bluuebunny/crossref_metadata_2025 huggingface dataset:
Insertion/Deletion Polymorphism of the Angiotensin Converting Enzyme Gene and Breast Cancer Risk
Authors: Javaid Naila Khatija , Fathima Husna , Abdulla Gulnaz , Al‐Ammari Abeer Saleh , Abbasi Fatma Ahmad
Published: June 2021

Background

Renin‐angiotensin converting enzyme (ACE) polymorphism has recently been linked to the pathogenesis and prognosis of human cancers. This study aims to evaluate the association of ACE insertion or deletion (I/D) polymorphism with the risk of breast cancer incidence and the effect of genotype prognostic criteria of the tumor.

Method

The study included 36 breast cancer female patients and 46 healthy control females. DNA was extracted from tissue and blood samples, and polymorphism was detected by polymerase chain reaction followed by gel electrophoresis.

Results

The distribution of the ACE genotype in the malignant group was 55.6% DD, 44.4% ID and 0% II. The DD genotype showed a significantly higher risk for the incidence of breast cancer (OR=3.9, 95% CI=1.5–10.2, p<0.05). Moreover, there was an association between the DD genotype and the risk for tumor metastasis (OR=1.7, 95% CI=0.3–11), poor differentiation (OR=1.5, 95% CI=0.4–5.8) and larger tumor size (OR=1.3, 95% CI=0.3–5.6).

Conclusion

Our findings suggest a probable prognostic value of the DD genotype in breast carcinoma.

Research Supporter: Dubai Medical College for Girls, Dubai
Analysis Of Ciprofloxacin Induced Evolutionary Changes In E. coli
Authors: Santos Elana , Khalpari Omid , Holden Todd , Gadura Nidhi
Published: June 2021

This study is designed to look at microbial evolutionary changes in E. coli brought about using a controlled dosage of the antibiotic ciprofloxacin as the selective pressure. The evolutionary changes were studied using bioinformatics. Wildtype (drug sensitive) E. coli was grown in a medium containing a low concentration of ciprofloxacin in order to keep the population stable over the course of several hundred generations. Ciprofloxacin is a type of quinolone antibiotic that binds to and interferes with the function of topoisomerases. Gyrase is a type of topoisomerase, an essential enzyme that unwinds DNA during replication. Mutations in genes GyrA or GyrB can prevent DNA replication. We used PCR to amplify both of these genes and analyze their sequences to reveal if long term exposure to ciprofloxacin has induced any mutations. Various drug resistant strains will also be isolated, the GyrA and GyrB DNA sequenced and analyzed to look for mutations. The presenter is a NIH‐Bridges student and the project is partially funded by CUNY – CCIRG grant.
Interactions of human mismatch repair proteins MutSalpha and MutLalpha with proteins of the ATR‐Chk1 pathway
Authors: Liu Yiyong , Fang Yanan , Shao Hongbing , Lindsey‐boltz Laura , Sancar Aziz , Modrich Paul
Published: June 2021

At clinically relevant doses, chemotherapeutic SN1 DNA methylating agents induce an ATR‐mediated checkpoint response in human cells that is dependent on functional mismatch repair activities MutSα and MutLα. To better understand the mechanisms linking mismatch repair to ATR‐Chk1 checkpoint activation, we have systematically examined interactions of human MutSα and MutLα with proteins of the ATR‐Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co‐immunoprecipitation, we have detected interaction of MutSα with ATR, TopBP1, BRCA1, Claspin and Chk1, and interaction of MutLα with TopBP1, BRCA1 and Claspin. We were unable to detect interaction of MutSα or MutLα with Rad17, Rad9 or RPA in the extract system. Use of purified proteins confirmed direct interaction of MutSα with ATR, TopBP1 and Chk1, and MutLα with TopBP1. Use of a modified chromatin immunoprecipitation assay showed that PCNA, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLα‐ and MutSα‐dependent fashion after treatment with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. While our failure to observe chromatin enrichment of RPA, Claspin, Rad17‐RFC, and Rad9‐Rad1‐Hus1 could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation. This study was financially supported by the National Institute of General Medical Sciences and the Howard Hughes Medical Institute.
The transcriptional factor, DACH1, functions as tumor suppressor by antagonizing FOX protein signaling
Authors: Zhou Jie , Wu Kongming , Casimiro Mathew C. , Popov Vladimir M. , Pestell Richard G. , Wang Chenguang
Published: June 2021

The Dachshund gene (dac) was originally cloned in Drosophila as a dominant inhibitor of ellipse and encodes a key component of the RDGN. Altered expression or function of mammalian RDGN components have been described in human tumors. DACH1 is thought to regulate gene expression by complexing with DNA‐binding transcription factors. Crystal structure reveals that DACH1 containing a DNA binding motif similar to that found in the winged helix/forkhead subgroup of DNA binding proteins, however, no sequence‐specific DNA binding of DACH1 has been identified so far. Herein, CAST identified a DACH1 DNA‐binding sequence which resembles the FOX (Forkhead Box containing protein) binding site. Genome‐wide in‐silico promoter analysis of DACH1 binding sites identified gene clusters populating cellular pathways associated with the cell cycle and growth factor signaling. ChIP‐Seq mapped DACH1 binding sites to corresponding gene clusters predicted in silico and identified as weight matrix resembling the CAST‐defined sequence. DACH1 antagonized FOXM1 target gene expression, promoter occupancy in the context of local chromatin, and contact‐independent growth. Proteomic analysis identified CA150 as the transcriptional co‐regulator of DACH1 activity. Attenuation of FOX function by the cell fate determination pathway has broad implications given the diverse role of FOX proteins in cellular biology and tumorigenesis.
Pokeweed antiviral protein (PAP) exhibits variable binding affinity to uncapped mRNA with differing structural elements
Authors: Friedland Diana E , DeGrazia Jeannine , Toney Alexandra
Published: June 2021

Ribosome‐inactivating proteins (RIPs) exhibit N‐glycosidase activity by depurinating a surface exposed adenine of the α‐sarcin/ricin loop (SRL) of the large rRNA, ultimately causing cell death. They are found in numerous plant, fungi, and bacterial species and are powerful antiviral agents. PAP is a single‐chain RIP and also possesses mRNA recognition, cap binding, and depurination activities that are novel to RIPs. PAP's recognition of certain secondary structural elements of uncapped mRNA are of key interest in elucidating its enzymatic mechanism.

