bluuebunny/crossref_metadata_2025 huggingface dataset:
Determining chromatin structure genome wide in yeast (564.2)
Authors: Kaster Kyle , Grilley Dan
Published: June 2021

The linear DNA in a human cell is compacted over a million fold, so much that, when compacted, 60,000 feet of DNA can fit on the tip of a needle. The first stage of compaction, nucleosome formation, is already known to impact gene regulation. The further compaction of nucleosomes into a chromatin fiber has also been shown to limit accessibility of DNA to DNA binding proteins. Despite the nucleosome crystal structure and decades of biochemical assays, the structure of this fiber is still a matter of debate. There are two competing structures proposed for the chromatin fiber: a two start and a one start model. A major difference between these models is the path of the linker DNA. Computational studies of chromatin structure suggest that details such as the length of linker DNA between nucleosomes can cause changes in the type of structure adopted by the fiber. Experiments using different linker lengths and different solution conditions support this claim and provide evidence in support of each model of the chromatin fiber. No study, however, has unambiguously accounted for the in vivo variability of linker lengths found in natural chromatin. We are developing a protocol that will allow us to assay the structure of chromatin in vivo using yeast DNA to account for the true variability of in vivo nucleosome spacing. We have shown our assay is sensitive to the structure of chromatin and are assaying how that structure differs at different genomic locations. We anticipate that the overall structure of the fiber at each genomic location is important for regulation. Our future aim is to explicitly test this prediction by first using our models to correlate changes in the fiber structure to differences in transcriptional regulation.
Genome‐wide mapping of large organized heterochromatin domains reveals hotspots of epigenetic and transcriptional changes associated with myeloid differentiation and acute myeloid leukemia (565.1)
Authors: Grigoryev Sergei , Salzberg Anna , Berg Arthur , Harris‐Becker Abigail , Loughran Thomas , Claxton David
Published: June 2021

Acute myeloid leukemia (AML) is a highly malignant blood cancer with the lowest survival rate among the four major types of leukemia. The objective of this study was to conduct a genome‐wide mapping of the epigenetic states in the normal myeloid and leukemia cells and to identify the chromosomal domains undergoing epigenetic and transcriptional destabilization. We hypothesized that large‐scale epigenetic changes manifested by heterochromatin markers, histone H3 di‐ and tri‐methylation at lysine 9, may interfere with transcription and promote AML. We used ChIP‐sequencing to conduct a detailed whole‐genomic characterization of H3(K9) methylation. For this study, we used primary AML myeloblasts, normal granulocytes, CD34+ hematopoietic progenitors, and cultured K562 cells as an epigenomic reference. Using ChIP‐sequencing, we mapped genome‐wide topography of histone H3(K9) methylation and its transitions in the process of myeloid differentiation and characterized spatial and functional relationship between histone H3(K9) methylation and transcriptional changes in AML. Aided by application of genome‐wide bioinformatic analysis using a hidden Markov model we were able to reveal multiple associations of chromatin modification topographies with transcription in leukemia and specific chromosomal domains undergoing both epigenetic and transcriptional changes. We expect that the observed epigenomic transitions may serve for AML prognosis as well as targets for its epigenetic therapy.

Grant Funding Source: Supported by Pennsylvania Department of Health
A genetic code with only 32 codons in LUCA (569.1)
Authors: Duax William , Redlinski jocelyn
Published: June 2021

We have found that the over 600 actinobacteria whose genomes have been reported lack ribosomal protein S21. Actinobacteria include several species in which the GC content of their DNA is greater than 74%. In several of these species the GC content in the wobble base position is greater than 97%. In these species codons ending in A or T are confined to hypothetical proteins having no homologues in the gene bank. Nine of the codons ending in A or T do not appear in any ribosomal proteins or tRNA sythetases of these species. No tRNAs cognate to these unused codons are found in these species. The reliably identified proteins have a marked amino acid bias favoring the four residues encoded by the eight GC‐only codons (Gly, Ala, Arg, and Pro). The vast majority of the putative proteins in these six species have full length multiple open reading frames making hypothetical protein gene identification particularly ambiguous. The absence of S21, the sever codon bias, and the absence of tRNAs corresponding the unused codons, suggest that these species are closely related to the last surviving universal common ancestor of all species. The data also suggest that a genetic code of 32 codons ending in G and C may have prefigured the modern code.
A mitochondrial bioenergetic hypothesis for autism spectrum disorder (570.3)
Authors: Chalkia Dimitra , Derbeneva Olga , Lvova Maria , Lakatos Anita , Leipzig Jeremy , Hadley Dexter , Hakonarson Hakon , Wallace Douglas
Published: June 2021

We are testing the hypothesis that a significant proportion of the risk for developing Autism Spectrum Disorder (ASD) is the result of inheriting partial defects in genes for mitochondrial bioenergetics. To test our hypothesis, first we determined if ancient human mtDNA variation is associated with ASD risk. To accomplish this, we used two independently generated and genotyped cohorts, the Autism Genetic Resource Exchange (AGRE) and the CHOP Autism Case‐Control (ACC). These pre‐existing mtDNA SNP data were generated by Illumina chip analysis. We deduced the mtDNA haplogroups for all samples and utilized cases and controls that harbored European haplogroups. Generalized linear modeling analysis of the AGRE cohort suggests that J mitochondrial lineage and T2 haplogroup are risk factors for ASD (J lineage: OR = 2.22, CI=1.21‐4.05, p=0.0094; T2 haplogroup: OR= 1.79, CI= 1.26‐2.55, p=0.0012). Congruently, in the ACC cohort we found that haplogroup T2 is strongly associated with ASD and represents a risk factor (OR=1.45, CI=1.04‐2.03, p=0.0284). These data demonstrate that ASD risk is modified by mtDNA haplogroup thus supporting our mitochondrial bioenergetic hypothesis of ASD.

