P467L) exacerbates atherosclerosis in ApoE knockout mice. However, the mechanism remains unknown. We hypothesize that mutant PPARG promotes inflammation because its stimulation of p65 degradation is impaired. To test this, we co‐transfected HEK293T cells with vectors encoding p65 and either empty vector, wild type (WT) PPARG, or different PPARG mutants (P467L, V290M, R165T, S273A, K268R and K293R). We showed that the level of p65 protein expression was significantly decreased in WT‐PPARG‐transfected cells compared to empty vector. Compared with WT‐PPARG, there was an increase in p65 in P467L transfected cells. Using immunoprecipitation, we also demonstrated that WT‐PPARG directly interacts with p65 and induces p65 of ubiquitination. However, this interaction is impaired with P467L PPARG.

We conclude that p65 turnover may be regulated, at least in part by PPARG and its mutation can result in impaired p65 degradation. Loss of PPARG‐mediated p65 degradation may contribute to the increased inflammation and formation of atherosclerotic lesions found in S‐P467L mice.

Grant Funding Source: Supported by NIH
Dysfunction of polyubiquitin binding by ABIN1 as a basis for lupus nephritis (930.3)
Authors: Korte Erik , G'Sell Rachel , Sheehan Ryan , Caster Dawn , Powell David
Published: June 2021

It is estimated that at least five million people worldwide have a form of systemic lupus erythematosus (SLE). Lupus Nephritis (LN) occurs in about 50% of SLE patients and represents a major source of morbidity and mortality. Recent genome‐wide association studies have linked mutations in the A20‐binding inhibitor of NF‐κB (ABIN1) gene to increased risk of developing LN. The molecular signalling events involved in the pathogenesis of LN in SLE patients are not well defined, but ABIN1 has been shown to have a role. We aim to describe the specific role for ABIN1 in the development of SLE and LN using ubiquitin binding deficient mutants in both humans (ABIN1[D472N]) and the orthologous mutation in transgenic mice (ABIN1[D485N]). Urinary protein loss measured by ELISA and gross pathological findings in sectioned kidneys of transgenic animals expressing ABIN1[D485N] mutation depict progressive nephritis which is apparent at 3 months and severe at 5‐6 months of age. Immune deposits in the kidneys were detected via immunofluorescence in the ABIN1[D485N] mice but were not seen in wild type mice. These data dramatically exhibit the importance of ABIN1 function in the maintenance of normal immune activity relative to the kidney. Furthermore, we illustrate the immunoprotective function of ABIN1 in kidney cells themselves. RT‐PCR, western blot and ELISA depict exaggerated immunostimulatory and inflammatory responses of cultured kidney cells expressing ABIN1[D472N] (including podocytes, tubular cells and mesangial cells) to TNF‐α and IL‐1β when compared to wild type ABIN1. Increased production of cytokines and chemokines suggests an increased propensity to recruit immune cells to target tissue which may lead to the inflammatory states characteristic of LN. Collectively, we have shown in both in vivo and in vitro models that ABIN1‐mediated inhibition of NF‐κB signalling is required in the kidney for the prevention of LN.

Grant Funding Source: National Institutes of Health
Identifying transcriptional changes in an oligodendrocyte‐specific conditional knockout mouse (934.6)
Authors: Stefanik Taylor , Yu Gang , Dries Daniel
Published: June 2021

gamma‐secretase is a proteolytic complex involved in development and implicated in the etiology of Alzheimer’s disease. We recently developed a conditional knockout mouse in which gamma‐secretase was specifically deleted from oligodendrocytes, the myelinating cells of the brain. The conditional knockout mouse had a profound neurological phenotype, including hyperactivity and decreased prepulse inhibition. Here, we use RNA‐seq to compare the transcriptional profiles of conditional knockout and control mice in an effort to identify a set of candidate genes that may explain the complex neurological phenotype of our conditional knockout mouse.