Here, constructs of tobacco etch virus (TEV) mRNA, each containing specific structural elements, were used to study equilibrium binding. Steady state fluorescence spectroscopy was used to study binding between PAP and three distinct mRNA constructs. The Kd values for the TEV mRNA constructs are given here: Full length 5′‐leader (3 pseudoknots) (FL) = 63nM, PK1 (pseudoknot 1) = 183nM and S2‐3 (a mutation in PK1) = 85nM. These results suggest that PAP recognizes specific structural elements within the mRNA for binding. The data here will be compared to equilibrium binding between PAP and FL, PK1, and S2‐3 capped TEV mRNA. This will show whether the cap structure recruits PAP at a higher affinity than the uncapped mRNA in order to place PAP within the molecular reading frame for scanning and and possibly subsequent depurination.
Kinetic Analysis of Iron Responsive Element (IRE) mRNA with Iron Regulatory Protein (IRP1)
Authors: KHAN MATEEN A , Walden William E , Theil Elizabeth C , Goss Dixie J
Published: June 2021

In mRNA a family of 5′‐noncoding structures, iron response elements (IRE), control mRNA translation. When iron concentrations decrease, IRP protein binds to the IRE‐RNA and inhibits formation of polysomes. To gain insight into the dynamic process of IRE‐mRNA/IRP regulation and the mechanism of binding, the kinetics of IRE mRNA binding to IRP1 protein were examined. The association rate constant for Ferritin IRE‐RNA was about 6‐fold faster as compared to mitochondrial IRE‐RNA (kon = 400 μM−1s−1 for Fer‐IRE and kon = 50 μM−1s−1 for mt‐IRE). However, the dissociation rate constant was similar for the two IRE‐RNAs binding to IRP1 protein. Addition of 50 μM magnesium ions (a model for air sensitive Fe2+) decreased the rate of association 5‐fold and increased the rate of dissociation 3‐fold for Ferritin IRE‐RNA and for mitochondrial IRE‐RNA the rate of association decreased <3‐fold and the rate of dissociation increased ~2‐fold. Metal ion selective destabilization of Ferritin and mitochondrial aconitase RNA/protein complexes as reported here explain in part quantitative differences in signal response to iron in vivo and indicate possible new regulatory interactions. These results demonstrate the first direct kinetic measurements of IRP1 protein binding to IRE‐RNA.

NSF: MCB‐ 0814051 (to, D.J.G.)

NIH: DK20251 (to E.C.T.)
The electrode‐electrolyte impedance spectroscopy measurements of cancer cell culture media and KCl (potassium chloride) while calibrating a system throughout a frequency range
Authors: Richardson Clarice , Bhansali Shekhar , Vuppala Geetha
Published: June 2021

The study and application of how systems are calibrated throughout a frequency range and impedance measurements are vital skills. The research objectives were to analyze how to calibrate a system to obtain correct readings throughout a frequency range and record the impedance of standard KCL (Potassium Chloride) and different cell culture media. Two methods that were used to calibrate the system were: 1. Open and short compensation and 2. Open, short and load compensation. We identified a system, Multi Micro‐Electrode Bio‐Sensor, located at Univ. of South Florida's Electrical and Biomedical Engineering Laboratory and conducted tests to study electrode‐electrolyte interface of impedance spectroscopy measurements. To understand how to calibrate the system, we built a circuit using the leads that come from the PG Stat 30 instrument which supplies the voltage and current and placed a load (10.7 KΏ resistor) across the leads. We found at lower frequencies, we obtained the correct value of the load resistor; however, at high frequencies, we obtained incorrect results. We also used KCL (Potassium Chloride) to study and understand the physical aspects of the Biosensor System. The Biosensor is also used to measure Electrode‐Electrolyte Impedance Spectroscopy of Cancer Cell Culture Media and KCL. Future work will focus on the long‐term analysis of the Multi Micro‐Electrode Bio‐Sensor System.
Metabolomic analysis of the Hepatocyte Nuclear Factor 1 Alpha knockout mouse
Authors: Bonzo Jessica A , Patterson Andrew D , Krausz Kristopher W , Gonzalez Frank J
Published: June 2021

Genomic mutations in the transcription factor hepatocyte nuclear factor 1 alpha (HNF1α) result in maturity‐onset diabetes of the young type 3 (MODY3), a form of non‐insulin dependent diabetes mellitus (NIDDM). In addition, Hnf1α was identified as a critical factor in the expression of many transporters of the liver, intestine, and kidney. In order to identify the physiological impact of Hnf1α regulation of transporter proteins, metabolomic analysis was performed on urine from Hnf1α+/+ and Hnf1α −/− using ultra‐performance liquid chromatography (UPLC) coupled with accurate mass determination by time‐of‐flight mass spectrometry (TOFMS). Multivariate analysis revealed clear separation of urinary metabolites between Hnf1α +/+ and Hnf1α −/− mice. Amino acid derived compounds were elevated in the urine of Hnf1α −/− mice suggesting a defect in apical reabsorption in the proximal tubules of the kidney. mRNA expression of members of the imino‐glycine family of transporters was also reduced in Hnf1α −/− mice. In addition to amino acid excretion, steroid hormones associated with adrenal gland function were elevated in the urine of knockout mice. Adrenal glands of Hnf1α −/− were 3‐fold heavier than Hnf1α +/+ and histology showed hyperplasia and reduced lipid content indicating a role for Hnf1α in the endocrine system. Research funded by NIGMS PRAT program and NCI Intramural Research Grants.
Regulation of hepatic CYP2B enzymes: Translational control via the heme‐regulated inhibitor (HRI)
Authors: Acharya Poulomi , Correia Maria Almira
Published: June 2021