Grant Funding Source: Supported by Simons Foundation Autism Research Initiative and National Institutes of Health
Lipoic acid induced weight loss (575.6)
Authors: Sanchez‐Cardenas Marina
Published: June 2021

Lipoic Acid Induced Weight Loss Over 1.4 billion adults worldwide are considered overweight or obese. Obesity occurs when a person’s body initiates a cycle in which glucose imbalances promote lipogenesis and increases oxidative stress which may cause certain diseases. Prior research has demonstrated that Lipoic Acid (LA) has the ability to act as an insulin mimic as well as an antioxidant. Its insulin mimic capabilities allow it to regulate glucose levels and its antioxidant potential may protect against oxidative damage. However, very little research has been done on its possible beneficial effects in combating obesity. This study aims to demonstrate how supplementation with LA assists in weight loss with no change in life style. The results showed a significant decrease in body mass (in weight) in mice supplemented with LA when compared to those that were not exposed to LA. This study proposes the initial steps to facilitating the prevention of obesity and its related health complications through supplementation.

Grant Funding Source: Supported by Title V STEM
Distinct gene expression pattern of osteoblastic cells derived from bone marrow and calvaria of osteoporotic rats (576.1)
Authors: Bombonato‐Prado Karina , de Azevedo Fernanda , Semeghini Mayara , Rosa Adalberto , Beloti Márcio , Siéssere Selma
Published: June 2021

Osteoporosis is one of the most important diseases associated with aging. The aim of this study was to determine if there is a distinct expression of genes associated with osteoblast differentiation in bone marrow mesenchymal stem cells (MSCs) and calvaria osteoblastic cells (COs) of rats induced to osteoporosis through ovariectomy. Cells from bone marrow and calvaria of control and osteoporotic rats were cultured in osteogenic medium and divided into four different groups: calvaria/control (CC); calvaria/osteoporosis (CO); bone marrow/control (MC) and bone marrow/osteoporosis (MO). After 3, 7 and 10 days, real‐time RT‐PCR for key bone markers was carried out in quintuplicate (n=5) and data were compared by Kruskal‐Wallis and Mann‐Whitney tests (p<0.05). Alkaline phosphatase and runt‐related transcription factor 2 expressions were significantly increased in MO and decreased in CO in all periods. Osteocalcin expression was significantly increased in MO at 7 and 10 days, and decreased in CO in all periods. The ratio of receptor activator of nuclear factor kappa‐B ligand /osteoprotegerin expression was significantly decreased in MO at 7 and 10 days and in CO in all periods. Our results show a distinct gene expression pattern for MSCs and COs after osteoporosis induction and suggest that MSCs undergo a metabolic adjustment in order to compensate the effects of estrogen decrease in this osteoporosis model.

Grant Funding Source: Supported by FAPESP
GRK5 knockout mice created by TALEN‐mediated gene targeting (576.2)
Authors: Min Kwan‐Sik , Nanjidsuren Tsevelmaa , Park Chae‐Won , Seo Eun‐Bi , Kang Myung‐Hwa
Published: June 2021

G protein‐coupled receptor kinase 5 (GRK5) is one of the seven GRK family members whose primary function is to desensitize G protein‐coupled receptors (GPCRs). In recently, GRK5 deficiency has been linked to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may accelerate to AD pathogenesis remains elusive. The GRK5 mRNA is expressed widely in brain and peripheral tissues, with highest expression evident heart, lung, and placenta. In cellular model systems, GRK5 can phosphorylate several neuronal GPCRs including ß2 adrenergic, M2‐muscarinic, secretin, angiotensin AT1, and thyroid stimulating hormone receptors. Transcription activator‐like effector nucleases (TALENs) are programmable nucleases that join FokI endonucleases with the modular DNA‐binding domain of TALEs and highly effective in inducing mutations at specific genome loci. TALEN‐mediated mutagenesis in zygotes is a potential alternative to conventional gene targeting in mice. In the presented study, we report the generation of mice with genetic knockout of the GRK gene using TALENs. We designed TALEN vectors for exon 1, 3 and 5 of mouse GRK5 gene and tested their ability to alter the each surrogate vector in 293T cells. We prepared of mRNAs for the linearized TALEN using the mMessage mMachine T7 Ultra kit. mRNAs (4ng/μl) was injected into cytoplasm of 180 one‐cell embryos. After incubation for 24 hours, the selected two‐cell embryos transferred into the oviduct of seven pseudopregnant C57BL/6 mice. We confirmed the genotype of Fo mice by sequencing and T7E1 assay.

Grant Funding Source: Supported by BK 21 plus program, the Ministry of Education, Science and Technology, Korea
Impact of resistance training on testosterone levels in individuals with moderate to severe chronic obstructive pulmonary disease (576.3)
Authors: Kuzel Aaron , Kaelin Mark
Published: June 2021

Purpose: Individuals with chronic obstructive pulmonary disease (COPD) exhibit decreased airflow and loss of diffusion area in the respiratory tree. Furthermore, current research suggests that low testosterone is a significant risk factor for these individuals. As testosterone levels decrease, the number and intensity of disease exacerbations significantly increases. The purpose of this study is to determine the impact of resistance training on testosterone levels in patients with COPD.

Methods: Bellarmine University’s Institutional Review Board approved the study in October 2013. Six individuals (1 male, 5 females) with moderate to severe COPD enrolled in an 11 week resistance training program. Average FEV1% and pack years of the subjects are 57 + 17 and 59.3 + 43.5, respectively. Subjects initial evaluation consisted of the Senior Fitness Test (SFT), the St. George’s Respiratory Questionnaire (SGRQ) and salivary assays of testosterone to determine baseline functional mobility, health related quality of life and free testosterone levels.

