bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a Translational Control Element Involved in KLF4 Posttranscriptional Regulation
Authors: Lin Chen‐Chung , Ruppert John Michael
Published: June 2021

Krüppel‐like factor 4 (KLF4) is a transcription factor regulating cell growth and differentiation. An important feature of KLF4 is its induction upon growth‐arrest in normal cells. In cancer cells this regulation is disrupted, as proliferating cancer cells show increased levels of KLF4 compared with primary epithelial cells. However, the mechanisms regulating the induction of KLF4 upon growth arrest in normal cells or its upregulation in cancer cells remain largely unclear. We recapitulated the proliferation‐dependent regulation of KLF4 using E1A‐immortalized rat kidney epithelial cells, RK3E cells. In these cells, the endogenous Klf4 RNA and protein was low in proliferating cells but high in post‐confluent cells that underwent a process similar to epithelial differentiation, with upregulation of cell cycle inhibitors such as p21/Waf1 and p27/Kip1. To map potential regulatory elements in KLF4 transcripts, we established an assay system by inserting full‐length or partial‐length fragments of human KLF4 cDNA into the 3′ UTR of firefly luciferase. By measurement of luciferase mRNA and protein levels, we successfully identified a novel, cell‐cycle‐dependent translational‐control element (TCE) in KLF4. This element mediates translational inhibition in proliferating cells. Current studies are focused on possible factors that can bind to TCE and regulate KLF4 expression.
Engineering mRNA structural regulation of transcription using an RNA‐sensing riboregulator
Authors: Lucks Julius Beau , Qi Lei S , Mutalik Vivek , Arkin Adam P
Published: June 2021

The control of gene expression lies at the heart of every genetic engineering enterprise. However, current strategies to manipulate gene expression are dominated by inefficient trial and error screening approaches. Here we experimentally investigate an RNA‐based transcription attenuation mechanism in E. coli with the aim of engineering a family of mutually orthogonal riboregulators that form a platform for the forward design of expression architectures. We focus on the antisense‐RNA‐mediated transcription regulator of S. aureus plasmid pT181 as the basis for this family of control units. Comparative sequence information from similar RNA‐mediated translation mechanisms suggest hypotheses about the detailed binding pathway between antisense and sense RNAs that enable us to investigate the mechanistic origin of specificity in this system with mutational analysis. We demonstrate how these investigations allow us to change specificity in this system, and construct orthogonally‐acting transcription regulators. Furthermore, we show how these transcriptional regulators can be composed in series on the same transcript to give increased cooperativity and the ability to integrate signals on a single transcriptional locus. Finally, we demonstrate how they can be chained together to create a two‐stage RNA‐mediated transcriptional cascade that opens the door for creating general feedback loops with this system. This work is funded as a part of the NSF Synthetic Biology Engineering Resource Center grant and the Miller Institute for Basic Research in Science.
Dynamics of the tRNase Z Flexible Arm
Authors: Wilson Christopher , Levinger Louis , Chang Emmanuel , Lama Neema , Sabry May , Rai Rita , Paul Sabina
Published: June 2021

Pre‐tRNAs are transcribed as precursors which undergo maturation including cleavage of 5′ and 3′ extensions and 3′ end CCA addition. The 3′ trailer is endonucleolytically removed by tRNase Z, a member of the β‐lactamase superfamily of metal‐dependent hydrolases. The body of the enzyme consists of a four‐layer alpha‐beta sandwich including the conserved metal‐coordinating HxHxDH motif. tRNAse Z features a unique protruding flexible arm (FA), an ααββ hand connected to the body of the enzyme by ascending and descending stalks, involved with recognition and binding of the elbow of pre‐tRNA. Deletion of the FA hand increases KM ~70‐fold relative to wild‐type tRNase Z. A 40‐residue Ala‐scan revealed two regions that are critical for binding (Figure), a leucine at the ascending stalk/α4 boundary at which Ala substitution increases KM ~40‐fold, and a glycine‐proline (GP) rich loop that connects α4/α5. Vertebrates have a conserved GP loop TAAIA insertion which could affect the FA‐substrate interaction based on homology modeling. The stalk is structured by an inter‐strand H‐bond network. Hand–stalk geometry is maintained in free enzyme by a conserved H‐bond between a lysine in the hand and a glycine in the stalk. The stalk and first α helix swing back from the body of the enzyme when tRNA is bound. Upon tRNA binding, the ε‐NH2 group of the lysine flips toward substrate, breaking its bond with glycine in the stalk and forming a bond with a T‐loop phosphate. The lysine is susceptible to trypsin, suggesting that its exposure to solvent could be a useful reporter of FA structure. Supported by the NIH.
Mouse lipocalin‐2 aptamer: an RNA probe for molecular discrimination
Authors: Zhai Lijie , Wang Tianjiao , Nilsen‐Hamilton Marit
Published: June 2021

High‐resolution molecular discrimination is a feature of aptamers that can be applied to molecular recognition. Aptamers can recognize molecules for which antibodies are lacking or confirm the molecular species recognized by existing antibodies. To develop the latter approach, an RNA aptamer was selected against mouse lipocalin‐2 (mLcn2) and found highly specific for mLcn2, not binding the human and chicken orthologs. The majority of bases in contact with the protein are located in the loop regions of the proposed aptamer structure as proved by sequence variants and RNA foot printing. Charged surface amino acids replaced by alanine did not change aptamer affinity for mLcn2, while mutation of three conservative basic core amino acids decreased or abolished the interaction. The mutants showed no evidence of global conformational changes as reflected by their unchanged Tmâ€™s from the wild‐type protein. Sequence alignment with the human Lcn2 (hLcn2) showed that residues surrounding these three conserved amino acids are also conserved, which suggests that the molecular discrimination of the aptamer derives from an interaction with another feature of mLcn2 that differs in hLcn2. The high degree of molecular discrimination of this aptamer, a general characteristic of aptamers, will be applied to sharpening the discriminatory capabilities of antibody‐based analysis. This work was funded by Iowa State University.
Effects of Viral Genome‐Linked Protein on Depurination of Viral RNA by Pokeweed Antiviral Protein
Authors: Domashevskiy Artem , Friedland Diana E , Goss Dixie J
Published: June 2021