Phenobarbital (PB)‐induction of hepatic CYP2B synthesis entails a well‐characterized constitutive androstane receptor‐mediated transcription/translation process. Our objective was to show that heme could regulate PB‐mediated‐induction of hepatic CYP2B synthesis at the translational level, most likely via the hepatic heme‐regulated inhibitor (HRI). We have shown that hepatic HRI exists in mice, rats and humans and functions as a heme‐sensitive eIF2α (eukaryotic translation initiation factor) kinase. Acute heme depletion of rat or mouse hepatocytes results in activation of HRI via autophosphorylation. Once activated, it phosphorylates eIF2α which in turn results in global translational arrest of hepatic proteins including that of CYP2B enzymes. Heme repletion reverses these effects. To further document a direct role for HRI in translational control of hepatic CYP2B enzymes, we have used a HRI (−/−) knockout (KO) mouse model. Acute heme depletion of KO‐mouse hepatocytes results in constitutive heme‐insensitive PB or Dexamethasone‐mediated induction of CYP2B and CYP3A enzymes respectively, unlike in the corresponding wild type [WT; HRI (+/+)].Thus HRI is a key heme‐sensitive translational regulator of hepatic P450 syntheses. Additionally we found that HRI content and phosphorylation is increased by P450 inducers such as PB, rifampin and barbituric acid. Collectively, our findings indicate that HRI activation after acute hepatic heme depletion in genetically predisposed individuals can result in global translational shut‐off of physiologically critical proteins leading to the severe and often fatal clinical symptomatology of acute hepatic porphyrias the severity of which could be further exacerbated upon HRI induction by cytochrome P450 inducers. Supported by NIH grants DK26506 and DK26743.
Mechanism‐Based Inhibition of Human Cytochrome P450 2D6 by Schering 66712
Authors: Mocny Catherine S. , Arthur Evan J. , Butler Brendan F. , Diffenderfer Laura E. , Palamanda Jairam R. , Guengerich F. Peter , Nomeir Amin A. , Furge Laura Lowe
Published: June 2021

Schering 66712 (5‐fluoro‐2‐[4‐[(2‐phenyl‐1H‐imidazol5‐yl)methyl]‐1‐piperzainyl]pyrimidine) is a mechanism‐based inactivator of human cytochrome P450 2D6 (P450 2D6) that displays Type I binding with KS of 0.39 ± 0.10 μM. The partition ratio is approximately 300. Inactivation was not prevented by addition of an exogenous nucleophiles including potassium cyanide. Within 15 minutes of incubation with Schering 66712 and NADPH, ~100% of P450 2D6 activity was lost with ~40% loss in ability to binding CO. These findings support inactivation primarily by adduction of protein. ESI‐LC‐MS analysis of whole protein also indicates the presence of a protein adduct. Modeling of Schering 66712 in the active site of P450 2D6 suggests interaction between the phenyl group of Schering 66712 and the heme iron. (Supported by: NIH GM086767‐01 and RGM086767Z, a grant from the Howard Hughes Medical Institute to Kalamazoo College, and the Hutchcroft Fund of Kalamazoo College).
Kinetic analysis of steps involved in oxidation by cytochrome P450 19A1
Authors: Sohl Christal Dyane , Guengerich F. Peter
Published: June 2021

Cytochrome P450s are heme‐containing monooxygenases that catalyze the oxidation of endogenous compounds (e.g. vitamins and steroids) and exogenous compounds (e.g. drugs and pollutants). P450 19A1, or aromatase, is responsible for the conversion of androgens (androstenedione or testosterone) to estrogens (estrone or 17β‐estradiol). The reaction proceeds in three sequential steps, generating first a 19‐hydroxy‐ and then a 19‐aldo‐intermediate prior to the final aromatization step. Much study has focused on the inhibition of P450 19A1 because aromatase inhibitors have proven to be successful therapy for estrogen‐dependent breast cancer. However, few steady‐state studies on catalysis involve endogenous substrates and pre‐steady‐state kinetic characterization has not been done. We have developed a procedure for consistent heterologous bacterial expression and purification of P450 19A1. Kd values for androstenedione, 19‐hydroxyandrostenedione, 19‐aldoandrostenedione, and estrone were measured to be in the very low μM range. Stopped‐flow spectroscopy indicated that binding of these ligands includes a fast spectroscopically silent (in absorbance) step, followed by a slower conformational change (E + L ⇔ EL ⇔ LE), which can be fit to double exponential curves. Single turnover (rapid chemical quench) yield progress curves that show temporal loss of substrate and formation of products and intermediates, generating rate constants for the sequential steps of catalysis. Global fitting of these steady state and pre‐steady state data allow the generation of the first kinetic models of the overall mechanism of P450 19A1 catalysis. Supported by USPHS R37 CA090426, T32 ES001590.
Acetylbritannilactone inhibits smooth muscle cell proliferation through suppression of cell cycle progression, ERK1/2 activation, and induction of apoptosis
Authors: Liu Bin , Han Mei , Sun Rong‐hua , Wang Jun‐jie , Liu Yue‐ping , Wen Jin‐kun
Published: June 2021