Grant Funding Source: Supported by National Institute of Neurological Disorders and Stroke grant 1R01NS079796
Is Arabidopsis thaliana gamma‐secretase a functional protease? (934.7)
Authors: Lee Jamie , Dries Daniel
Published: June 2021

gamma‐secretase is a multisubunit proteolytic complex responsible for the final step of the formation of beta‐amyloid peptides. Neurotoxic beta‐amyloid peptides are the primary components of amyloid plaques that, along with neurofibrillary tangles of tau protein, are found in the brains of Alzheimer’s disease patients. The gamma‐secretase complex contains four proteins _ presenilin, nicastrin, Aph‐1, and Pen‐2 _ and the presence of all four components is required for proteolytic activity of the complex. Surprisingly, all four proteins co‐evolved in plants, suggesting that a shared function was also conserved. However, since plants do not have nervous system, nor do they suffer from Alzheimer's disease, the function of plant gamma‐secretase is still a mystery. In this study, we subclone four components of Arabidopsis thaliana gamma‐secretase into a single plasmid in order to generate recombinant baculovirus for expression of Arabidopsis gamma‐secretase in Sf9 host cells. This allows for the simultaneous recombinant expression of four proteins in the same cell and from a single plasmid, thereby ensuring 100% co‐infection by all four gamma‐secretase components. This, then, gives us a powerful tool to study the putative proteolytic function of recombinant Arabidopsis gamma‐secretase in vitro.
Enhancing key radiation induced phenotypic changes by inhibition of the 26S proteasome in order to better stimulate immune responses (935.2)
Authors: Cacan Ercan , Kumari Anita , Garnett‐Benson Charlie , Greer Susanna
Published: June 2021

Radiotherapy has been extensively used for cancer therapies and sub‐lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T‐cell‐mediated immune attack. Inhibition of the ubiquitin‐proteasome system causes inhibition of cell proliferation in all cells, including cancer cells, which is a promising strategy of cancer therapy. The objective of this study is to investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in colorectal and breast cancer cells. Here, two colorectal carcinoma cell lines (HCT116 and SW620) and two breast cancer cell lines (MDA231 and MCF7) were examined for changes in expression of multiple co‐stimulatory molecules and death receptors. Our results indicate a combination of 26S proteasome inhibition and sub‐lethal radiation significantly increases the sensitivity of carcinoma cells to apoptosis. Combination treatment upregulates cell surface expression of multiple death receptors (DR4, DR5 and CD95) and co‐stimulatory molecules (OX40L and 41BBL) by increasing transcriptional activation of each gene. Our studies show for the first time that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor‐specific T‐cell survival and activation.
Investigating the DNA binding and transactivation activity of rdyCRX as a molecular basis for retinal dysplasia in Felis catus (943.1)
Authors: Paszek Miles , Lipsett Koren
Published: June 2021

In a pedigree developed from affected Abyssinian cats, an early onset rod‐cone dysplasia (Rdy) was identified and demonstrates an autosomal dominant inheritance. This is an excellent model for inherited retinal disorders such as retinitis pigmentosa (RP), cone‐rod dystrophy (CoRD), and Leber’s congenital amaurosis (LCA) because these lead to early developmental blindness via rod and cone photoreceptor dysfunction. The putative mutation for the Rdy phenotype is a single nucleotide deletion in the cone rod homeobox (CRX) protein, causing a frame shift that introduces a premature stop codon. Both normal CRX and rdyCRX proteins are expressed in vivo, suggesting a competitive interaction as the mechanism of pathology. The premature stop codon causes the truncation of the last third of the CRX protein containing the transactivational domains required for functional activity, while retaining the DNA binding domains allowing the rdyCRX to potentially compete with the normal CRX protein for binding to promoter regions. This could result in disrupting transcription initiation of genes necessary for the development and maintenance of the eye. EMSA analysis demonstrates competitive binding to Bat‐1 promoter oligonucleotide. Wild type CRX, rdyCRX, NRL, and NR2E3 have been successfully cloned in preparation of the development of a transcriptional activity assay.

Grant Funding Source: Supported by HHMI
Characterization of RAP1A and RAP1B expression across cell types and identification of their core promoter elements (946.1)
Authors: Painter Mark , Augustin Ryan , Niemela Amy , Cruise Jennifer
Published: June 2021

The RAP1A and RAP1B genes are members of the RAS oncogene family of GTPases and are implicated in many processes, including cell adhesion, growth, and signal transduction. Cell type‐specific expression of these genes and their regulatory elements, however, have been poorly investigated. Our lab has worked to define the levels of RAP1A and RAP1B native expression in a variety of human cell lines and to characterize the cis‐regulatory elements controlling this expression. In investigating native expression, RNA was isolated from eight different lines and analyzed using qRT‐PCR. Our findings suggest that RAP1A is expressed at statistically similar levels in all lines tested, while RAP1B shows more variable expression. Putative promoter fragments for both genes were identified using the UCSC genome browser, cloned by PCR, and inserted directly upstream of the Green Fluorescent Protein (GFP) gene. Plasmids were transiently transfected into a variety of cell lines, and GFP expression was analyzed by fluorescence microscopy. Activity of the core promoter for RAP1A appears to explain the observed native expression, and a possible repressor element has been identified. For RAP1B, the identified core promoter fails to explain cell type‐specific native expression, requiring further analysis of potential enhancer regions. Additionally, lack of RAP1B core promoter function in an osteosarcoma line may be SP1‐mediated.
Identification of distal regulatory regions for the human LAT gene (946.11)
Authors: Finco Timothy , Glowka Lena , Son Geurim
Published: June 2021