Results: Initial evaluations revealed below average functional mobility in these subjects compared to their age‐related peers in the following tests: Chair Stand Test ‐21% + 0.14%, Arm Curl ‐7% + 0.14%, Back Scratch ‐14% + 0.35, 8ft Up and Go Test ‐22% + 0.15%, 6‐minute walk ‐22% + 0.33%. Scores for the SGRQ came in at 37.74 points higher than those reported by healthy respondents. At the time of submission, initial testosterone collection remains in progress.

Conclusions: Results of the SFT and SGRQ clearly demonstrate impaired functional mobility and self‐reported quality of life in this population.
Cerebrospinal fluid sphingolipids and acid sphingomyelinase change with beta amyloid levels in Alzheimer’s disease (576.5)
Authors: Ormseth Cora , Chiang Jiarong , Arakaki Xianghong , Harrington Michael , Fonteh Alfred
Published: June 2021

Background: Sphingolipids (SP) and sphingomyelinase (SMase) play a role in amyloid processing. Thus, their cerebrospinal fluid (CSF) levels may change with beta amyloid concentration in Alzheimer’s disease (AD).

Methods: Using liquid chromatography tandem mass spectrometry, we identified SP classes and species in two CSF fractions: nanometer‐sized particles (NP) representative of brain membrane metabolism and the NP‐depleted supernatant fluid (SF) representative of interstitial metabolism. We compared SP levels and SMase activity between 70 cognitively healthy CH study participants, 40 participants with mild cognitive impairment (MCI), and 29 with probable AD.

Results: CSF fractions are differentially enriched with the SP classes and molecular species. In AD, SP metabolism was altered in both CSF fractions. The NP fraction had the greatest change in sphingomyelin (SM) species: concentrations of three species [SM(34:2), SM(34:1), SM(35:1)] were lower in AD and three [SM(34:2), SM(40:1), SM(41:2)] were lower in MCI compared with CH controls. Acid SMase activity decreased by 44 % in AD and correlated positively with the amyloid β42 levels in CSF from CH controls.

Conclusions: Altered SP metabolism in dementia combined with decreased acid SMase activity and its lost association with CSF amyloid β42 levels in AD underscore these as potential disease biomarkers, and acid SMase as a target for AD research.

Grant Funding Source: L.K. Whittier and the Helen Posthuma Foundations
Influence of environmental factors on the activity of enzymes of purine nucleotide metabolism (577.1)
Authors: Ilderbayev Oralbek , Abduldayeva Aigul , Nurmukanov Dulat , Rachyzhanova Saule
Published: June 2021

There is an experimental study of combined influence of 2 Gy dose gamma‐radiation and chrysotile‐asbest dust in the remote period on activity of enzymes of the purine’s nucleotides metabolism: 5’‐nucleotidase (5’‐NT), adenosindesaminase (ADA) and adenilatdesaminase (AMF‐ase) in different organs and tissues.

Research results showed that combined effect of chrysotile dust and ionizing radiation in the remote period causes increase of 5'‐NT, ADA, AMF‐ase activity in the liver. In case of conjuncted action 5'‐NT, ADA, AMF‐ase’s activity increases in 1.6; in 2.5 and in 6.0 times accordingly. The activity of 5'‐NT, AMF‐ase in adrenal medulla and lymphocytes of animals’ peripheral blood credibly increased. The ADA’s activity had not changed.

Taking into account the obtained results we can say that adrenal medulla tissues and lymphocytes of blood after the combined action of radiation and dust responses considerable intensification of the processes of anabolism and catabolism in the remote period, which marked as the secondary character of changes.

The combined action of radiation and asbest dust causes considerable interruption of purine metabolism enzymes’ level, which characterized by strain of adaptive‐compensate mechanisms after the influence of dust‐radiation factor.
Novel ligands for human pregnane X receptor differentially regulate CYP3A4 promoter activity (579.1)
Authors: Banerjee Monimoy , Chen Taosheng
Published: June 2021

Human pregnane X receptor (hPXR) also known as xenobiotic sensing nuclear receptor (SXR) is a member of nuclear receptor family and a critical regulator of the expression of genes involved in drug metabolism and clearance. The pregnane X receptor (PXR) regulates drug metabolism by regulating the expression of drug‐metabolizing enzymes such as cytochrome P450 3A4 (CYP3A4), which is involved in the metabolism of >50% of clinically prescribed drugs. It also plays an important role in cancer drug resistance by regulating the expression of MDR1. Activity of PXR can be modulated by agonists or antagonists. There are several agonists for PXR have been reported but the reported antagonists for PXR are limited. Therefore, there is still need to develop novel ligands for PXR. Here, we report the virtual screening of ~25,000 natural product derivatives from the ZINC database using the MOE docking software tool against the PXR‐Rifampicin complex X‐ray crystal structure. Our screening resulted in identification of compounds based on the lowest S scores which measures binding energy. All of these ligands were docked within the ligand binding pocket forming interactions with several hydrophobic amino acid residues. Further, all these main compounds and analogs were synthesized to verify with biological assays. We found that the compounds that bind directly to PXR, as revealed in an intrinsic tryptophan fluorescence assay, modulate CYP3A4 promoter activity differentially in HepG2 cells. Mutational analysis and docking studies showed that these compounds bind broadly in the ligand binding pocket but interact with different amino acid residues. The mechanism of binding and the functional group of the ligands that are important for distinguishing agonists/antagonists were also investigated.