Smooth musclecell (SMC) proliferation and migration are responsible for restenosis after angioplasty and stent placement. Acetylbritannilactone (ABL) has been shown to have anti‐inflammatory and anticancer properties. To evaluate its therapeutic implication in atherosclerosis, we examined whether ABL could affect SMC proliferation and apoptosis in vivo and in vitro. ABL treatment inhibited platelet‐derived growth factor (PDGF) or fetal bovine serum (FBS)‐induced DNA synthesis and proliferation in cultured SMC. Such growth‐inhibitory effects were associated with G1 phase arrest. When analyzing the expression of cell cycle‐related proteins, we found that ABL reduced the expression of cyclins and cyclin‐dependent kinases (CDKs) and increased p53‐p21cip1. In addition, ABL induced apoptosis in SMCs, as evidenced by the activation of caspase‐3 and the cleavage of endogenous substrate Poly (ADP‐ribose) polymerase. ABL also increase the expression of Bax with a concomitant decrease Bcl‐2 expression. Furthermore, the antiproliferative and pro‐apoptotic effects of ABL were associated with the downregulation of extracellular signal‐regulated kinase (ERK) 1/2 signaling pathways. In vivo, ABL reduced injury‐induced ERK1/2 phosphorylation and proliferating cell nuclear antigen (PCNA), increased p53‐p21cip1 expression and SMC apoptosis 14 days after balloon injury. Taken together, these findings suggest a potential use for ABL in the prevention of restenosis after angioplasty.
Molecular Mechanism of Nanodiamonds Attacking Cancer Cell
Authors: Chen Hong‐Wei , Cheng Tsai‐Mu , Chu Hsueh‐Liang , Chang Chia‐Ching
Published: June 2021

Nanodiamond (ND) surrounds with layers of graphite on its surface. This unique structure feature creates unusual fluorescence spectra which can be used as an indicator to monitor its surface modification. Meanwhile, it contains nitrosol (C‐N=O) inside the ND and this impurity can absorb nanosecond laser radiation to cleavage the ‐C‐N=O chemical functional group into ‐C +N≡O. Moreover, the NO gas molecules are heated by hot phonon of ND and generate huge pressure inside the diamond core to facilitate the sp3 diamond core transforming into sp2 graphite structure as a nanoblast. Therefore, the growth hormone receptor (GHR) of cell surface can be destroyed by growth hormone conjugated‐ND (NDGH) and then be triggered the process of cell apoptosis. This photo‐trigger nanoblast can be used as nano‐knifes in nano‐medicine application.
A role for c‐Jun N‐terminal kinases in apoptosis triggered in human macrophages by carboxydextran‐coated superparamagnetic iron oxide nanoparticles
Authors: Lunov Oleg , Syrovets Tatiana , Buechele Berthold , Tron Kyrylo , Nienhaus G. Ulrich , Simmet Thomas
Published: June 2021

Superparamagnetic iron oxide nanoparticles are widely used for cell labeling and as diagnostic contrast media. The aim of this study was to investigate how carboxydextran‐coated superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) nanosized particles used for magnetic resonance imaging might affect signaling and functions of human macrophages. Within 1 h, both SPIO and USPIO were rapidly taken up by macrophages leading to increased intracellular iron contents. One day after treatment, most nanoparticles were localized within the lysosomal compartment. Prolonged exposure of the macrophages to SPIO and USPIO led to a significantly enhanced production of reactive oxygen species associated with a long‐lasting activation of JNK kinases and induction of apoptosis as measured by surface expression of phosphatidylserine and caspase 3 activation. USPIO elicited a more pronounced cytotoxicity than SPIO. Treatment of macrophages with Trolox® or N‐acetyl‐L‐cysteine, two strucurally different scavengers of reactive oxygen species, or with the JNK inhibitor V abolished the JNK activation as well as the cytotoxicity caused by the iron oxide nanoparticles. These data indicate that nanosized contrast media hamper macrophage function, an effect that can be antagonized with suitable oxygen radical scavengers. This work was supported by the DFG.
Quantitative interactome analysis of RGS9‐2 degradation mechanisms in mammalian brain.
Authors: Posokhova Ekaterina , Uversky Vladimir N , Martemyanov Kirill A
Published: June 2021

RGS9‐2 is a striatal‐specific protein involved into regulation of movement and reward through negative regulation of muopioid and D2 dopamine receptor signaling. Changing RGS9‐2 expression is an important way to achieve the plasticity of signaling regulation and behavioral outcomes. R7 binding protein (R7BP) is an interaction partner of RGS9‐2 that sets the expression of RGS9‐2 by controlling proteolytic stability of the complex. Uncoupling of RGS9‐2 from R7BP is thought to be a mechanism of RGS9‐2 levels regulation.

The aim of the study was to elucidate the molecular mechanism of RGS9‐2 degradation upon loss of R7BP.

We developed a proteomic approach for quantitative analysis of interactome changes in mouse knockout models and used it to identify interactions of RGS9‐2 upregulated in R7BP knockout mice. The screen resulted in the identification of 21 proteins among which chaperone Hsc70 was found to exhibit one of the biggest changes in binding to RGS9‐2. The interaction was validated both in vivo and in transfected 293 cells and was found to be mediated by the intrinsically disordered C‐terminal domain of RGS9‐2. Down‐regulation of Hsc70 expression using siRNA and a dominant negative approach increased levels of RGS9‐2.

We conclude that Hsc70 is a negative regulator of RGS9‐2 levels in the striatum.