The human LAT gene encodes a transmembrane adaptor protein linking receptor engagement to the activation of downstream signaling pathways. This gene, highly conserved among mammals, is critical for the development and/or function of T cells, mast cells, Natural Killer cells, and megakaryocytes/platelets. Previously, we characterized the LAT promoter region and identified members of the Ets and Runx family of transcription factors as important regulators of LAT gene expression. In the present study, we sought to identify and characterize distal control regions that also contribute to LAT gene expression. Using a variety of criteria, including DNase hypersensitivity, sequence conservation, the presence of various histone modifications, and RNA polymerase binding, we identified three regions, either within introns or downstream of the gene, that displayed features consistent with enhancer elements. One of these regions displayed significant enhancer activity in a transient transfection assays. Further characterization of this region as well as the identification and testing of additional potential LAT enhancer regions will be presented.

Grant Funding Source: NIH grant #11255497
Hereditary kidney cancer models for understanding the cancer genome (95.2)
Authors: Spellman Paul
Published: June 2021

Von Hippel‐Lindau syndrome manifests primarily as hereditary kidney cancer. The syndrome is caused by single loss of function alleles in the VHL tumor suppressor that act in an autosomal dominant pattern. Patients with the syndrome develop clear cell kidney cancers in which the wild‐type germline allele is mutated or lost. The penetrance of the phenotype is so severe that most patients develop multiple independent tumors because of the somatic mutations are distinct.

My group in collaboration with Marston Linehan's group have sequenced the genomes of 43 tumors from seven individuals as well as matched normal DNA. I will present an analysis of mutation spectra in the samples and show patterns that suggest individual level biases in those spectra. Further, I will present analyses of the genomes that suggests guiding biases that have influenced the development of tumors guided by the host genome.
Filamin A is required for HIV‐1 Vpu to promote HIV‐1 release (950.2)
Authors: Dotson Dominique , Liu Ling , Sutton Jessica , Dong Xinhong
Published: June 2021

The interferon‐induced tetherin is a restriction factor that inhibits the release of HIV‐1 and other enveloped viruses from infected cells. The unique topology of tetherin, featured with two different membrane anchors at the opposite ends, enables it to capture the mature virions at the cell surface through a direct tethering mechanism. HIV‐1 has evolved the strategy to overcome tetherin restriction by encoding an accessory protein, Vpu. HIV‐1 Vpu neutralizes tetherin through multiple mechanisms. Recently, we identified that the host cytoskeletal component, filamin A (FLNa) acts as a cofactor in HIV‐1 assembly and release. By cell‐based assays, we demonstrated that FLNa was capable of binding tetherin. FLNa depletion increased steady‐state and cell surface levels of tethrin expression, whereas FLNa overexpression reduced tetherin expression. Furthermore, FLNa depletion or overexpression redistributed tetherin subcellular localization. These findings suggest that FLNa is involved in tetherin turnover by mediating tetherin trafficking pathways. Transmission electron microscopy studies illustrated that FLNa depletion induces a defect in HIV‐1 particle release, characterized with virions tethered to each other as well as to cell surface. Finally, virus release experiments revealed that FLNa depletion abolished the activity of Vpu to enhance particle release. Taken together, our results suggest that HIV‐1 Vpu takes advantage of the FLNa‐dependent tetherin trafficking pathways to neutralize tetherin and promote virus release. These studies shed new insights into the mechanisms of Vpu‐mediated counteraction of tetherin restriction.