Grant Funding Source: Supported by the ALSAC, St. Jude CRH, NIH, NCI.
Circadian clock control of hepatic metabolism via nuclear receptor SHP (579.3)
Authors: Lee Sang Min , Zhang Yuxia , Tsuchiya Hiroyuki , Smalling Rana , Jetten Anton , Wang Li
Published: June 2021

Objective: In mammals, circadian rhythms are essential for coordinating the timing of various metabolic processes. SHP interacts with many nuclear receptors to regulate the expression of multiple metabolic genes in the liver. This study investigates the role of SHP in controlling the rhythmicity of metabolites and liver clock genes. Methods: RNA‐sequencing (RNA‐seq) and qPCR were used to examine the rhythmic expression of liver clock and metabolic genes in Shp+/+ and Shp−/− mice on a C57BL/6 background (n=3‐5/group). GC/MS was used to identify the oscillation of metabolites in serum and liver. In vitro assays include: transient transfection, luciferase reporter assay, ChIP assay, gel‐shift assay, Co‐IP, Western blots, adenovirus overexpression and siRNA knockdown. Results: The transcriptomics and metabolomics analyses revealed a global alteration in the rhythm of metabolic genes and intermediate metabolites in glucose, lipid, amino acid, and bile acid homeostasis in the Shp−/− mice. Several components of the liver clock including Npas2 and Rorα/γ were sharply induced in Shp−/− liver. SHP inhibited Npas2 transcription through direct interaction with Rorγ to repress Rorγ transactivation of the Npas2 promoter and by directly interacting with Rev‐erbα to enhance its inhibition on the Npas2 promoter activation by Rorα. Conversely, Npas2 controlled the circadian rhythm of SHP by binding rhythmically to the SHP promoter. The Shp−/− mice exhibited higher locomotor activity which was consistent with their increased Pgc‐1α and energy metabolism. Conclusions: These novel findings identified SHP as an integral component of the liver circadian network.

Grant Funding Source: Supported by AHA post‐doc fellowship (S.L), and DK080440 and AHA GIA (L.W).
Three polymorphisms and susceptibility to breast cancer development: a Turkish study population (58.1)
Authors: Bayram Banu , Özkurt Mete , Erkasap Nilüfer , İhtiyar Enver , Erkasap Serdar
Published: June 2021

Objective: Genetic variations like polymorphisms underlie differences in our susceptibility to diseases. According to our literature reviews we have found three polymorphisms which contribute to breast cancer development from different pathways. However genetic variations of polymorphism vary between populations according to gene pools, life style and gene‐environment interactions. Based on these findings we aimed to investigate the association between p53 gene codon 72 polymorphism, MTHFR gene C677T polymorphism and PAI‐1 gene 4G/5G polymorphism and breast cancer in a Turkish study population.

Material and Methods: Genomic DNA was extracted from 100 women (52 with breast cancer and 48 controls) in the study. PCR technique was used to amplify extracted DNA with proper primers for each polymorphism. For identifying genotypes PCR products were assessed with UV transilluminator by being exposed to agarose gel electrophoresis.

Results: According to genotype distributions and allele frequencies of three polymorphisms there were no statistical differences between breast cancer patients and control group.

Conclusion: As a conclusion of our study we may assert that p53 gene codon 72, MTHFR gene C677T and PAI‐1 gene 4G/5G polymorphisms should not be considered as susceptibility factors for breast cancer development. However polymorphism studies with larger numbers of the population provides more meaningful results. The significance of our result remains to be further investigated in different and even larger populations, combined with other polymorphisms considered as susceptibility factors for breast cancer.
An innovative role of thyroid receptor β in triple‐negative breast cancer (58.4)
Authors: Gelsomino Luca , Guowei Gu , Kyle Covington , Babarinde Toyin , Zhou Jianlin , Rechoum Yassine , Corona‐Rodriguez Arnoldo , O'Malley Bert , Mangelsdorf David , Minna John , Webb Paul , Ando' Sebastiano , Fuqua Suzanne
Published: June 2021

Background Currently endocrine treatment is the most effective therapeutic strategy in patients affected by estrogen‐receptor (ERα) positive breast cancer cells. In contrast, treatment of triple‐negative breast cancer (TNBC) is a major clinical problem due to the lack of useful therapeutic targets. Microarray analysis demonstrated that THRβ is one of the most expressed receptors in TNBC cells. Aim of our study was to evaluate THRβ’s role in TNBC.

Experimental Design and Methods Microarray and predicition analysis, qRT‐PCR, THRβ shRNAs for stable clones using pGIPZ lentiviral vectors, MTT, soft‐agar assays, immunoprecipitation and proximity ligation assays.

Results Statistical analysis using a published dataset1 showed that patients with low THRβ have a poor clinical outcome. ShTHRβ stable clones were resistant to chemodrugs (doxorubicin and docetaxel), while specific THRβ agonists increased sensitivity to these agents. Interestingly, THRβ agonists through post‐traslational modifications enhanced ERα expression and activity rendering the cells sensitive to the inhibitory effects of the antiestrogen tamoxifen on growth.

Conclusion THRβ might represent a predicitive and prognostic marker in TNBC patients. THRβ agonists can enhance chemosensitivity and by restoring ERα expression make cells targetable for hormone therapy.
Development and characterization of a multi‐drug resistant Her‐2/neu positive breast cancer cell line (58.6)
Authors: Tang Yuan , Lamberti Giuseppina , Curran Elizabeth , Kiani Mohammad , Wang Bin
Published: June 2021

Research has shown that ~ 25% ‐ 30% breast cancers overexpress Her‐2/neu receptor. The major problem in the management of Her‐2/neu overexpressed breast cancer is its multi‐drug resistantce (MDR). P‐glycoprotein (P‐gp) mediated MDR has been recognized as one of the major mechanisms in drug resistance in breast cancer. Therefore, developing a breast cancer cell line with both Her‐2/neu and MDR positive becomes necessary.

Dual positive breast cancer cell line was developed by transfection of the multidrug resistance gene (MDR1 gene) to human breast carcinoma cell line BT‐474 (Her‐2/neu +), followed by selection on clochicine. The presence of membrane protein Her‐2 and P‐gp were tested by immunohistochemical methods. The multidrug resistance was tested by the treatment efficacy of doxorubicin.