NIH NIDA DA021743
Production of recombinant dengue virus‐like particles by COS‐1 cells in a serum‐free defined medium
Authors: Simonian Michael Haig , Nair Mahalakshmi , Jacky Lucien , Chang Gwong‐Jen
Published: June 2021

COS‐1 cells transfected with recombinant genes from the four dengue virus serotypes (DENV‐1, ‐2, ‐3 and ‐4) produce and secrete virus‐like particles (VLP). The recombinant DENV envelope and premembrane or membrane proteins are translated and assembled by the transfected cells into VLPs, which are noninfectious because they lack the genomic RNA. The four DENV VLPs are combined and used as antigens to clinically diagnose IgM and IgG antibodies to DENV infection in human serum by enzyme‐linked immunosorbent assays (ELISA). A serum‐free defined medium was developed to replace the fetal bovine serum (FBS) supplemented medium currently used to produce DENV VLP in COS‐1 cells, which was done on a four day harvest cycle. Different supplements in Dulbecco's Modified Eagle Medium (D‐MEM) were evaluated for COS‐1 production of DENV‐1, ‐2, ‐3 and ‐4 VLP by Western blot analysis and ELISA of the culture medium. D‐MEM supplemented with lipid‐rich bovine serum albumin (BSA) supported secretion of the four DENV VLPs for up to eight harvest cycles that was equivalent to FBS supplemented medium. Neither D‐MEM alone nor supplementation with fatty acid‐free BSA supported VLP secretion. This serum‐free defined medium will reduce cost of production, minimize variability between batches due to FBS and to facilitate DENV VLP purification for the ELISA kits. Source of research support: Focus Diagnostics, Inc.
Dabcyl/Fluorescein‐based Probes for Detection of Thiols and Disulfides: Proteomic Application to Discovery of Reactive Disulfides in Live Cells
Authors: Jonhera Timothy , Pullela Phani Kumar , Bassam Wakim T , Carvan Michael J , Sem Daniel S
Published: June 2021

Most diseases produce some perturbation of the levels and redox state of cellular thiols. Herein, we report an improved version of our previously reported fluorescent‐dithio probes for detecting cellular thiols, referred to as PMR‐CYS‐FITC, where PMR is p‐methyl red (aka dabcyl), a fluorescein quencher. Unlike most thiol‐reactive probes, PMR‐CYS‐FITC can also detect certain disulfides, making it useful for ‘disulfide proteomics’. PMR‐CYS‐FITC was used to identify new disulfide‐containing protein(s) in the zebrafish chorion, with potential role(s) in protecting the embryo during development. Confocal microscopy of the embryo, followed by in‐gel staining and imaging of proteins, then tandem MS, led to our discovery of two novel disulfide‐containing proteins in the fish chorion, lipovitellin and C‐reactive protein (CRP). CRP is a well‐known biomarker for infection or inflammation (associated with oxidative stress) in humans, so discovery of CRP in the zebrafish has relevance for its use as a model organism. In vitro biophysical characterization of this probe provides further explanations for the differential reactivity of disulfides, and enhanced fluorescence for some fluorescein‐protein mixed disulfides. Research was supported by BTA grant #01‐KMS LEG FY06‐12368.
Antioxidant and anti‐inflammatory effects of black rasperries in a dextran sodium sulfate (DSS) model of colitis
Authors: Horelik Nicole Annette , Montrose David C , Madigan James , Pleau Devon , Rosenberg Daniel W , Stoner Gary D
Published: June 2021

Previous studies suggest the antioxidant and anti‐inflammatory effects of anthocyanins—antioxidants abundantly found in berry fruits such as black raspberries. In our study, a dextran sodium sulfate (DSS)‐induced model of inflammatory colitis was used in mice in order to study the effects of black raspberries. Mice were administered DSS in their drinking water and treated with either a control or black raspberry powder diet. Cellular markers of reactive oxygen species were investigated as biochemical indicators of oxidative damage. Although there was evidence of oxidative damage, our results indicated no significant differences. Proinflammatory cytokine expression levels as well as immunohistochemical analysis of the NF‐kappaB signaling pathway through western blots were used to study the biochemical indicators of inflammation. The relative mRNA expression levels of cytokines, interleukin‐1β and tumor necrosis factor‐α increased after DSS treatment, but showed suppression after treatment with black raspberries. Western blot analysis demonstrated an increase in the proinflammatory NFkappaB signaling pathway in mouse colon tissue after DSS treatment. Furthermore, colon tissue from mice treated with black raspberry displayed a decrease in signaling through the NFkappaB pathway after DSS treatment. Our results provide evidence of the anti‐inflammatory effects of black raspberries.
Synthesis and the Biological Evaluation of Arylnaphthalene Lignans as Anti‐hepatitis B Virus Agents
Authors: Yeh Sheau Farn , Lin Chih Hsiu , Janmanchi Damodar , Tseng Ya Ping
Published: June 2021

We have previously shown that helioxanthin can suppress human hepatitis B virus gene expression. A series of helioxanthin analogues were synthesized and evaluated for their anti‐hepatitis B virus activity. Modifications at the lactone rings and methylenedioxy unit of helioxanthin can modulate the antiviral activity. Among them, compound 32 is the most effective anti‐HBV agent. Compound 32 can suppress the secretion of viral surface antigen and e antigen in HepA2 cells with EC50 values of 0.06 and 0.14 μM, respectively. Compound 32 not only inhibited HBV DNA with wild‐type and lamivudine‐resistant strain but also suppressed HBV mRNA, core protein and viral promoters. In this study, a full account of the preparation, structure‐activity relationships of helioxanthin analogues, and the possible mechanism of anti‐HBV activity of this class of compounds are presented. This type of compounds possesses unique mode of action differing from existing therapeutic drugs. They are potentially new anti‐HBV agents.
DNA Analysis Using Polymerase Chain Reactions and Micro‐Electrophoresis
Authors: Hunley Ryan Timothy , Harwood John , Boles Jeffrey O. , Mancuso Matt
Published: June 2021

Polymerase Chain Reaction (PCR) is used in a variety of applications in forensics, medicine, and research. PCR relies on the ability to thermally break apart a DNA strand, and replicate selected portions of the DNA (if present) through addition of “primer” coding for the selected DNA segment. Following amplification through 30–40 precisely controlled thermal cycles, samples are analyzed for the DNA segments by electrophoresis. We use a bioanalyzer “lab‐on‐a‐chip” instrument in the analysis, which represents an example of a micro‐total analysis system (u‐TAS).