Pokeweed antiviral protein (PAP) is a ribosome inactivating protein and is an N‐glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA and arrests protein synthesis at the translocation step. PAP, a potent antiviral agent against many plant, animal, and human viruses, inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNA depurinating them at sites downstream of the cap structure. To understand the molecular basis of PAP's antiviral activity, using fluorimetric methods, we examine interactions occurring between PAP and Potyvirus genome linked protein (VPg) that is shown to perform as a cap analog in viral cap‐independent translation of RNA bearing internal ribosome entry site (IRES). VPg interacts with cap‐binding proteins, stimulates translation of uncapped IRES‐containing RNA, and inhibits capped RNA translation in wheat germ extract. We have characterized interaction of PAP with VPg, and structured RNA derived from tobacco etch virus (TEV). When VPg complexes with PAP, the affinity for uncapped TEV RNA tripled compared with PAP alone, whereas affinity of PAP for capped RNA was not altered significantly. This selective preference of PAP for uncapped RNA in the presence of VPg stimulates the idea of creating a chimeric PAP‐VPg mutant that possesses selectivity to depurinate uncapped viral RNA by directing it to viral IRES.
Chronic β3 adrenergic stimulation induced remodeling of white adipose tissue in mice
Authors: Lindholm Chris R , Bauwens Jake D. , Ertel Rebecca L. , Mulligan Jake D. , Schmuck Eric G. , Saupe Kurt W.
Published: June 2021

Obesity and associated co‐morbidities are major health concerns. Here we define how a potential pharmacological treatment for obesity (in vivo chronic stimulation of β3‐adrenergic receptors with CL 316,243) affects remodeling of intra‐abdominal fat and specific serum markers closely related to obesity‐associated co‐morbidities. Male C57Bl/6 mice (n=8/group) were studied during 14 days of either no intervention (controls) or CL infusion (1mg/kg/day via Alza pump) with epididymal fat pads collected at sacrifice. CL treatment had no effect on food intake or body weight but decreased epididymal fat pad mass from 250±29 to 121±19 mg (p<0.01). While CL treatment did not change the protein concentration of whole adipose tissue or the non‐adipocyte cell fraction, it increased protein concentration in the adipocyte fraction > 4‐fold (0.2±0.04 to 0.9±0.09 mg/ml, p<0.01). CL treatment decreased blood glucose from 202±10 to 144±10 mg/dl (p<0.01) and serum leptin from 2.0±0.2 to 1.3±0.2 ng/ml (p<0.01). The cardioprotective adipokine adiponectin was increased from 6.2±0.3 to 8.3±0.3 μg/ml (p<0.01) by CL treatment. We conclude that the favorable serum changes induced by β3‐adrenergic stimulation are not dependent on decreased body weight but instead are likely linked to a remodeling of adipose tissue involving primarily the adipocytes.
Association of the Anillin‐related Protein Mid1 With Actin
Authors: Jakubowski Jennifer L , Hart Dawn M Clifford
Published: June 2021

Schizosaccharyomyces pombe, like human cells, divide symmetrically through constriction of an acto‐myosin ring. The conserved protein Mid1 plays a key role in the early assembly of the ring. Cells lacking mid1 form acto‐myosin rings that are non‐centered and disorganized, resulting in unequal division of the cellular contents. In human cells a Mid1 related protein called anillin regulates contraction of this ring. Anillin contains an actin binding domain that is necessary for the protein's direct association with actin. Unlike anillin, Mid1 does not contain an actin binding domain, and its ability to interact directly with actin has not been previously reported. However, previous studies show that anillin depleted and Mid1 depleted cells show similar defects in ring placement, ring formation, and cytokinesis. To assess whether or not Mid1 directly associates with actin, in vitro actin binding assays were performed. To further characterize the relationship between Mid1 and the acto‐myosin ring, we tested the ability of actin to bind GST‐Mid1 fragments with and without targeted mutations. Determining which members of the acto‐myosin ring associate directly with actin, an essential ring component, will provide significant insight into the molecular details of cytokinesis.

Research supported by NSF‐Advance and GVSU.
Bardet‐Biedl Sydrome (BBS) proteins play a role in Centrosome Amplification
Authors: Teixeira Miguel G , Zhang Qihong , Sheffield Val C , Kooyman David L
Published: June 2021

Many cancers result from mitotic or apoptotic abnormalities. We and others have previously shown that BBS proteins are involved in centrosome organization and regulation. We observed centrosomal clusters in renal epithelial primary cells obtained from BBS mutant mice. Along with the extra centrosomes, we observed a marked increase in the amount of gamma‐tubulin in BBS renal epithelial cells compared to wild type controls. It was unclear if these phenomena were related to the cell culture or a hallmark of BBS mutations. Therefore, we compared BBS mutant and wild type primary renal epithelial cells in culture with kidney sections of the same mice. Although the culture of cells exacerbates the phenotype, we demonstrate a definitive increase in centrosome number within cells in fixed kidneys obtained from BBS mutant mice as compared to wild type controls. We hypothesize that BBS proteins are involved in the regulation or transport of proteins destined for proteosome degradation. Specifically targeting cancer cells with multiple centrosomes presents a viable and novel therapy. Confirming BBS proteins role in centrosome regulation may help to better understand tumorigenesis.