The overexpression of p‐glycoprotein in multidrug resistant cells and the presence of membrane protein Her‐2 were confirmed by immunocytochemistry staining as well as Western Blot. Fluorescence imaging study showed that drug resistant BT‐474 cells uptake much more Her‐2/neu tagged nanoparticles than non‐tagged ones. Cytotoxicity test shows that the transfected BT‐474 is 10 times more resistant than its drug sensitive counterpart.

In conclusion, we have successfully developed and characterized Her‐2/neu and MDR dual positive breast cancer cell line which can serve as a platform for testing new therapeutic strategies.

Grant Funding Source: Supported by Susan G. Komen for the Cure foundation
PMPMEase inhibitors as potential therapies for castration‐resistant prostate cancer (585.7)
Authors: Poku Rosemary , Amissah Felix , Duverna Randolph , Byron Aguilar , Lamango Nazarius
Published: June 2021

Castration resistance continues to pose major challenges in the management and treatment of prostate cancer. Monomeric G‐protein signaling is critical in prostate carcinogenesis. These proteins are polyisoprenylated and regulated by polyisoprenylated methylated protein methyl esterase (PMPMEase).

It was hypothesized that PMPMEase regulates cell viability and migration. The purpose was to determine the effect of PMPMEase inhibition on the malignant characteristics of prostate cancer cells.

The effects of PMPMEase inhibition on cell viability, proliferation, migration and gene profiling were determined with the specific PMPMEase inhibitors, L‐28 and NSL‐RD‐036.

PMPMEase specific activities were 1.5 and 4.5‐fold higher in androgen‐dependent 22Rv1 and LNCaP cells and 1.5‐ and 9.8‐fold higher in androgen‐independent DU 145 and PC‐3 prostate cancer cells compared to normal WPE1‐NA22 prostate cells. Treatment of the cells for 48 h with L‐28 induced apoptosis with EC50 values ranging from 1.8 to 4.6 µM. The PMPMEase activity in the treated cells followed a similar profile with EC50 values from 2.3 to 130 µM. L‐28 treatment significantly disrupted F‐actin cytoskeleton in PC‐3 cells and inhibited cell migration. NSL‐RD‐036 inhibited PC‐3 proliferation with an EC50 of 2 μM. The expression of cell proliferation, metastasis, apoptosis and DNA synthesis and repair genes were significantly altered by specific PMPMEase inhibition.

PMPMEase inhibition induces apoptosis, disrupts F‐actin and suppresses the migration of androgen‐independent prostate cancer cells. Inhibition and measurement of PMPMEase has the potential to serve in the effective management of aggressive prostate cancer.

Grant Funding Source: Supported by National Center for Research Resources and the National Institute of Minority Health an
Altering substrate specificity of aldehyde dehydrogenase 3A1 to enhance acetaldehyde metabolism, in vivo (585.9)
Authors: Chen Che‐Hong , Cruz Leslie , Mochly‐Rosen Daria
Published: June 2021

Acetaldehyde is a well‐know cytotoxin, and carcinogen. Detoxification of acetaldehyde, which is mainly carried out by the mitochondrial aldehyde dehydrogenase 2 (ALDH2), is crucial to human health. High levels of acetaldehydes are present in the saliva and in circulation after ethanol ingestion and likely contribute to the high incidence of esophageal cancer in alcoholics and in carriers of a common ALDH2 mutation (ALDH2*2) found in 540 million East Asians. Acetaldehyde is also partly responsible for behavior impairment following ethanol consumption. Therefore, means to accelerate acetaldehyde clearance will be beneficial for human health. We previously identified an activator of ALDH2 that accelerated acetaldehyde metabolism. Here we describe the recruitment of an additional ALDH isozyme, ALDH3A1, to accelerate the clearance of acetaldehyde by the use of a small molecule. ALDH2 is the main isozyme that metabolizes acetaldehyde in liver. In contrast, ALDH3A1, which is present in the stomach and mucosa of the esophagus, normally metabolizes aromatic aldehydes and is ineffective in metabolizing acetaldehyde. We identified a small molecule activator, Alda‐89, which increased the Kcat of ALDH3A1 for acetaldehyde and enhanced its catalysis. We showed that when given together with an ALDH2‐specific activator, Alda‐1, Alda‐89 induced a rapid reduction in blood ethanol and acetaldehyde levels after acute ingestion of ethanol in mice. Importantly, treatment with Alda‐1 and Alda‐89 also reduced behavioral impairment in both wild‐type and ALDH2*2 mice. The ability to recruit an enzyme to metabolize a substrate that it usually does not metabolize using a pharmacological agent may represent a novel strategy to increase metabolism of toxic agents, in vivo.

Grant Funding Source: Supported by NIH AA11147 to DM‐R
Isolation and characterization of two cDNAs expressed in the Harderian gland of the anole lizard (Anolis carolinensis) (589.4)
Authors: Prokopiak Zoey , Steglich Carolyn
Published: June 2021

The Harderian gland (HG) is a secretory gland in the eye of most land animals. Its structure has been studied, but little is known about the gland's actual function, which varies depending on species. Structural observations have suggested multiple possible functions (lubrication of the eye, sensing of chemical signals via the vomeronasal organ, or an immunological role) but have little supportive experimental data. To investigate the HG’s function in an organism such as the Anole lizard (Anolis carolinensis), cDNAs were isolated from the HG and sequenced. Two cDNAs (E1 and M1) that code for proteins of unknown structure or function (no significant hits in a GenBank search) were selected for further study. Using RT‐qPCR (reverse transcriptase quantitative polymerase chain reaction), the expression of the cloned genes (cDNAs) that had been isolated from the HG of the Anole lizard were compared to other known Anolis genes (in this case, genes encoding ribosomal proteins) that are expressed in all cells. The E1 cDNA is expressed at high levels in the HG but also expressed at background levels in liver and tongue tissues. The M1 cDNA is expressed at extremely high levels only in the HG. These cDNAs have been subcloned into the pET‐22 expression vector for expression in bacterial cells to provide an unlimited source of the proteins. The functional characterization of the proteins can then be directly investigated.

