bluuebunny/crossref_metadata_2025 huggingface dataset:
Influx of acetyl‐CoA into the ER lumen regulates the induction of autophagy during the UPR
Authors: Puglielli Luigi , Pehar Mariana
Published: June 2021

One of the main functions of the UPR is to ensure disposal of large protein aggregates that accumulate in the lumen of the ER while avoiding, under non‐lethal levels of ER stress, cell death. When tightly controlled, ERAD(II) allows the cell to recover from the transient accumulation of protein aggregates; however, when unchecked it can be detrimental and cause autophagic/type 2 cell death. Here we report the results of a proteomic study showing that a large number of ER‐resident and ‐transiting proteins undergoes Nε‐lysine acetylation in the lumen of the organelle. The list of ER‐resident proteins includes chaperones and enzymes involved with post‐translational modification and folding. We also show that during ER stress IRE1/XBP1 controls the influx of acetyl‐CoA into the lumen of the ER by inducing the ER membrane acetyl‐CoA transporter SLC33A1/AT‐1. Failure to induce AT‐1 and maintain the acetylation status of the ER leads to widespread autophagic cell death. Mechanistically, the regulation of the autophagic process involves Nε‐lysine acetylation of Atg9A. In fact, a gain‐of‐acetylation mutant form of Atg9A protected from autophagic cell death while a loss‐of‐acetylation mutant achieved the opposite effect. Finally, we report that a mutant form of AT‐1 that is associated with Autosomal Dominant Spastic Paraplegia is unable to form homodimers and transport acetyl‐CoA across the membrane.

Supported by NIH
Using bakersˈ yeast to identify genetic modifiers of alpha‐1 antitrypsin deficiency
Authors: Gelling Cristy L , Brodsky Jeffrey L
Published: June 2021

Around 1 in 2500 individuals in the US have inherited alpha‐1 antitrypsin deficiency (ATD), which manifests as lung and liver disease. The most common cause of ATD is homozygosity for the Z‐variant of AT (ATZ), which accumulates in the endoplasmic reticulum of liver cells as a native polymer. ATZ is inefficiently secreted to the bloodstream, resulting in AT deficiency in the lungs and polymer accumulation in the liver. However, only a subset of ATD patients suffer from liver disease: 10% of patients show liver dysfunction at infancy, and 50% show signs of cirrhosis in adulthood. The factors that predispose this subset of individuals to liver disease are unknown.

We have developed a yeast antitrypsin expression system to find candidate genetic factors that affect the risk for ATD‐associated liver disease. This approach takes advantage of the genetic tools available for yeast, as well as the conservation of pathways implicated in ATD‐associated liver disease. Important disease‐associated characteristics of antitrypsin were recapitulated in yeast: the wildtype form was secreted, while mutant ATZ was not. We then screened mutants representing all non‐essential genes in the yeast genome for increased ATZ secretion. Current work is focused on confirming the effect of the candidate genes in a hepatic cell model.

Funding from the Alpha‐1 Foundation, Australian Research Council, and the American Australian Association.
Inhibitor mediated protein degradation
Authors: Hedstrom Lizbeth , Long Marcus J.C. , Coffey Rory , Gollapalli Deviprasad R.
Published: June 2021

Several drugs cause the degradation of their target proteins. The discovery of compounds with this property has been serendipitous. We have discovered that the tert‐butyl carbamate‐protected arginine (Boc3Arg) moiety is a degron that can be used to construct inhibitors that induce degradation of their target proteins. Boc3Arg‐linked ethacrynic acid and thiobenzofurazan cause the specific degradation of glutathione‐S‐transferase (GST) isozymes in lysates and whole cells. Similarly, the degradation of Escherichia coli dihydrofolate reductase (eDHFR) is induced when treated with trimethoprim (TMP)‐linked Boc3Arg. Importantly, TMP forms a noncovalent complex with eDHFR, so covalent attachment of the Boc3Arg degron is not required for degradation of the target protein. These Boc3Arg‐inhibitors induced degradation of 30–80% of their abundant target proteins within 2–5 hours. The proteasome is required for Boc3Arg‐mediated degradation but the ubiquitin pathways do not appear to be involved. These results demonstrate that the Boc3Arg moiety provides a general strategy for inducing protein degradation that should be useful in therapeutic and conditional protein knockdown applications. This work was supported by NIH GM54403 (LH) and AI075466 (LH).
Essential role of the ubiquitin‐selective chaperone CDC48 in a poikilothermic vertebrate in cold adaptation
Authors: Imamura Shintaro , Yabu Takeshi , Yamashita Michiaki
Published: June 2021