This work was supported by the NIH and HHMI.
Intracellular growth of Toxoplasma gondii requires association with respiration‐competent mitochondria
Authors: Andrews Joel F , Barik Sailen
Published: June 2021

Toxoplasma gondii (Tg) is an intracellular parasite that causes the disease toxoplasmosis. Tg grows within a parasitophorous vacuole (PV), a parasite‐modified space surrounded by the parasitophorous vacuolar membrane (PVM), to which host mitochondria and other organelles are recruited. It is our objective to characterize the mechanism and significance of mitochondrial association with the PVM (mito‐PVM association). Initial studies showed that mito‐PVM association is microtubule‐dependent. Using chemical inhibitors, protein over‐expression constructs, and siRNA we have shown that microtubule‐dependent mito‐PVM association requires a functional host dynein/dynactin complex. Tg growth is reduced when mito‐PVM association is inhibited. Tg scavenges lipids from mitochondria, but the significance of host mitochondrial ATP production for Tg is unknown. To address mitochondrial ATP production, a cell line defective in mitochondrial respiration was infected with Tg tachyzoites. Tg growth, development, and egress were significantly inhibited in the respiration‐defective cell line, despite no difference in mito‐PVM association or cellular ATP levels. These results suggest that Tg growth is dependent on association with aerobically respiring mitochondria. Supported by Alabama EPSCoR and NIH Grant A1059267
Flavin‐dependent N‐hydroxylating enzymes from Mycobacterium smegmatis and Aspergillus fumigatus
Authors: Sobrado Pablo , Chocklett Wyatt , Robinson Reeder
Published: June 2021

Siderophores are iron‐binding molecules required for virulence by several human pathogens. M. tuberculosis and A. fumigatus siderophores contain hydroxamate groups that are essential for iron binding. Hydroxamate‐containing siderophores are synthesized via the hydroxylation of the amino groups on the side chains of lysine and ornithine by flavin‐dependent N‐hydroxylating enzymes. The N6‐lysine hydroxylase from Mycobacterium smegmatis (MbsG) and the N5‐ornithine hydroxylase from Aspergillus fumigatus (SidA) have been shown to be essential for virulence in these organisms and, thus, represent attractive drug targets. Currently, there is no biochemical or structural information about these enzymes. We present the cloning, expression, and characterization of MbsG and SidA. These enzymes are expressed in the active form containing tightly bound FAD. Kinetic characterization with NADPH, NADH, and various amine containing substrates is also presented and we demonstrate that dinucleotide‐induced conformational changes are required to stabilize the peroxyflavin in this family of enzymes.

This research was partially funded by grants from the Oak Ridge Associated Universities and the Virginia Academy of Sciences.
Creation of cell models from confocal microscopy data files using rapid prototyping
Authors: Paliwal Vipin , Konieczny John , Eliceiri Kevin , Reifschneider Isaac
Published: June 2021

A novel process was developed to convert digital data files from confocal microscopy of cells into three‐dimensional models. This process produces accurate models of cells as compared to existing methods that primarily rely on renditions. Data sets form Confocal Laser Scanning Microscopy (CLSM) and Multiphoton Laser Scanning Microscopy (MPLSM) for C. elegans embryo were converted to TIFF files using open source program Image J. Following importing files in Mimics, a medical imaging program, a slice distance of 4uM was chosen for modeling. Following enhancement, a Boolean operation was performed creating 2 separate segmentation masks, one for cellular structures and the other for exterior wall of embryo. The files were then exported into Freeform Modeling program which allowed us to convert 3‐D computer images into virtual clay. Files were further converted to a .stl format which can be read by rapid prototyping machine spectrum ZTM 510 (Z Corporation) 3D printer. Another build using 3D systems 250 SLA employing the DSM Somos Watershed XC 11122 resin was accomplished. The novel process described for building accurate 3‐D models of cells could find use in producing specimens for educational, research and clinical applications. Supported by National Science Foundation, grant EEC‐0648845 awarded to Subha Kumpaty, Department of Mechanical Engineering, Milwaukee School of Engineering.
A rapid method for removal of detergents from proteins and protein digests in a spin column format
Authors: Antharavally Babu S , Salunkhe Ashok M , Mallia Krishna A , Rosenblatt Michael M , Rogers John C , Haghdoost Navid R , Haney Paul J , Benton Betsy , Bell Peter A
Published: June 2021

We have developed a high‐performance resin that offers exceptional detergent removal for proteins and peptides. The easy‐to‐use spin format dramatically improves results over standard drip column or batch methodologies with >95% removal from 1–5% solutions of detergents such as SDS, sodium deoxycholate, CHAPS, Triton X‐100, Triton X‐114, NP‐40, Brij‐35, octyl glucoside, octyl thioglucoside and lauryl maltoside with high recovery of proteins and peptides. Post‐column LC‐MS/MS analysis of BSA trypsin digests followed by analysis of the data using MASCOT revealed excellent sequence coverage, and the high MASCOT scores obtained indicate successful removal of detergent from the digested peptides. MALDI‐MS analysis of unprocessed and processed samples further confirmed efficient removal of detergents. The advantages of this method include speed (less than 15 minutes), efficient detergent removal, and high recovery of proteins/peptides.
Mass Spectral Analysis of Mutant and Wild‐Type Selenomethionyl‐Dihydrofolate Reductase
Authors: Boles Jeffrey Oakley , Broderick Kathleen M , Broderick Molly
Published: June 2021