Grant Funding Source: AI089330, GM059994
Effects of avocado oil on kidney mitochondrial membrane potential of L‐NAME‐treated rats (953.4)
Authors: Hernandez De La Paz Lucio , Ortiz‐Avila Omar , Canizal‐Garcia Melina , Calderon‐Cortes Elizabeth , Clemente‐Guerrero Monica , Saavedra‐Molina Alfredo , Cortes‐Rojo Christian
Published: June 2021

Hypertension is an important risk factor for the development of terminal kidney disease. Mitochondria have been proposed as a novel target for the treatment of hypertensive complications because has been demonstrated that the prevention of mitochondrial dysfunctions through the improvement of redox status by antioxidant therapy ameliorates vascular dysfunction caused by angiotensin II. Recently, we have reported that avocado oil intake has beneficial effects in kidney mitochondria of diabetic rats by improving the function of the electron transport chain (ETC) and decreasing ROS generation. Since kidney mitochondrial dysfunctions in both diabetes and hypertension shares enhanced oxidative stress features and avocado oil is a rich source of antioxidants, we aimed to test the effects of 90‐days avocado oil supplementation on kidney mitochondrial membrane potential of L‐NAME‐treated rats, a model of hypertension. No significant differences were observed in the phases of hyperpolarization and depolarization of membrane potential when succinate plus rotenone or glutamate‐malate plus succinate were used as respiratory substrates. However, when glutamate‐malate was used alone, a severe inhibition of membrane potential of control and L‐NAME rats was observed, suggesting impairment in complex I function. These effects were partially prevented by avocado oil supplementation. Interestingly, a protective effect of similar magnitude was obtained when L‐NAME rats were treated with losartan, an AT‐1 receptor antagonist. These data suggest that avocado oil may protect from mitochondrial ETC dysfunction in hypertensive kidney by decreasing pro‐oxidative effects of angiotensin II.

Grant Funding Source: Supported by CONACYT (130638 to CCR) and CIC‐UMSNH (16.12 to CCR) grants
Maternal inheritance of circulating irisin in humans (954.1)
Authors: Al‐Daghri Nasser , Al‐Attas Omar , Alokail Majed , Alkharfy Khalid , Yousef Mansour , Vinodson Benjamin , Amer Osama , Alnaami Abdullah , Sabico Shaun , Tripathi Gyanendra , Piya Milan , McTernan Philip , Chrousos George
Published: June 2021

Background The recently discovered myokine irisin has been implicated in several observational studies, suggesting a potential therapeutic target for obesity‐related diseases. However, no information is available as to the heritability of this hormone. This study aims to fill this gap.

Methods A total of 120 families (N=254;121 adults and 133 children) were included in this study taken from the Biomarkers Research Program cohort. Information included anthropometrics, glycemic, lipid and adipocytokine profiles. Irisin was measured using ELISA.

Results The most significant heritable traits in sons included irisin (p=1.6*10‐5), systolic blood pressure (p=3.6*10‐4), LDL‐ (p=1.2*10‐6) and total cholesterol (p=3.5*10‐7). Heritable traits between mother and daughter again included irisin (p<0.002), as well as waist (p<0.01), hips (p<0.005) and blood pressure (p<0.002); HDL‐cholesterol was the single most significant predictor for irisin in adults, explaining 17% of variance, whilst in children angiotensin II was the most significant predictor of irisin explaining 19% of variance (p=0.003).

Conclusion Circulating irisin appears to be maternally inherited and is predicted by HDL‐cholesterol in adults and angiotensin II in children, both factors influenced by energy expenditure and regulation. Taken together, these findings suggest a significant role of irisin in energy‐generating processes.
Physical activity may protect against low grade systemic inflammation in young adults (954.2)
Authors: Escalante Priscilla , Vera Desiree , Weaver Kathleen , Garcia Jerome , Dunn Sarah
Published: June 2021

Physical activity may positively impact low grade systemic inflammation by stimulating the upregulation of the multifactorial cytokine, Interluekin‐6 (IL‐6), secreted from muscle cells following chronic exercise ultimately decreasing ones risk for disease. Whether there is a significant difference in inflammatory (C‐Reactive Protein, IL‐6) markers and the gene expression molecule nuclear factor kappa B (NF‐κB) between young trained and untrained males and females is unknown. To examine the differences between C‐reactive protein (CRP), IL‐6, and NF‐κB in young (21.1 ± 2.1 yr) trained (n = 9) and untrained (n = 7) males and females. Fasting venous whole blood (40 ml) and a finger prick (triglycerides) along with body composition measurements (percent body fat, fat mass, etc…), and a medical history were obtained from young apparently healthy endurance trained (NCAA student athletes) and untrained individuals (less than three hours a week of physical activity). Enzyme‐linked immunosorbent assays were conducted to analyze fasting plasma (CRP and IL‐6) and isolated lymphocyte NF‐κB activation. Lymphocytes were isolated from whole blood samples through differential centrifugation with the use of ficoll. An Independent T‐test was used to identify differences (p<0.05) between trained and untrained groups (N = 16). Significant differences exist in body composition (p<0.05), triglycerides (p<0.05), and CRP (p<0.01). Finally, no significant difference in NF‐κB rels (p50 and p65) and IL‐6 was found between trained and untrained individuals. A significant difference exists between trained and untrained male and female participants for low grade systemic inflammation (CRP) levels despite no difference in the precursor gene expression molecule, NF‐κB or IL‐6. This suggests that physical activity has a positive protective impact on low grade systemic inflammation levels possibly through an anti‐inflammatory mediated pathway and may help minimize disease risk in a young population.