We are developing experiments to help chemistry students in the second semester quantitative analysis course become more familiar with PCR and with micro‐total analysis analyzers. We use a commercially available kit to provide needed PCR primers and reagents, and standard reference corn and soya powder samples containing the gene modifications. Components of “Round‐up Ready” and Bt‐producing crops, the 35S promoter of the cauliflower mosaic virus and the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, are amplified. Students are assigned to bring in food samples of their own choosing for analysis. We are investigating the feasibility of performing analyses for other gene modifications in foods as experiments in undergraduate analytical and biochemistry courses.
Team Based Learning Activities in the Academic Research Laboratory
Authors: Stahelin Robert V
Published: June 2021

Learning for entering the culture and practice of science takes place in three program‐embedded‐areas: laboratory‐centered, program centered, and peer‐centered. Additionally, group‐based work brings together skills at acquiring, communicating, and integrating information, while prospective employers support programs that allow the growth of interpersonal, organizational, and teamwork skills that will better prepare students for the scientific workforce. Team Based Learning (TBL) originated with Larry Michaelsen in the late 1970's. TBL is an especially powerful form of learning as it creates learning teams, which are distinct from other group learning activities. Based upon our preliminary studies and the current state of literature in the education field, we hypothesized that high efficiency learning teams could be created in the academic laboratory and be used to revitalize group meetings. Mentoring of undergraduate, graduate, and postdoc research teams performing research in the Stahelin lab now utilizes TBL for efficient scientific discovery. The outline, assessment, advantages and disadvantages for including these learning modules in productive academic research groups will be presented.
Zebrafish as a model to determine the mechanisms of vitamin E function
Authors: Miller Galen W , Labut Edwin M , Lebold Katie , Floeter Abby , Tanguay Robert L , Traber Maret G
Published: June 2021

Vitamin E (alpha‐tocopherol, AT) is required to prevent fetal resorption in rodents. To study its role in fetal development, a non‐placental model is required. Therefore, we have developed an AT‐deficient zebrafish model by feeding a defined diet (with or without AT) that we created. Zebrafish (age: 1 m) were fed the deficient (E−), sufficient (E+), or Lab diet up to 1 y. All groups showed similar growth and normal development. When compared to E+, the E− fish had lower (p<0.05) AT levels within 3 w; by 112 d the E− fish (3±1 nmol/g) contained 100 fold less AT than the E+ fish (375±100, p<0.001). The rate of depletion was 0.029 ± 0.006 pools/d. At age ~4 m, E‐depleted adults were spawned and produced viable embryos with similar AT concentrations to those of their parents. The E− embryos exhibited a higher mortality at 24 h post fertilization (hpf) and a higher combination of malformations and mortality at 120 hpf than the control diets. This study documents for the first time that vitamin E is essential for normal embryonic development. It also shows that the zebrafish is an exceptional model to define the molecular functions of vitamin E.

Grant Funding Source: NIH #HD062109 and #ES00210
Chronic alcohol consumption causes hyperlipidemia and depleted vitamin E status in cynomolgus monkeys
Authors: Lebold Katherine , Leonard Scott W , Miller Galen , Grant Kathleen , Traber Maret G
Published: June 2021

Chronic ethanol consumption induces oxidative stress; thus to evaluate the relationship between alcohol consumption and α‐tocopherol (AT) status, monkeys voluntarily drinking ethanol (1.36 to 3.98 g/kg for 15 months, n=11) were compared with non‐drinkers (n=5). Drinking induced hyperlipidemia in three of the alcoholic monkeys, such that plasma triglyceride (TG) levels (1.8 ± 0.9 mmol/L) were double (p<0.05) those of alcoholics (0.6 ± 0.2) or controls (0.6 ± 0.3); plasma cholesterol levels (3.6 ± 0.5 mmol/L, p<0.05) were also elevated compared with alcoholics (2.3 ± 0.2) or controls (2.9 ± 0.3). Plasma AT concentrations were positively correlated with both TG (R=0.492, p<0.0001) and cholesterol concentrations (R=0.504, p<0.0001). Plasma AT per TG (14.8 ± 7.1 mmol/mol) were lower (p<0.05) than in controls (29.3 ± 9.0) and alcoholics (27.5 ± 7.8). Liver α‐tocopherol concentrations (47 ± 8 nmol/g) were somewhat lower (p=0.068) than those of alcoholic (69 ± 5) or control monkeys (70 ± 6). Thus, alcohol drinking monkeys with hyperlipidemia have an apparent defect in lipoprotein metabolism resulting in lower plasma AT per TG and depleted tissue α‐tocopherol suggesting increased oxidative stress.
Endothelial protective effects of erythritol
Authors: den Hartog Gertjan , Berger Alvin , Bast Aalt
Published: June 2021

Vascular health is strongly dependent on endothelial state, which regulates vascular tone and inflammatory responses. High glucose‐induced endothelial cell apoptosis, likely via reactive oxygen species (ROS), has a pivotal role in diabetes‐associated vascular diseases, including atherosclerosis. We evaluated the anti‐oxidative and endothelium‐protective properties of erythritol (ERT), a non‐caloric sweetener which is well tolerated, and whose chemical structure resembles mannitol, a hydroxyl radical scavenger. In competition assays, ERT was an excellent hydroxyl radical scavenger (inert towards superoxide radicals); and inhibited radical–induced hemolysis. ERT reacted with hydroxyl radicals forming erythrose and erythrulose by carbon‐bound hydrogen atom abstraction. In streptozotocin‐induced diabetic rats, ERT had endothelium‐protective effects and erythrose was detected in urine. The clear endothelial protective effects of ERT cannot be explained by hydroxyl radical scavenging only. We are currently investigating mechanisms underlying protective effects of ERT on endothelium dysfunction in various model systems.