The biosynthetic incorporation of unnatural amino acids, primarily selenomethionine, has been increasingly used for over a decade to facilitate structural determination of proteins through the utilization of multi‐wavelength anomalous diffraction (MAD). This substitution has been shown to lead to instability and incomplete incorporation in some target proteins. In this study, the stability, level of incorporation and catalytic activity of selenomethionyl dihydrofolate reductase (SeMet‐DHFR) and wild type DHFR have been analyzed for catalytic activity and stability. The sensitivity to cyanogen bromide and trypsin for mass spectral analysis has been examined. A methionine to leucine mutation at position 6, also subjected to these analyses for both the wild‐type and selenoprotein, suggests subtle but potentially significant perturbations which likely go undetected if the wild type structure is not also solved.
Electrochemical and Spectroscopic Studies of Stellacyanin of Japanese Lacquer Tree Sap from Oku‐kuji, Japan
Authors: Takahashi Rika , Terakado Hideto , Kohzuma Takamitsu
Published: June 2021

Stellacyanin from Japanese lacquer tree is a member of blue copper protein, which shows an intense absorption at ~600 nm. The electrochemical and spectroscopic properties of stellacyanin have been investigated by cyclic voltammetry, electronic absorption, and resonance Raman (RR) spectroscopy. Stellacyanin was extracted and purified from the acetone powder of the lacquer tree sap provided by Oku‐Kuji Laquer Tree Sap Union. Cyclic voltammograms of stellacyanin were recorded at various promoter modified gold electrodes to know the electron transfer manner of the protein. Stellacyanin showed well defined quasi‐reversible electrochemical behavior at 4, 4_f‐pyridinedisulfide (4‐PDS), 3‐dithiopropionate (3‐DTP), or mercaptosuccinate (MSA) modified Au electrode, and the reduction potential of stellacyanin from Oku‐Kuji was estimated to be 161 mV at pH 7.0. The quasi‐reversible behavior of stellacyanin was not observed at a diethylaminoethanethiol (DEAET) at pH 7.0 and 3‐DTP or MSA/Au electrode at pH 11.0. These results could be concluded that the surface conserved lysine residue of stellacyanin is important for the electron transfer. Related spectroscopic studies will be reported.

This work was supported by the Project of Development of Basic Technologies for Advanced Production Methods Using Microorganism Functions by the New Energy and Industrial Technology Development Organization (NEDO).
Development of an ELISA‐based screening assay for the selection of α‐Synuclein Phosphorylation modulators
Authors: Arteaga Alexandra , Luzardo Melissa , Segatori Laura
Published: June 2021

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminenergic neurons. Affected neurons typically present accumulation of misfolded proteins into insoluble amyloid‐like aggregates (Lewy bodies). The most abundant protein present in Lewy bodies is α‐synuclein (α‐syn), a naturally unfolded monomeric protein that at high concentrations oligomerizes to form protofibrils, which can further aggregate in the form of fibrils and precipitate into Lewy bodies. α‐syn is hyperphosphorylated in brain tissue of PD patients, suggesting a role for phosphorylation of α‐syn in PD pathogenesis. However, the causative correlation between α‐syn phosphorylation, the formation of soluble oligomers and insoluble deposits, and cellular toxicity is still unclear. In order to investigate the correlation between α‐syn phosphorylation and cellular toxicity, we are developing an ELISA based assay for the detection of phosphorylated and non‐phosphorylated α‐syn. This assay will be optimized for the screening of small molecule libraries, particularly kinase and phosphatase inhibitors.

This research was made possible by the Minority Access to Research Careers program for funding this wonderful opportunity, and the Alliances for Graduate Education and the Proffesoriate at Rice University.
Divalent interaction between HPS1 and HPS4 to form the Biogenesis of Lysosome‐related Organelles 3 (BLOC‐3)
Authors: Carmona‐Rivera Carmelo , Bonifacino Juan S , Gahl William A , Cadilla Carmen L
Published: June 2021

Hermansky‐Pudlak Syndrome (HPS) [MIM#203300] is a rare autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding tendency, and sporadic pulmonary fibrosis, granulomatous colitis or infections. HPS is a heterogeneous disorder; eight HPS‐causing genes have been identified in humans. HPS type 1 is the most severe subtype with a prevalence of ~1/1800 in Northwest Puerto Rico. Mutations in HPS genes affect the biogenesis of lysosome‐related organelles. The products of the HPS type 1 and 4 genes (i.e., HPS1 and HPS4) assemble to form a complex known as Biogenesis of Lysosome‐related Organelle Complex 3 (BLOC‐3). Here, we report the identification of two interacting regions in each HPS protein required to form the BLOC‐3. We found that HPS1 interacts with HPS4 in two regions, spanning amino acids 1–197 and 506–700 but not with the middle portion of HPS1 (250–505). In addition, HPS4 displays dual binding sites to HPS1, comprised by its N‐terminus (1–230) and a distinct region between residues 340–399. In vitro assays with truncated HPS1, mimicking mutations found in HPS1 patients, showed retention of some interaction with HPS4, suggesting the possibility to generate (a truncated form of) BLOC‐3 in these patients that could retain partial function and could possibly explain the variable progression of the pulmonary fibrosis in BLOC‐3 deficient patients.
“Analysis of Aromatic Analogs on Cell Proliferation of CRL2338, CRL2351, and CRL2315 Expressing Combinations of Her2/Neu receptor and ER and PR receptors”
Authors: Le HongVan Thi , Hopkins Nancy
Published: June 2021