Grant Funding Source: Supported by STEM
Antiatherosclerosis effect of pine nut oil in HCHF diet‐fed rats (959.12)
Authors: Lee Jae Sung , Kim Dae Jung , Kang Yun Hwan , Kim Tae Woo , Kim Kyoung Kon , Choe Myeon
Published: June 2021

The objective of this study was to determine the antioxidative and antiatherosclerosis effects of a pine nut oil on serum and abdominal aorta in high fat high cholesterol diet fed rats. 6 weeks old male Spraque‐Dawley rats were divided into 5 groups. First group was fed with normal diet (normal control group, ND), second group was fed with high cholesterol‐high fat diet (negative control group, HCHF), third group was fed with HCHF diet with simvastatin diet (positive control group, HCHFS), fourth group was fed with HCHF diet with pine‐nut oil 7% (HCHFP 7%), and fifth group was fed HCHF with pine‐nut oil 15% diet (HCHFP 15%) for 6 weeks. This study shows that harmful parameter such as liver thiobarbituric acid reactive substance (TBARS) level, fatty steak area of aorta and low‐density lipoprotein cholesterol (LDL‐c) concentration of HCHFP group lower than that of HCHF group and beneficial parameters such as paraoxonase activity and high‐density lipoprotein cholesterol (HDL‐c) concentration were higher in HCHFP group than that of HCHF group. Through these results, our studies suggest that pine‐nut oil ingestion prevents the oxidative stress and atherosclerosis via the improvement of serum lipoprotein and the inhibition of lipid oxidation.
Characterization of saccharogenic mixed grain beverages produced by various microbial species (959.13)
Authors: Lee Jae Sung , Kim Dae Jung , Kang Yun Hwan , Kim Tae Woo , Kim Kyoung Kon , Choe Myeon
Published: June 2021

The purpose of this study was to evaluate the functional characteristics of saccharogenic mixed grain beverages (SMGBs) obtained using different microbial species and to evaluate their suitability for consumption. Various SMGBs were prepared through saccharification with Aspergillus (A.) oryzae CF1003 (A), A. acidus KACC46420 (B), Rhizopus (R.) delemar KACC46149 (C), R. oryzae KACC45714 (D), R. oryzae KACC46148 (E), A‐E mixed strains (F), A. oryzae CF1001 (G), A. acidus CF1005 (H) and A+H mixed strains (I). The visual appearance, flavor, taste, and the antioxidant capacity of each SMGB were then assessed. The chromaticity and aesthetic quality of the SMGBs was measured and all SMGBs appeared yellow. The C‐, G‐, H‐ and I‐saccharogenic beverages received scores of 3.319, 3.206, 3.170 and 3.025 points, respectively, following a sensory evaluation, while the others received less than 3 points. The polyphenol content of the different beverages were similar, while the flavonoid content significantly differed. In particular, the flavonoid content of the C‐ and E‐saccharogenic beverages was significantly higher than other beverages. Although the electron donating ability and reducing power of the SMGB was very low, the superoxide dismutase (SOD)‐like activity of all beverages (except the E‐saccharogenic beverage) increased in a concentration‐dependent manner. Specifically, the SOD‐like activity from the F‐saccharogenic beverage at 10,000 ppm was more than 50%. Interestingly, the antioxidant activities of the beverages were unrelated to their polyphenol or flavonoid levels. This study also found that the aesthetic qualities of G‐ and H‐saccharogenic beverages were the highest and that this was completely independent of their antioxidant capacity. Therefore, our results suggest that further studies are required to develop mixed grain‐derived saccharogenic beverages that can also fulfill a useful functional purpose.
A study on the glucose‐regulating enzyme activities of water extracts from Korea medicinal herbs (959.18)
Authors: Kim Dae Jung , Kang Yun Hwan , Kim Tae Woo , Kim Kyoung Kon , Choe Myeon
Published: June 2021