The genome of the Anole lizard was completed in 2010 but is currently incompletely annotated. BLAST searches of the Anole's genome with the E1 and M1 sequences have returned some short matches for the sequences, which locates each of them to a particular scaffold in the genome, but no predicted genes are annotated for those regions. Additional alignment work with MegAlign locates the likely exons and introns of these genes more precisely, thereby providing additional annotation to those regions of the Anole genome.

Grant Funding Source: Supported by a Faculty/Student Research Grant and a Student Research Grant from Slippery Rock Univ.
Mutations of the LYL1 gene in patients with T‐cell acute lymphoblastic leukemia (59.7)
Authors: Chan Tsz Yin , Zohren Fabian , Ballard Brandon , Goodell Margaret , Rabin Karen , Lukov Georgi
Published: June 2021

The lymphoblastic leukemia ‐ 1 (LYL1) gene is a transcription factor believed, when upregulated, to support the formation of T‐cell Acute Lymphoblastic Leukemia (TALL). One possible cause for the upregulation of LYL1 is the presence of mutations, affecting the normal expression, processing or degradation of LYL1. The objective of our study is to identify if there are mutations in the LYL1 gene in patients with TALL, and determine the effect of such mutations on LYL1 expression. RNA, for quantitative PCR, and DNA, for LYL1 sequencing, were extracted from blood and bone marrow samples from twenty TALL patients. Ten samples showed high and intermediate LYL1 expression. Five of them had single nucleotide polymorphism with three of them being non‐silent. LYL1 variants with single or combined mutations were constructed, and their expression, in cultured CHO‐K1 and H441, was measured by western blotting. The LYL1 variants showed significantly increased expression, up to 3.6 fold, compared to the wild type form. Our findings suggest that these mutations may have a role in the expression and processing, or the degradation of LYL1, which not only adds to our understanding of the LYL1 biology, but it may offer one more clue why LYL1 is upregulated in some forms of TALL.

Grant Funding Source: BYUH Mentored Research & Student Research Associateship Funds
Elevated plasma biomarkers of chronic inflammation in Gulf War illness (591.3)
Authors: Johnson Gerhard , Slater Billie , Leis Linda , Bach Ronald
Published: June 2021

As many as 200,000 veterans of the 1990‐1991 Gulf War suffer from unexplained chronic multisymptom illnesses collectively known as Gulf War Illness (GWI). Symptoms of GWI may include muscle and joint pain, unexplained fatigue, and cognitive impairment. The etiology of GWI is undetermined, and no effective treatment has been defined.

Objective: To determine if blood abnormalities exist in veterans with GWI that could lead to the identification of potential therapeutic targets.

Methods: Eighty‐five gulf war veterans were enrolled. Subjects with GWI were identified by standard (CDC‐10) criteria. Data derived from subjects with GWI (58) were compared to those without GWI (27). Multi‐Analyte Profiling of plasma proteins was performed and analyzed by the Mann‐Whitney rank sum test.

Results: Five of 89 proteins were significantly elevated (p<0.05) in the GWI subjects: C‐reactive protein (CRP) (1.75‐fold), leptin (1.67‐fold), interleukin‐1 beta (1.66‐fold), brain‐derived neurotropic factor (1.59‐fold), and matrix metalloproteinase‐9 (1.14‐fold). The 14 individuals in this study with the highest CRP levels, as much as 10‐fold above the median, were all in the GWI group.

Conclusion: The biomarker evidence is consistent with activation of the innate immune system. It supports the hypothesis that chronic inflammation is a component of GWI pathophysiology.

Grant Funding Source: Supported by VA ORD and DoD CDMRP
Globotriaosylceramide and globotriaosylsphingosine mediated epithelial‐to‐mesenchymal transition in kidney cells: implication for Fabry disease nephropathy (600.1)
Authors: Jeon Yeo‐Jin , Jung Namhee , Jung Sung‐Chul
Published: June 2021

Fabry disease is a lysosomal storage disorder caused by deficiency of α‐galactosidase A (α‐gal A), resulting in deposition of globotriaosylceramide (Gb3) in the vascular endothelium. Deacylated Gb3, globotriaosylsphingosine (lyso‐Gb3), also accumulated in the tissues of Fabry disease patients. Lyso‐Gb3 has been suggested as a candidate biomarker. According to renal and cardiac pathological findings, epithelial‐to‐mesenchymal transition (EMT) could be a possible pathogenic mechanism in Fabry disease. However, the association of EMT with Fabry disease has not been elucidated yet. Expression level of EMT markers such as E‐cadherin, N‐cadherin, β‐catenin and α‐SMA in mouse renal glomerular mesangial cell (SV40MES13) significantly increased when treated with Gb3. Whereas, in human proximal renal tubular epithelial cells (HK‐2), expression of EMT markers were significantly altered when treated with lyso‐Gb3 rather than treated with Gb3. Additionally, we observed that the activation of TGF‐β, phospho‐AKT, and PI3K were highly elevated in these cells. When we treated recombinant α‐gal A with Gb3 or lyso‐Gb3, TGF‐β expression was reversed in HK2 cells. Our study demonstrates that Gb3 and lyso‐Gb3 can potentiate EMT‐like processes through AKT/PI3K signaling pathway in HK2 and SV40MES13 cells. Moreover, these findings suggest that lyso‐Gb3, rather than Gb3 may be a crucial role nephropathy of Fabry disease by inducing EMT and may contribute to a better understanding of the renal fibrosis.
Portal bile acid perfusion bypasses ileal FGF19 and suppresses hepatic CYP7A1 expression in rabbits (605.6)
Authors: Xu Guorong , Shang Quan , Guo Grace , Shi Daniel , Saumoy Monica , Salen Gerald
Published: June 2021