Several reports have cited that naturally occurring plant aromatic compounds such as curcumin, capsaicin, and eugenol have decreased the incidence of breast cancer in populations that incorporate the products in their diets. The objective of this project is to analyze the inhibitory effects of natural product analogs on breast cancer cell lines CRL2338, CRL2351, and CRL2315 that display different expression patterns of Her2/Neu receptor and estrogen and progesterone receptors. This project uses a modified version of the CellTiter96 Aqueous assay to screen for inhibiting analogs. Cells are seeded into 96 well plates, and concentrations of test compounds at 10 uM, 20 uM, and 50 uM are added per well. Blank wells and vehicle control wells are also included. Viability of cells is determined based on absorbance detected by the Spectramax150 plate reader at 490 nm. Comparisons of data between cell lines are carried out to establish the inhibitory effects of aromatic analogs in relations to the cells’ holistic receptors patterns. Findings obtained from this research may be useful in future considerations for employing natural products as a basis for breast cancer drug treatment.
Superior Efficacy of Phosphoplatins: Novel Non‐DNA‐Binding Platinum Drugs for Human Ovarian Cancer
Authors: Moghaddas Shadi , Majmudar Pooja , Bose Rathindra N.
Published: June 2021

Platinum (Pt) drugs are effectively used as cancer chemotherapy. These drugs form intra‐ and inter‐strand Pt‐DNA cross links leading to transcription inhibition process leading to cell death. Over 10% patients develop resistance to Pt drugs mainly due to the efficient removal of Pt from DNA by NER enzyme. The new class of Pt‐(II) and ‐(IV) pyrophosphato complexes developed in our laboratory, exhibit superior tumor‐specific toxicity and higher efficacy in human ovarian cancer (HOC) in vivo and head and neck cancer cell lines in vitro. Their mechanism of cytotoxicity does not involve covalent binding of Pt to DNA, but rather activate the Fas receptor signaling pathway leading to cell death. Clonogenic assay demonstrated that the phosphoplatins are more effective towards both cisplatin (CP)‐sensitive and ‐resistant HOC cell lines at lower concentrations as compared to CP and carboplatin (CR). In vivo studies in SCID mice, exhibit superior tumor regression and reduced toxicity of HOC compared to CP and CR. Apoptosis was established as the primary mechanism of cytotoxicity by cell cycle analysis and induced by activating cell‐surface receptors genes targeted towards cancer cells. Superior pharmacokinetic properties, reduced toxicity, better efficacy, ability to overcome CP‐resistant and activation of apoptotic genes make phosphaplatins the promising next generation of anticancer drugs.
Determination of the drug binding site within integrin CD11b/CD18
Authors: Rands Keith , Stoub Darren , Gupta Vineet
Published: June 2021

α/β integrin heterodimers are cell surface proteins involved in cellular communication. The integrin CD11b/CD18 is the predominant integrin found in neutrophils, macrophages and monocoytes and has been implicated as a signaling protein for the inflammation response. Recently, a class of small molecule drugs that activate the integrin CD11b/CD18 was reported. Using molecular docking experiments and site‐directed mutagenesis, we have identified a putative drug binding site in the ligand‐binding aA domain of CD11b, which suggest an allosteric activation of the integrin.
Mechanistic studies of the enoyl‐ACP reductase from Yersinia pestis and its inhibition by diaryl ether compounds
Authors: Ng Shuying , Lu Hao , Bostrom Kathleen , Slayden Richard , Tonge Peter J.
Published: June 2021

Yersinia pestis is a gram‐negative bacterium which causes bubonic and pneumonic plague. Due to the microbe's ability to spread rapidly and cause high mortality, the Centers for Disease Control and Prevention (CDC) have classified this organism as a Category A pathogen as it can be easily utilized as a biological weapon. Currently, streptomycin or gentamicin is used for the treatment of this disease. However, neither of the drugs can be orally administered, thus limiting their potential usefulness. The bacterial fatty acid synthesis (FAS‐II) pathway is essential for bacterial viability and it has also been validated as an attractive target for the discovery of novel antibacterials. We have cloned, over‐expressed and purified FabV, the FAS‐II enoyl‐ACP reductase from Yersinia pestis (ypFabV). In vitro kinetic experiments demonstrated that the recombinant enzyme can efficiently catalyze the reduction of the synthetic substrate using Trans‐2‐dodecenoyl‐CoA (DD‐CoA) as the substrate and NADH as the cofactor. Structural activity relationship (SAR) studies have lead us to the identification of a series of diaryl ether inhibitors with Ki values as low as 0.50 μM and MIC values as low as 0.5 μg/ml.
POST‐GENOMICS ERA TOPICS IN MULTIDISCIPLINARY BIOLOGY COURSES
Authors: Candas Mehmet
Published: June 2021

The advent of genomics followed by post‐genomics era advances has changed the ways in which biology is practiced. These changes lay the foundation for “new biology” in the 21st century and promise to bring solutions to problems across a broad spectrum of fields. With these changes, undergraduate students are expected to possess knowledge related to post‐genomics era concepts. To develop students understanding of concepts involving genomics, proteomics, metabolomics, bioinformatics and systems biology and increase awareness to new paradigms for research and technology applications, we incorporated an upper‐level elective course, Introduction to Biotechnology. The course examines biotechnology as it relates to basic and applied research as well as practices utilized in the industry. Topics include an overview of the biological macromolecules with reference to isolation and detection techniques as well as instruments for studying the molecules in a number of context including disease research, drug discovery, molecular diagnostics, clinical trials, forensic analysis, biosensors, transgenic plants, biofuels, and bioengineering applications. Incorporation of contemporary concepts of the post‐genomic era into appropriate courses with multidisciplinary content should enhance students’ post‐graduation prospects in graduate school, academic research, biotechnology and biomedical industry.
Stability and clinical determinants of long‐term warfarin therapy: a retrospective study
Authors: Ferland Guylaine , Desjardins Katherine , Presse Nancy , Pharand Chantal
Published: June 2021