We selected Korea medicinal herbs having potential anti‐diabetic effects by activation of hepatic glucose‐regulating enzymes, such as glucokinase (GK). The glucokinase(GK) activity using the cytosol fraction in liver of a type II diabetic animal (GK rat, Goto‐kakizaki) was measured by spectrometric procedure. The selected Korea medicinal herbs were CM(Cordyceps militaris), MWY(Paeonia suffruticosa Andrews), SSY(Cornus officinalis), JSY (Perilla sikokiana), MWR(Angelica utilis Makino), SJH(Rehmannia glutinosa), DMH(Scrophularia buergeriana MIQUEL), JGP(Lycium Chinense), JC(Panax notoginsengs), YSB(Pyrus pyrifolia), PBG(Chamaecyparis obtusa), CWJ(Cucumis melon L. ), GSO(Acanthopanax senticosun), SY(Discorea Japonica Thunb.), YM(Coix Lachryma‐jobi L.) and DS(Salvia miltriorrhiza Bunge). Additionally, glucose‐uptake activity was measured by detection of 2DG accumulation in HepG2 cell. As a results of study, the GK activity in cytosol fraction of liver was higher at the 1mg/ml CM(2.99 fold induction) water extract than the control, and glucose‐uptake effect was higher at the MWY, CJW and MWR water extracts than at the control.
Aqueous Maté Extract Decreases the Viability Human Breast Cancer through Apoptosis and Cancer Repression (959.21)
Authors: Nisbett Kenisha , Sadik Amina
Published: June 2021

Maté is a subtropical plant native to South America where this plant is used as herbal tea. Several studies have been conducted to elucidate the beneficial effects of Yerba Maté tea. A study showed that saponins isolated from Maté extract induce apoptosis in human colon cancer cells. Another study indicated that the consumption of Maté tea causes higher incidence of esophageal cancer. This study aimed to determine the effects of Yerba Maté extract on breast cancer cells. Our findings show a decrease in viable T47D breast cancer cells after treatment with Maté extract in a concentration‐dependent manner. Caspase3‐AF647 assay allowed the detection of an increase in caspase 3 activity. The gene expression of p53 and caspase 3, two apoptotic markers, were assessed using RT‐PCR. The results showed a slight increase in the expression of the p53 gene but no significant change in the expression of caspase 3. Further investigation into the effect of Maté on the expression of BCl2 is underway.
Inflammatory bowel disease is a disorder of ganglioside metabolism (959.4)
Authors: Miklavcic John , Hart Tasha , Lees Gordon , Schnabl Kareena , Thomson Alan , Mazurak Vera , Clandinin M
Published: June 2021

AIM To demonstrate that the metabolism of ganglioside is altered in inflammatory bowel disease (IBD). METHODS Sections of intestinal mucosa from terminal ileum and/or colon were obtained from subjects with ulcerative colitis or inflammatory Crohn’s disease undergoing surgical resection for complications related to IBD. Control groups consisted of tissue samples of apparently normal intestinal mucosa from subjects with colorectal cancer and apparently normal intestinal mucosa from subjects with benign adenoma polyposis. Gangliosides of the intestinal mucosa were characterized using LC‐QQQ‐MS and compared among subject groups. Ganglioside catabolism enzymes HEXA and NEU3 were measured in the intestinal mucosa by western blot. RESULTS GM3 was significantly higher (P<0.01) by 2.1 fold in IBD intestine compared to apparently normal intestinal mucosa. IBD intestine exhibited 2.7 fold lower (P<0.001) GD1a content than control intestine. The mean relative expression of HEXA in IBD intestine was increased 1.7‐fold (P<0.08) and the mean relative expression of NEU3 in IBD intestine was increased 8.3‐fold (P< 0.001) compared to normal intestine. CONCLUSION Metabolism of gangliosides is altered in the intestinal mucosa of subjects with IBD. Investigation into utility of dietary ganglioside as a potential treatment of IBD is warranted.

Grant Funding Source: Supported by Broad Foundation and CIHR
Nuclear sphingosine 1‐phosphate and lung cancer (96.3)
Authors: Ogretmen Besim
Published: June 2021