This research project was financially supported by Cargill Inc.
Press Juices from Hamour and Kingfish exhibit Antioxidant Properties
Authors: Waly Mostafa I , Al‐Sabahi Jamal N , Al‐Bulishi Mohamed , Soussi Bassam
Published: June 2021

Many recent studies have suggested that regular consumption of antioxidant‐rich foods improve the cellular antioxidant capacity and hence reduce oxidative stress and related chronic diseases occurrence.

The main aim of this study was to evaluate the antioxidants contents of the press juices extracted from Hamour and King Fish, locally available fishes in Oman.

Press juices (PJs) filtrate was prepared by centrifuging the fish's light muscle portion at 18368 g for 2 hrs at 4 °C. For the measurements, 10 filtrates were prepared. Using HPLC, vitamin C was measured for Hamour PJ (89.22 μM ± 1.2) and for kingfish PJ (85.13 μM ± 1.2), p >0.05.

Total antioxidants content was quantified using a commercial kit (RANDOX, UK) and the measurements were significantly higher for Hamour PJ (1.32 mmol/Liter ± 0.6) as compared to kingfish PJ (0.55 mmol/liter ± 0.8), p <0.05. Glutathione (GSH) was measured using fluorescent ELISA (APO‐GSH, USA) assay kit and the GSH content of Hamour fish PJ; 412.5 μg ± 9.3 was significantly higher than that of kingfish 269.43 μg ± 13.7, p <0.05. All results were the mean ± SD and student t‐test, unpaired test, was used for the statistical analysis.

This study highlights the potential of aqueous of PJs filtrates from Hamour and kingfish as natural powerful antioxidants. Human cell culture studies are on‐going to shed further light on the protective health aspects of Press Juice.

This study was supported by HM Trust Fund for Strategic Research, SR/AGR/FOOD/05/01.
Zip5: a candidate protein for zinc transport from maternal circulation into the mammary gland
Authors: Velasquez Vanessa Renee , Kelleher Shannon L
Published: June 2021

An extraordinary amount of zinc (Zn) moves through the mammary gland (MG) and into milk during lactation, but the mechanism by which the MG accumulates Zn from maternal circulation is not yet understood. We postulate that a Zip protein is responsible for importing Zn into the MG. Zn accumulation in the MG in vivo was visualized using X‐ray fluorescence microscopy. Candidate Zip proteins (Zip1, 3, 5, 6, 8, 10) were identified by real‐time PCR in secreting mouse mammary epithelial cells (HC11). This screen identified Zip5 by a 10‐fold increase in mRNA expression relative to non‐secreting cells. To confirm Zip5 protein expression during lactation, we assessed expression by immunblotting. Zip5 protein expression was ~3 fold higher in lactating compared with non‐lactating MG, further supporting the hypothesis that Zip5 is important for Zn accumulation in the MG during lactation. To confirm that Zip5 was appropriately localized to transport Zn from maternal circulation, we successfully localized Zip5 to the serosal membrane of epithelial cells in mouse MG using immunohistochemistry, and by cell surface biotinylation and confocal microscopy in HC11 cells. In conclusion, our data supports the postulate that Zip5 is a candidate for Zn accumulation from maternal circulation as it is localized appropriately and protein expression increases during lactation in the MG.

Supported by intramural funds to SLK

Grant Funding Source: intramural funds to SLK
Sequence of the peptide component of low‐molecular‐weight chromium‐binding substance
Authors: Vincent John B. , Chen Yuan , Watson Heather , Cassady Carolyn J.
Published: June 2021

Two biomolecules are known to bind chromium in the body: transferrin and low‐molecular‐weight chromium‐binding substance (LMWCr). While transferrin is responsible for the transfer of Cr from the bloodstream to the tissues, LMWCr has been proposed to have a function in the removal of chromium from the body and in the enhancement of insulin signaling. LMWCr is comprised of a peptide that can tightly bind four equivalents of Cr(III). While reports on the magnetic and spectroscopic properties of the chromic centers have appeared, research has been hampered by difficulties in characterizing the organic portion of the biomolecule. Treatment of LMWCr with trifluoroacetic acid releases the Cr, converts the amino terminal glutamate of the peptide to pyroglutamate, and generates a heptapeptide. This procedure has allowed the isolation of the chromium‐free peptide component of LMWCr from a variety of sources; this peptide can be sequenced by mass spectrometry.
Hemoglobin and blood lead associated with mothers’ perceptions of the relationship with their children
Authors: Kordas Katarzyna , Queirolo Elena I , Ardoino Graciela , Manay Nelly
Published: June 2021

Iron‐deficient (ID) mothers are less emotionally available to children; maternal behavior may be related to child ID and behavior. It is unclear how mother or child iron status affects maternal perception of the mother‐child relationship. No studies also exist on child or mother blood lead level (BLL) and maternal perceptions of the mother‐child relationship. We examined these questions in 118 mother‐child pairs participating in a study of ID and BLL and cognition of Uruguayan children (31.2±9.9 mo). Hb and BLL were measured in venous blood. Mothers (28.9±7.2 y) were assessed on depression, IQ, and statements about mother‐child relationship. Two factors were identified from statements: limit setting (LS) and emotional‐cognitive support (ECS). Hb_child was 13.7±1.6 g/dL (15.7% were <11) and Hb_mom 12.2±1.7. BLL_mom was 5.9±2.7 μg/dL and BLL_child 6.3±3.3 (15.7% were ≥10). For kids <24 mo, Hb_child was related to ESC scores (β=1.9, p=.063), adjusting for covariates. BLLs were related to LS (β=−0.5, <.01 child; β=−0.4, <.05 mom). Hb and BLL were associated with how mothers perceived the mother‐child relationship. Altered perceptions could impact maternal behavior and child development.
The effect of soy diets on obesity‐related pathologies in lean and obese Zucker rats.
Authors: Cain James , Davis Jeremy , Strader April , Butteiger Dustie
Published: June 2021