Anticoagulation (AC) with vitamin K antagonists (VKAs), notably warfarin, is the therapy of choice for the prevention and treatment of thrombotic complications. However, stability of treatment with VKAs remains a daily challenge with bleeding a common side effect of the treatment. To this day, causal factors for bleeding episodes remain ill defined. Here we present a study that aimed to characterize the stability and complications of patients undergoing AC therapy, and identify associated risk factors. The study included 1003 patients treated at the AC clinic of a Montreal university hospital between April 1 2008 and March 31 2009. Socio‐demographic and medical information were extracted from medical charts. Stability of anticoagulation was based on the International Normalized Ratio (INR). Half of patients spent 85% of the time within their respective therapeutic range and factors investigated explained only ~ 8 % of the treatment variance, with intensity of treatment, patient's health condition and non‐compliance to treatment contributing the most. Nutritional information was largely missing from the medical charts and could not be assessed as part of this study. Complications were few and minor in nature. This study highlights the lack of attention paid to nutritional factors, notably those related to vitamin K status, which could contribute to the unexplained treatment variance. Supported by CIHR.
Antioxidant effect of ethanol extract of powdered grape skin in C57BL/6J mice fed high‐fat diet
Authors: Park Hae‐Jin , Choi Su‐Jung , Park Yong‐Bok , Choi Myung‐Sook
Published: June 2021

Grape seeds and skins are good sources of phytochemicals such as gallic acid, catechin, and epicatechin and are suitable raw materials for the production of antioxidative dietary supplements. And grape skins were a much more potent source than either grape pulp, juice or seeds. This study investigated the effect of ethanol extract of powdered grape skin antioxidant metabolism in high fat fed C57BL/6J mice. Five‐week‐old male C57BL/6J mice were randomly divided into two groups and fed high‐fat diet (HF control, 20%fat(w/w)) and with ethanol extract of powdered grape skin(GS) for 9 weeks. The plasma total cholesterol concentration was significantly higher in the HF group. Hepatic glutathione reductase, erythrocyte superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase activities were significantly higher in the GS group than in the HF group. Erythrocyte thiobarbituric reactive substances (TBARS) level was tended to be lower in the GS group than in the HF group. Therefore, we suggest that grape skin is effective material on improvement of antioxidant metabolsim because GS supplementation induced the elevation of hepatic antioxidant enzyme activities, the reduction of erythrocyte lipid peroxide levels and hypocholesterolemic effects in high‐fat‐fed mice.
Angiogenic and Inflammatory activities are modulated in vivo by polyphenol supplemented beer
Authors: Negrão Rita , Costa Raquel , Duarte Delfim , Taveira‐Gomes Tiago , Mendanha Mário , Soares Raquel
Published: June 2021

Angiogenesis is deregulated in pathologies like cancer, diabetes and cardiovascular diseases. Hops used in beer production contain xanthohumol (XN), converted to other products during beer production. XN seems to influence inflammation and angiogenesis. The aim of this study was to evaluate the in vivo effect of a XN supplemented stout beer on angiogenesis and inflammation. Wistar rats drank stout beer or stout beer supplemented with XN, during 30 days. Then longitudinal incisions were created on the dorsal skin. Animals continue to ingest the beverages for 7 days. Treatment with stout beer supplemented with XN seem to diminish the number of vessels formed in wounded tissue (vWF) and decrease N‐acetylglucosaminidase activity, NO release and also IL‐1fÒ in the serum. These findings suggest that XN supplemented beer may affect the wound healing process, diminishing inflammation and angiogenesis, by gastrointestinal administration, resulting in systemic effects. They also provide clues to the development of useful preventive or therapeutic nutritional supplementation, against inflammation‐ and angiogenesis‐associated pathologies.

Supported by FCT (SFRH/BD/41888/2007), iBeSa (P10‐08) and University of Porto/Santander Totta.
Vitamin D Status of Florida College Students
Authors: Dahl Wendy J , Langkamp‐Henken Bobbi , Foster Lauren , Stolarz Jamie , Whiting Susan J
Published: June 2021

Vitamin D insufficiency is common in the US. The purpose of this study was to evaluate the relationships between serum 25(OH)D levels of University of Florida students (18–30 y) and their dietary intake of vitamin D, skin color, tanning, race, BMI and body composition. Methods included vitamin D food frequency, sun exposure and demographic questionnaires, colorimetry for skin color and tanning determination and Bioelectric Impedance Analysis. 133 students (99F; 34M)(20.9±1.7 y) participated in the study in January 2009. Serum 25(OH)D (mean 62.5±20.3 nmol/L) was associated with skin color (p<0.001), but not with intake, tanning, sun exposure, race, BMI or % body fat. Inadequacy (<80 nmol/L) was found in 68% of students. In conclusion, our results show that skin color is the prime determinant of vitamin D inadequacy in college students in Florida.
Effects of lutein and β‐cryptoxanthin on MMP‐13, PGE2 and cytokine production in human chondrosarcoma cells stimulated with IL‐1α in vitro
Authors: Chew Boon P , Di Filippo Michael M , Mathison Bridget
Published: June 2021