Mechanisms that selectively regulate canonical telomere‐replication and non‐canonical oncogenic functions of telomerase reverse transcriptase (hTERT) are largely unknown. Here, we show that sphingosine kinase‐2 (SK‐2)‐generated nuclear sphingosine 1‐phosphate (S1P), a bioactive sphingolipid, interacts with hTERT, involving the C3‐OH of S1P and the D684 residue of hTERT, which then prevents E3 ubiquitin ligase MKRN1‐dependent hTERT degradation. Accordingly, molecular, genetic or pharmacologic targeting of SK‐2, or inhibition of the S1P/hTERT interaction, decreased hTERT stability, and suppressed orthotopic or xenograft‐driven lung tumor growth, without altering telomere length, by activation of a tumor suppressor TCF21. Expression of wild‐type or catalytically inactive‐hTERT mutant that increased or decreased telomere length, respectively, but not the D684A‐hTERT with altered S1P binding, prevented lung tumor suppression by SK‐2/S1P inhibition.
The role of bacterial cell wall dimers in the innate immune response (970.1)
Authors: Genova Lauren , Melnyk James , Mohanan Vishnu , Grimes Catherine
Published: June 2021

The innate immune system is the body’s first line of defense against pathogens. The innate immune system is triggered by pathogen associated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs) such as Toll‐like receptors (TLRs) and Nod‐like receptors (NLRs). This research project focuses on providing a better understanding of how the innate immune system senses and responds to the presence of bacteria. Specifically, our group is interested in the relationship between Nucleotide‐binding oligomerization domain‐containing protein 2 (Nod2), an NLR protein found in the cytosol of mammalian host cells, and muramyl dipeptide (MDP), the smallest bacterial cell wall fragment known to elicit an immunological response. When Nod2 is mutated, the signaling pathway becomes disrupted and uncontrollable inflammation arises, leading to chronic inflammatory bowel disorders such as Crohn’s disease. In order to discover how to better treat these diseases, it is imperative to learn more about how Nod2 and MDP interact, a mechanism which is currently unknown. The Grimes Lab has previously shown that Nod2 binds to MDP in vitro; however, research suggests that a heightened immunological response may be elicited in a host if molecules containing multiple MDP’s are used, suggesting multivalency is at play. This project highlights my current research progress to synthesize a variety of novel MDP dimers to test for a multivalent interaction between Nod2 and MDP.
Antibacterial activities of new coumarine heteroaryl derivatives (973.6)
Authors: Govori Odai Sevdije , Haziri Arben , Spahiu Sabrie , Ibrahimi Hamide
Published: June 2021

The antibacterial activities of new heteroarylderivatives with coumarine moieties were tested against several types of microorganisms: Staphylococcus aureus, Streptococcus pneumoniae, Aeromonas salmonicida, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Hafnia alvei and Bacillus cereus.

The antibacterial activities of the synthesised derivatives were compared with the antibacterial activity of some standard antibiotics such as Novobiocine, Cephalexine and Streptomycin. Synthesized compounds show different bacteriostatic and bactericidal activities.
Pomegranate (Punica granatum) fruit polyphenols, punicalagin, ellagic acid and gallic acid, inhibit formation of advanced glycation endproducts by scavenging reactive carbonyl species (975.10)
Authors: Liu Weixi , Ma Hang , Dain Joel , Seeram Navindra
Published: June 2021

Advanced Glycation Endproducts (AGEs) are a heterogeneous group of molecules produced from the non‐enzymatic condensation with reactants typically including a reducing sugar and a protein. The accumulation of AGEs in vivo plays an important role in the pathology of long‐term complications in diabetes. Inhibitors of AGEs, especially polyphenols which are antioxidants from dietary sources, have shown immense potential for diabetes mellitus management. The objectives of this study were: 1) to evaluate the inhibitory effects of pomegranate (Punica granatum) fruit polyphenols, namely, punicalagin (PA), ellagic acid (EA) and gallic acid (GA), on the glycation of bovine serum albumin (BSA) using D‐fructose and methylglyoxal as glycating agents, and 2) to investigate the mechanisms of anti‐glycation effects of these pomegranate polyphenols by quantifying the levels of reactive dicarbonyl species in glycated BSA solutions. The HPLC and fluorescence spectroscopy data revealed that the pomegranate compounds showed more potent inhibitory effects on BSA glycation than the positive control aminoguanidine. Moreover, pomegranate compounds also exhibited carbonyl scavenger reactivity by reducing the level of methylglyoxal, a dicarbonyl compound known as an AGE precursor. In summary, our study suggests that pomegranate compounds reduce the formation of AGEs and one of the mechanisms of such anti‐glycation effect could be related to their trapping abilities of reactive dicarbonyl species.
The licorice (Glycyrrhiza glabra) root extract alleviated TCDD‐induced toxicity in primary rat hepatocytes (975.5)
Authors: Chu Xiaoting , dela Cruz Joseph , Hwang Seong Gu
Published: June 2021