This study challenged the hypothesis that CYP7A1 expression is regulated by a gut‐hepatic signaling pathway FGF15/19‐FGFR4 initiated from the ileum but not the liver. We would like to show that increase of portal bile acid flux alone should be able to suppress CYP7A1 expression. Methods: We developed a rabbit model to infuse glycodeoxycholic acid (GDCA) via the splenic vein to bypass ileal FGF19. Four study groups of rabbits (n=8/group): Control; bile fistula (BF); BF+GDCA (by portal perfusion); and BF+ GDCA‐f (by femoral perfusion). Results: Portal GDCA perfusion in the BF+GDCA group for 6 hours resulted in significant reduction in CYP7A1 mRNA but femoral perfusion in the BF+GDCA‐f group showed no change with the same perfusion rate of GDCA. In both GDCA perfusion groups, there was no increase in ileal FGF19 expression nor portal FGF19 protein levels, but similar increase in biliary bile acid outputs. Conclusions: This study demonstrated that portal bile acid flux alone inhibited CYP7A1 expression without increase of FGF19 in the ileum. However, the same bile acid flux through the liver via the hepatic artery (femoral perfusion) did not suppress the CYP7A1 expression. We propose that bile acid flux through the portal venous system may be the so called “intestinal factor” that suppresses CYP7A1 expression.

Grant Funding Source: VA Merit Review Grant
Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in Apo E‐/‐ mice and rabbits fed a high fat and cholesterol diet (607.11)
Authors: Amuzie Christine , Mishra Sumita , Bedja Djahida , Avolio Alberto , Kass David , Berkowitz Dan , Renehan Mark , Chatterjee Subroto
Published: June 2021

Objective ‐‐ Glycosphingolipids are integral components of the cell membrane and have been shown to serve as messengers, transducing growth factor initiated phenotypes. Here we have examined whether inhibition of glycosphingolipid synthesis could ameliorate atherosclerosis and arterial stiffness in transgenic mice and in rabbits.

Methods and Results ‐‐ Apolipoprotein E‐/‐ (apoE‐/‐) mice (12 weeks of age, n = 6) were fed regular chow or a western diet (1.25% cholesterol, 2% fat). Mice were fed 5mg/kg (mpk) or 10 mpk of an inhibitor of glycosphingolipid synthesis inhibitor D‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (D‐PDMP) solubilized in vehicle (5% Tween‐80 in PBS) and the placebo group received vehicle only. At 20 weeks and 36 weeks of age, serial echocardiography was performed without sedation to measure aortic pulse wave velocity (PWV). Feeding mice a western diet markedly increased aortic PWV, aortic intima medial thickening (IMT), oxidized LDL, Ca2+ deposits, and glucosyl‐ and lactosylceramide synthase activity. These were dose‐dependently decreased by feeding D‐PDMP. In liver, D‐PDMP decreased cholesterol and triglyceride levels by raising the expression of SREBP‐1, LDL‐r, HMGCo‐A reductase, and cholesterol efflux genes (e.g., ABCG5, ABCG8). D‐PDMP affected VLDL catabolism by increasing the gene expression for LPL and VLDLr. Rabbits fed a western diet for 90 days had extensive atherosclerosis accompanied by a 17.5‐fold increase in total cholesterol levels and a 3‐fold increase in lactosylceramide levels. This was completely reversed by feeding D‐PDMP.

Conclusions ‐‐ Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in ApoE‐/‐ mice and rabbits. Thus, inhibition of glycosphingolipid synthesis may be a novel approach to treating atherosclerosis and arterial stiffness.

Grant Funding Source: Supported by P01HL107153
Inhibiting glycosphingolipid glycosyltransferase activity prevents cardiac hypertrophy in apoE‐/‐ mice fed a western diet (607.12)
Authors: Bedja Djahida , Mishra Sumita , Amuzie Christine , Kass David , Avolio Alberto , Chatterjee Subroto
Published: June 2021

Background: Although diet enriched in high fat and cholesterol can induce cardiac hypertrophy, little is known about the role of glycosphigolipids and glycosyltransferases contributing to this phenotype. Herein, using transgenic apo E‐/‐ mice fed a high fat and cholesterol diet we show that treatment with a glycosphingolipid inhibitor blunts cardiac hypertrophy.

Methods and Results: Male apoE‐/‐ mice were fed a western diet (20% fat and 1.25% cholesterol) from 12 weeks of age till 36 weeks with and without D‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (D‐PDMP) daily by oral gavage (vehicle alone, 5 mpk and 10mpk) . At 12 week, 20 week and 36 week, trans ‐thoracic echocardiography were performed in conscious mice using an ultrasound system (n=5 in each group). Mice were euthanized once maladaptive cardiac hypertrophy time point is reach. Tissues such as the heart were harvested for were harvested for histochemical, biochemical, and molecular analysis to ascertain the effects of treatment on glycosphingolipid glycosyltransferase activity, gene/protein expression.

We observed an age‐dependent increase in the left ventricular mass (LVmass) and decline in fractional shortening indicative of cardiac hypertrophy and dysfunction which worsens with western diet. This was accompanied by increased gene expression for atrial natriuretic protein, Brain‐type natriuretic protein, and increased protein expression of transforming growth factor, MAPK and AKT‐1. Masson’s trichrome staining revealed hypertrophy in cardiomyocytes and extensive fibrosis. In contrast, treatment with D‐PDMP preserved fractional shortening and the left ventricular mass to control levels over a 6 month treatment period. Also, the level of various cardiac glycosphingolipids and glycosyltransferases were decreased to control levels.