Studies have shown that lutein (Lu) and β‐cryptoxanthin (βCr) may down‐regulate factors involved in inflammation associated with osteo‐ and rheumatoid arthritis. We studied the possible protective effects of Lu and βCr against human chondrocyte dysfunction using the SW‐1353 human chondrosarcoma cell line. Cells were cultured for 24 hr in complete medium containing 0, 0.01, 0.1 or 1.0 μmol/L of Lu or βCr and subsequently stressed with 10 ug/L IL‐1β (final concentration) for 24 hr. Conditioned medium was analyzed for matrix‐metalloproteinase‐13 (MMP‐13), cytokines (IL‐1α, IL‐2, IL‐4, IL‐10, IFN‐γ, IL‐6, IL‐8, and TNF‐α), and PGE2, and the nuclear extract analyzed for NFκB. Lu (1.0 μmol/L; P<0.05) but not βCr decreased MMP‐13. Both Lu (1.0 μmol/L; P<0.05) and βCr (0.1 and 0.01 μmol/L; P<0.01) inhibited PGE2 production. All concentrations of βCr suppressed (P<0.05) IL‐1α, IL‐2 and IFN‐γ production while Lu increased concentrations of these cytokines. Lu increased (P<0.05) while βCr decreased IL‐4 and IL‐10 concentrations. NFκB p50 production was suppressed (P<0.01) by both Lu and βCr, with Lu being more inhibitory. Therefore, Lu and βCr protected against IL‐1β‐induced chondrocyte dysfunction by down‐regulating NFκB activation and inhibiting inflammatory response, albeit through somewhat different pathways.
Comparison of Lycium barbarum‐containing Liquid Dietary Supplements to Caffeinated Beverages on Energy/Caloric Metabolism Activity and Salivary Adrenocortical Hormone levels in Healthy Human Adults
Authors: Amagase Harunobu
Published: June 2021

Impact of L. barbarum‐containing liquid dietary supplements on energy metabolism was compared to caffeinated beverages (tea, coffee and other commercially available products). The tested products were GoChi® (non‐caffeinated L. barbarum juice); Chi3® (caffeinated L. barbarum juice with B vitamins, folate, taurine, L‐phenylalanine, N‐acetyl‐L‐tyrosine, glucuronolactone and green tea extract); and TAIslim® (L. barbarum juice with caffeinated standardized tea polyphenols, soluble dietary fiber, L‐phenylalanine and N‐acetyl‐L‐tyrosine). Subjects (age=34.5 y) took single bout of 1 serving of test samples after a 12 h fast. Resting metabolic rate (RMR) was measured by indirect calorimeter immediately before (baseline), 1, 2 and 4 h after sample intake. Salivary cortisol and DHEA levels were analyzed by EIA one day prior and post intake. While non‐L. barbarum‐containing caffeinated beverages did not increase area under the curve (AUC, 0–4 h) of RMR from the control level (395 kcal‐h), all L. barbarum‐containing products increased RMR AUC by 752–927 kcal‐h over baseline (1,761 kcal), higher than the control or other beverages (P<0.05). Unlike caffeinated beverages, cortisol and DHEA were significantly reduced by GoChi intake. These results suggest that L. barbarum‐containing products exhibit energy stimulating effect, which may be related to acute adrenocortical hormonal reduction, a different mechanism than that of caffeine.
Preventing bone loss & weight gain with combinations of Vitamin D & phytochemicals
Authors: Lai Ching‐Yi , Ambati Suresh , Della‐Fera Mary Anne , Hamrick Mark W , Rayalam Srujana , Wolfe Marissa , Yang Jeong‐Yeh , Hartzell Diane L , Lewis Richard , Baile Clifton A
Published: June 2021

Vitamin D (VD) & certain flavonoids have been shown to act directly on adipocytes & osteoblasts. In this study, we investigated whether a diet using a combination of flavonoids with VD would inhibit bone loss & decrease adiposity to a greater extent than control or VD alone diets. Ovariectomized (OVX) female rats (12 mo old, N=10, initial BW=240g) were given control (AIN‐93M diet alone), VD (10 IU/kg BW/d) or VD + resveratrol (R: 4, 20, or 100 mg/kg BW/d) + quercetin (Q: 20, 125 or 625 mg/kg BW/d) + genistein (G:16, 64 or 256 mg/kg BW/d) in AIN‐93M diet for 8 wk. After rats were killed, inguinal (I) & retroperitoneal (RP) fat pads were weighed & bones were collected for measurements. The high dose combination treatment (VD+R100+Q625+G256) significantly reduced BW gain (79±5 vs 62±3g, p<0.05), weight of RP+I fat pads (21.8±1 vs 19.8±0.8g, p<0.05) & RP+I fat pads as % of BW (7.5±0.4 vs 6.9±0.4%, p<0.05). Bone mineral density (BMD) & content (BMC) of femora were significantly increased by the high dose combination, although they weren't different from VD alone. However, BMD corrected for BW was significantly greater in the high dose treated group compared to both control & VD groups (4.7±0.05 vs 5.0±0.2 vs 5.6±0.2, p<0.05). We conclude that VD when combined with R, Q & G improved bone density & reduced weight gain & adiposity in OVX female rats compared to control & VD alone. Supported by the GA Research Alliance Eminent Scholar endowment (CAB).
Tea and policosanol act through different mechanisms to activate AMP‐kinase and suppress HMG‐CoA reductase to inhibit cholesterol synthesis
Authors: Banerjee Subhashis , Porter Todd D
Published: June 2021