The purpose of this study was to compare the effects of soy versus dairy whey and caseinate diets on obesity‐related pathologies such as non‐alcoholic fatty liver and impaired glucose homeostasis in an insulin resistant rodent model. Male lean (L) and obese (O) Zucker rats were randomly assigned to three diet groups for 17 weeks: sodium caseinate control (C), dairy whey control (W) and soy protein (S). Physiological data were collected throughout the study and at week 14 animals were subjected to an oral glucose tolerance test (OGTT). As previously shown, OS animals had greater final body weights and body fat percentages, and exhibited an increased food intake vs the caseinate group. OS rats had a significantly lower liver weight and percent liver lipid content versus OC and OW groups. There was no significant difference in non‐fasted blood glucose levels or OGTT between diet groups. Nonetheless, OS rats had significantly lower OGTT plasma insulin concentrations at 15 and 30 min compared with OC. OS and OW groups had significantly lower insulin:glucose ratios than OC at all OGTT time points and for AUC, indicating higher insulin sensitivity in these groups. No statistically significant differences were identified in the lean groups. Further molecular and biochemical analyses are currently under examination. Preliminary results demonstrate benefits of a soy protein diet in amelioration of obesity‐related pathologies such as non‐alcoholic fatty liver and impaired insulin sensitivity.

Grant Funding Source: ISA
Impact of Orally Consumed Lycium barbarum on Various Urinary Neurotransmitter Levels in Healthy Human Male Adults
Authors: Amagase Harunobu
Published: June 2021

Standardized Lycium barbarum (L. barbarum) juice, GoChi® has previously shown statistically significant clinical improvements in weakness, stress, mental acuity, ease of awakening, shortness of breath, focus on activity, sleep quality, daydreaming, fatigue, depression, circulation, calmness and overall feelings of health and well‐being. We investigated the impact of L. barbarum on various neurotransmitters related to the above clinical effects. Subjects (n=5, age=44.6 y) consumed single bout of L. barbarum juice (120 ml) after at least 12 h fast. Urinary neurotransmitter levels were analyzed by EIA one day prior and post‐intake. Compared to pre‐intake, serotonin and taurine levels were significantly increased by 12% and 25% respectively, and 5‐HIAA was decreased by 16% (P<0.05). Epinephrine and norepinephrine levels tended to be elevated, but dopamine, DOPAC, glycine, GABA, PEA, histamine and creatinine levels had no changes. Resting metabolic rate measured by indirect calorimeter was increased by L. barbarum intake, and area under the curve (0–4 hr) over baseline (1,761 kcal) was 927 kcal‐h, higher than the control level (395 kcal‐h) (P<0.05). These results suggest that L. barbarum exhibits stimulus effects on various neurotransmitters and energy/caloric metabolism in humans, which may be related to the improvements in the above subjective symptoms.
Comparison for bioavailability of 1‐deoxynojirimycin with mulberry water extract in rat
Authors: Kim Ji Yeon , Kwon Hye Jin , Jung Ji Young , Kim Do Ram , Kim Young Suk , Kwon Oran
Published: June 2021

The bioavailability of dietary phytochemicals may be influenced by the food matrix in which they are prepared. In this study we compared the intestinal absorption and excretion of orally administered 1‐deoxynojirimycin, which is well known as a potent glucosidase inhibitor, as isolated DNJ and mulberry water extract. When the rats sacrificed after administered mulberry water extract or isolated DNJ at 0, 15, 30, 45, 60, 120, 240, and 360 min, plasma DNJ levels reached a maximum at 30 min and then rapidly decreased. The DNJ levels of plasma 30 min after administration of mulberry leaf extract were lower than isolated DNJ with statistical significance (p<0.05). Moreover, the DNJ was dose‐dependently absorbed from the digestive tract into rat plasma (p<0.0001). We analyzed the concentration of DNJ in plasma, feces, and urine when administered as isolated DNJ as well as mulberry water extract. The absorption and excretion pattern was similar between them. These findings indicate that orally administered mulberry water extract and isolated DNJ are absorbed and excreted as similar pattern and the absorption rate for isolated DNJ is higher than water extract.

Grant Funding Source: ASN
Hepatoprotective Effects of Soybean Varieties Containing Different Levels of Isoflavones and Anthocyanins against Carbon Tetrachloride‐Induced Liver Injury in Mice
Authors: Kim Jon‐Sang , Park Jia , LIM Ji Sun , Nam Dae Hwan , Hong Ye Seul , Han Min Young , Kim Hyo Jung , Seo Ji Yeon
Published: June 2021

Considering the high lethality rate of high isoflavone variety of soy (Aga No. 3) group on mice insulted with CCl4, the potential risk of the excessive intake of isoflavones was examined using four different varieties of soybean (Aga No. 3, Aga No. 4, DaeWon, ChungJa) and 3 different levels of Aga No. 3 with varying concentrations of isoflavones and anthocyanins, 110 mice were divided into 10 experimental groups including control, CCl4–treated group, four soybean + CCl4 groups, and vitamin E + CCl4 group. Mice were fed experimental diet containing 30% soybean, except for Aga No. 3 (5%, 15%, 30%), vitamin E and isoflavone mixture for 13 days, followed by injection with CCl4 (0.6 ml/kg bw, day 1 & 12) and sacrificed at 13th day after injection. The antioxidative properties of soybean diets were compared based on FRAP value and concentration of plasma 8‐OHdG. The hepatoprotective effect of soybean varieties was evaluated by measuring levels of plasma enzymes such as GOT, GPT, and LDH which are biomarkers for hepatic inury. The hepatic levels of antioxidant enzymes including heme oxigenase‐1 and NQO1 collected from experimental groups were compared from each using western blot. Our data suggested that hepatotoxicity of CCl4 was ameliorated by feeding black soybean containing anthocyanins while yellow soybean with any level of isoflavones did not have protective effect.