2, 3, 7, 8‐Tetrachlorodibenzo‐p‐dioxin (TCDD) is the most toxic member of a class of planar, halogenated aromatic hydrocarbons. Recently it raises great public concern about its impact on human health. It has been suggested that TCDD induce hepatocyte toxicity by a mechanism involving generation of ROS through CYP1A1 (cytochrome P450 1A1) activation. Many studies indicated that licorice (Glycyrrhiza glabra) root extract (LRE) exhibits chemopreventive and detoxicative properties. The aim of this study was to assess the effect of ethanolic LRE against TCDD induced toxicity in a primary culture of rat hepatocytes. Primary cultured hepatocytes were treated with TCDD (10nM) alone or together with LRE (0‐400μg/ml) for 24 and 48 hours. Treatment with TCDD alone lowered cell viability, but the toxic effect of TCDD on cell viability was recovered by the treatment of LRE dose dependently. Antioxidant capability by DPPH assay showed that LRE have a strong antioxidant activity. RT‐PCR and Western Blot analysis both showed that TCDD toxicity related genes (AhR, ARNT and CYP1A1) were subsequently down‐regulated by LRE treatment in a dose dependent manner. DNA fragmentation assay showed the protective effect of LRE against TCDD mediated DNA damage. These data suggested that LRE can mitigate TCDD toxicity in cultured hepatocytes by strong antioxidant activity as well as the suppression of CYP1A1 expression followed by down regulation of AhR and ARNT genes. In conclusion, LRE can be used as a potential toxicity alleviating agent against TCDD induced hepatocyte toxicity.
Elucidation of cellular signaling triggered by Francisella infection by comparative phosphoproteomic analysis (981.2)
Authors: Ansong Charles , Nakayasu Ernesto , Tempel Rebecca , Heffron Fred , Smith Richard , Adkins Joshua
Published: June 2021

Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune response that normally leads to protection against the infection. To elucidate the cellular signaling underlying this muted recognition of Francisella by the innate immune system, we performed a large‐scale comparative phosphoproteomic analysis of RAW 264.7 cells infected with either Francisella tularensis novicida or Salmonella Typhimurium, which is well recognized by the innate immune system. From our phosphoproteomics analysis >3000 phosphopeptides from >1200 phosphoproteins were identified, of which 368, 169, and 332 phosphopeptides were differentially abundant comparing Francisella‐ or Salmonella‐ infected and control cells at 0, 1, or 4 h post‐infection, respectively. Striking differences in phosphorylation levels on proteins from a diversity of cellular pathways were observed comparing Francisella and Salmonella infections, including key players of the innate immune response, such as AKT and MAP kinases and E3 ubiquitin ligases. In conclusion, our comparative phosphoproteomic analysis of cells infected with Francisella and Salmonella shows differences between the cellular signaling triggered by either pathogen, which may be reflective of specific mechanisms to evade the host immune response.

Grant Funding Source: Supported by GM094623 and 8 P41 GM103493‐10
Targeted and multiplexed quantitation of CSF proteins by MRM and labeled peptide standards (981.5)
Authors: Percy Andrew , Chambers Andrew , Yang Juncong , Hardie Darryl , Borchers Christoph
Published: June 2021

Precise and accurate protein quantitation is essential for screening biomarkers for risk stratification, disease prognostication, and therapeutic monitoring. The most promising analytical strategy for quantitating unverified biomarkers in pre‐clinical biofluids relies on targeted MRM/MS with isotopically labeled standards. Through this approach, considerable effort has been extended toward verifying protein biomarkers of non‐communicable disease in the systematic circulation, but little in the way of accelerating the discovered markers of central nervous system‐related diseases in cerebrospinal fluid (CSF). We herein aimed to develop a rapid and robust, antibody‐free method to quantify a large panel of proteins in human CSF for disease biomarker verification and validation studies. Using pooled CSF and a complex isotopically coded peptide mixture, various denaturation/digestion approaches were evaluated within a bottom‐up proteomic workflow. For enhanced reproducibility and multiplexing, peptides were separated at standard‐flow rates and detected by dynamic MRM in a triple quadrupole mass spectrometer. The final method demonstrated excellent reproducibility (average retention times of 0.06% and signal of 5.8% CV) and enabled the quantitation of 155 protein biomarker candidates (inferred from 337 interference‐free peptides) in a 43 min run. The quantitative analyses of >100 CSF samples from spinal cord injury patients is currently being performed and will additionally be presented.

Grant Funding Source: Supported by Genome BC, Genome Canada, and the Western Economic Diversification of Canada.