Policosanol is a mixture of long‐chain primary alcohols that has been reported to decrease serum cholesterol in animals and in man. Similarly, green and black tea consumption has been associated with lower serum cholesterol levels in both clinical and epidemiological studies. Our laboratory has shown that these botanicals decrease cholesterol synthesis in cultured hepatoma cells by activating AMP‐kinase, a key metabolic regulatory enzyme, which then phosphorylates HMG‐CoA reductase, the regulatory step in cholesterol synthesis, to suppress its activity. The present studies were undertaken to identify the mechanism by which these substances activate AMP‐kinase. Treatment of rat hepatoma cells with green or black tea extract or policosanol increased the phosphorylation of AMP‐kinase by 6.6‐, 3.3‐ and 3‐fold, respectively, and increased the phosphorylation (inactivation) of HMG‐CoA reductase by 2.5‐fold. Phosphorylation of LKB1, thought to be the principal activator of AMP‐kinase, was increased by greater than 4‐fold by treatment with either tea extract, but was not significantly affected by policosanol. Current data further suggest that phosphorylation of LKB1 by green tea is mediated by PKCζ, while LKB1 activation by black tea involves PKA. In contrast, treatment of cells with policosanol increased the amount of phosphorylated CaMKK, which can directly activate AMP‐kinase. siRNA‐mediated suppression of fatty aldehyde dehydrogenase, fatty acyl CoA synthase 4, or β‐ketothiolase in hepatoma cells prevented the phosphorylation of AMP‐kinase and HMG‐CoA reductase by policosanol, indicating that metabolism of these long‐chain alcohols to fatty acids and subsequent peroxisomal β‐oxidation is necessary for their suppression of cholesterol synthesis.

Grant Funding Source: NIH AT 003488
High‐fat diet‐induced obesity in myostatin null mice
Authors: Dilger Anna Carol , Gabriel Savannah R , Kutzler Louis W , Boler Dustin D , Killefer John
Published: June 2021

The objective of this experiment was to examine the resistance to diet‐induced obesity of myostatin (Mstn) null mice and determine if genes involved in thermogenesis were altered in these mice. Four‐week‐old male Mstn null and wild type mice were given ad libitum access to diets with 10 or 60% kcal from fat for 12 weeks. Glucose tolerance was determined after 0, 4, 8 and 12 weeks on diet. Body composition, muscle and fat pad weights, as well as other markers of metabolic syndrome were recorded and mRNA expression of several genes was examined in muscle, fat and liver. High‐fat fed mice consumed more calories and deposited weight and calories more efficiently than control mice. After 12 weeks of high‐fat feeding, body weight, body fat, fat pad weight, fasting blood glucose, serum insulin and leptin expression in adipose tissue were increased in both wild type and Mstn null mice. However, for all these parameters, the increase in Mstn null mice was less pronounced than in wild type mice indicating partial resistance to fat gain with excessive caloric consumption. Contrary to previous research, glucose tolerance was not altered in Mstn null mice. Furthermore, expression of some thermogenic genes including UCP‐3 and PGC1β were increased in adipose tissue of Mstn null mice fed high‐fat diets which may account for differences in obesity susceptibility.
Regulation of cathepsins in adipose tissue and macrophages in obesity
Authors: Hannaford Jennifer Elyse , Chen Xiaoli
Published: June 2021

Obesity and its related chronic inflammation are major risk factors for developing metabolic disturbances. The roles of cathepsins, cysteine proteases, have been tied to adipogenesis, inflammation, and atherosclerosis. The objectives of this study were to examine the regulation of cathepsins in adipose tissue and macrophages in genetic and diet‐induced obesity, and explore cathepsin activity with cholesterol metabolism macrophages. We found cathepsins to be upregulated in obese Zucker rats compared to lean counterparts, especially in the stromal vascular (S‐V) fraction of adipose tissue. Mice fed high fat diet compared to low fat diet showed increased cathepsin protein expression in adipose tissue; this affect was attenuated with a PPARγ agonist. TNFα did not affect cathepsin L expression in 3T3‐L1 adipocytes, but LPS simulation significantly increased cathepsin L expression in Raw264.7 macrophages. This supported in vivo data that the S‐V fraction accounted for increased cathepsin expression in obesity. In regards to cholesterol metabolism, LPS inflammatory stimulation of macrophages lead to an increased expression of NPC2, a cholesterol trafficking protein; NPC2 expression was decreased with cathepsin inhibitors in basal and LPS‐stimulated states in macrophages. In conclusion, cathepsins appear to be involved in the inflammatory response and in cholesterol metabolism.

Grant Funding Source: National Institute of Diabetes and Digestive and Kidney Diseases Grant No. 2 P30DK050456 (Minnesota Obesity Center)
miR‐103 modulates G1/S transition in IGF‐1 stimulated mouse intestinal crypt cell proliferation
Authors: Liao Yalin , Lonnerdal Bo
Published: June 2021

microRNAs play extensive roles in cellular development. Analysis of the microRNA expression pattern during intestinal cell proliferation in early life is likely to unravel molecular mechanisms behind intestinal development and have implications for therapeutic intervention. In this study, we examined differences in microRNA expression and identified miR‐103 as a regulator in IGF‐1 stimulated proliferation of mouse intestinal crypt cells. Mouse intestinal crypt cells were cultured and treated with IGF‐1 for 24 h, microRNA microarrays showed that multiple microRNAs are regulated by IGF‐1, and miR‐103 was the most sharply down‐regulated among these microRNAs. Expression of miR‐103 in mouse intestinal crypt cells was confirmed by real‐time Q‐PCR. Sequence analyses showed that, among the 1040 predicted miR‐103 target genes, CCNE1, CREB1, IGFBP1, and CDKL2 contain complementary sequences to the miR‐103 seed region and are conserved between human and mouse. We further demonstrated that miR‐103 directly controls the expression level of the four genes by luciferase assay and immunoblotting assay, respectively. Taken together, our data suggest that in mouse crypt cells, miR‐103 is part of the G1/S transition regulation network, which targets CCNE1, CREB1, and CDKL2 during IGF‐1 stimulated proliferation. (Supported by Mead Johnson Nutrition)