Conclusion: Treatment with a glycosphingolipid glycosyltransferase inhibitor blunts cardiac hypertrophy in apoE‐/‐ mice fed a western diet.

Grant Funding Source: Supported by NHLBI grant P01HL107153.
Site‐specific chemoenzymatic glycoengineering of cetuximab, a therapeutic monoclonal antibody (607.16)
Authors: Lomino Joseph , Giddens John , Wang Lai‐Xi
Published: June 2021

IgG type monoclonal antibodies are an important class of therapeutic glycoproteins used in the treatment of a wide range of maladies: cancer, auto‐immune disorders, and infectious diseases. Cetuximab is a therapeutic monoclonal antibody, which is glycosylated at both the FAB and FC domains of the heavy chain at Asn ‐ 88 and Asn ‐ 299 respectively. The FAB glycan is composed of ca. 30% non‐human α ‐ gal and/or N‐glycolylneuramic acid (NGNA), which have been shown to cause allergic reactions in some patient populations, while the FC domain glycans are not optimal for effector functions. In this work, we perform site‐specific glycan remodeling of cetuximab by taking advantage of the substrate specificity of two endoglycosidases and their cognate glycosynthases. Endo F3, from E. meningosepticum, was initially used to remove glycans from both the FAB and FC domains. A fully galactosylated complex type glycan was precisely transferred to the FC using the glycosynthase mutant of Endo S from S. pyogenes. Finally a fully sialylated N‐glycan was added to the FAB glycosylation site using the Endo F3 glycosynthase mutant. The glyco‐reengineered cetuximab is expected to possess enhanced therapeutic efficacy without the allergic side effects. Our site‐specific glycosylation approach should be generally applicable to other therapeutic monoclonal antibodies.

Grant Funding Source: NIH Grant R01 GM080374
Anti‐angiogenic and anti‐cancer property of a protein N‐glycosylation inhibitor (607.4)
Authors: Baksi Krishna , del Rosario Cristina , Banerjee Aditi , Zhang Zhenbo , Banerjee Dipak
Published: June 2021

Tumor growth is angiogenesis dependent. Current therapeutics targets either tumor cells or microvasculature but not both. The patients therefore enjoy disease free survival only for a limited period of time, irrespective of a good initial prognosis and pathological response rates. Aberrant glycosylation has been observed in tumors and we have hypothesized that an asparagine‐linked (N‐linked) protein glycosylation inhibitor could target both endothelial cells and tumor cells and eliminate tumor progression. Our objective is to test the efficacy of Tunicamycin. The results support Tunicamycin treatment causes cell cycle arrest, develops endoplasmic reticulum (ER) stress and induces unfolded protein response (upr)‐mediated apoptosis. Analysis of C=O stretching vibrations of Amide I of proteome by Raman spectroscopy concluded protein denaturation. Tunicamycin treatment (i) prevented the colony formation, (ii) failed to reverse the inhibition of angiogenesis by VEGF165; and (iii) indiscriminately inhibited the progression of double‐ and a triple‐negative breast cancers by ~55‐65% in three weeks. Proliferation of MDA‐MB‐231, MDA‐MB‐468, BT‐20, BT‐474 and ZR‐731 human breast cancer cells was also inhibited. Induction of apoptosis in MDA‐MB‐23 confirmed translational attenuation of upr. Supported in part by grants from Susan G. Komen for the Cure BCTR0600582 (DKB) and NIH/NIMHD 2G12MD007583 (KB).

Grant Funding Source: Supported in part by grants from Susan G. Komen for the Cure BCTR0600582 (DKB) and NIH/NIMHD 2G12MD0
TNFα /TNFα receptors in adult rat testis after ethylene dimethane sulfonate treatment (609.15)
Authors: Singhal Barkha , Park In , Sang Samuel , Talapatra Arpita , Dutta Dibyendu , Mills Nathaniel
Published: June 2021

Tumor necrosis factor (TNF‐α) is a multifunctional cytokine secreted in mammalian testis by germ cells, Sertoli cells and to some extent by macrophage; however TNF‐α receptors are largely confined to Sertoli cells. TNF‐α regulates different cellular processes pertinent to spermatogenesis including steroidogenesis, germ cell apoptosis, growth hormone function and inflammation. The Fas/FasL apoptotic pathway is the current model for depletion of Leydig cells with ethylene dimethane sulfonate (EDS) treated adult rat testis. We are investigating a potential role of TNF‐α in EDS Leydig cell depletion as well as other tissue correlates. At 6, 15 and 24 hr post‐EDS, using RT‐ and q‐PCR we find that mRNA expression of Leydig cell markers, Lhr and Insl‐3 declined by more than 85% and 3β‐HSD declined by 97% at 24 hr after EDS compared to controls. Using TUNEL, a DNA fragmentation detection assay, we observed a time dependent loss of Leydig cells by apoptosis. Next, genes associated with inflammation, TNF‐α was increased modestly and IL‐1β increased 3 fold, 24 hr post‐EDS. A three‐fold increase in TNF‐α receptor superfamily member 1A mRNA (Tnfrsf1a/ CD120a) was found. However, no change was observed in tumor necrosis factor receptor superfamily member 1B mRNA (Tnfrsf1b/CD120b) at 24 hr post‐ treatment. The mRNA of the two TNF‐α receptors has a differential expression, suggesting their presence on different cell types. ELISA detection of the testicular tissue showed an increased TNF‐α protein expression at 24 hr post‐EDS. Thus, with a high percentage of Leydig cell loss at a time when TNF‐α and its receptors are changing may imply TNF‐α activity is involved with Leydig cell apoptosis following EDS, however, stronger correlates may be found with other cell types. Future work is to localize TNF‐α receptors using immunohistochemistry.