Cold‐inducible proteolytic degradation mechanism by the ubiquitin‐proteasome system (UPS) and autophagy was identified and characterized in zebrafish embryos. Previously, we identified a homolog of the mammalian valosin‐containing protein (VCP)/p97 and the yeast cell division cycle gene 48 (CDC48) as the major cold‐inducible protein in fish cells. We characterized that CDC48 was essential for cell growth and cell death functions in zebrafish embryonic ZE cultured cells under cold adaptable temperature between 15–20 °C (Imamura et al., 2003). In order to elucidate protective roles of CDC48 for cold acclimation and adaptation in poikilotherm, the protein degradation by UPS and autophagy mediated by CDC48 were examined in zebrafish embryos. When we compared between wild type and CDC48 deficiency embryos that were treated with antisense morpholino oligonucleotides, the CDC48 deficient embryos showed lethal defects in neuronal formation with apoptosis induction, ROS production and the accumulation of poly‐ubiquitinated proteins in the central nervous system under cold conditions. In these embryos, the degradation of poly‐ubiquitinated proteins was suppressed under cold condition at 15 °C. Thus, UPS functions were impaired in zebrafish embryos under cold conditions. In addition, autophagic marker protein p62 was accumulated in autophagosomes, whereas autolysosomes were not produced under cold conditions irrespective of the presence of CDC48. Therefore, CDC48 activated the UPS functions and promoted the neural development under cold conditions that caused autophagy impairment.
Screening and kinetic studies of new cathepsin D inhibitors
Authors: Sheppard Donald R , Wen Lisa , Huang Jenq-Kuen , McConnell Rose M
Published: June 2021

Cathepsin D is a ubiquitous aspartyl lysosomal protease. Because it is associated with the mobility and invasiveness of various cancers, inhibitors of this enzyme have the potential for use as chemotherapeutic drugs. Previously prepared synthetic compounds are being evaluated for their ability to inhibit cathepsin D by determining their IC50 values. The studies were performed using human liver cathepsin D and a fluorogenic substrate (Mca‐Gly‐Lys‐Pro‐Ile‐Leu‐Phe‐Phe‐Arg‐Leu‐Lys(Dnp)‐D‐Arg‐NH2).

Several of these compounds were found to be strong inhibitors of cathepsin D. The kinetic properties of four potential inhibitors were characterized. These data will be used in the development of new and more effective inhibitors. Supported by National Cancer Institute at NIH (3R15CA08933‐04 and 3R15CA86933‐04A2S1) and Western Illinois University.
Evaluation of synthetic thiosemicarbazone containing compounds as inhibitors of human recombinant cathepsin B
Authors: Lucas Joseph Anthony , Yelma Niranjan Reddy , Wen Lisa , Huang Jenq-Kuen , McConnell Rose
Published: June 2021

The objective of this study was to test the inhibitory effects of eight different synthetic compounds on recombinant Cathepsin B, a lysosomal cysteine protease that plays roles in processes such as protein degradation, bone resorption, and antigen processing. This protein has been found in abnormally high levels in the bloodstream of both Alzheimer's and cancer patients. In order to investigate the veracity of synthetic Cathepsin B inhibitor candidates, recombinant procathepsin B protein was obtained by overexpression of previously constructed E. coli transformant followed by purification of inclusion bodies, solubilization, and refolding. After refolding, the procathepsin B was autoactivated by lowing the pH to 4 with formic acid. Finally, the effectiveness of each inhibitor candidate was determined via colorimetric enzymatic assay using N‐Carbobenzoxy‐L‐Phenylalanyl‐Arginine‐4‐nitroanilide hydrochloride as the chromogenic substrate. The results of each inhibitor candidate are presented as an IC50 (50% Inhibitory Concentration). Funding provided by National Cancer Institute at NIH (Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1) and Western Illinois University.
Hepatic HRI eIF2α kinase‐mediated translational suppression activates Nuclear Factor Kappa B in response to heme‐deficiency stress
Authors: Liu Yi , Correia M. Almira
Published: June 2021

Nuclear factor kappa B (NF‐κB), a key proinflammatory factor involved in inflammation, cancer, apoptosis and immunity, is activated in response to diverse cellular/environmental stresses including ER stress and UV irradiation that activate PERK and GCN2 eIF2α kinases, respectively. We have found that inhibition of heme synthesis in cultured mouse hepatocytes results in acute heme depletion and consequent autoactivation of heme‐regulated inhibitor HRI eIF2α kinase, an exquisite heme‐sensor. The 2‐fold increased eIF2α phosphorylation results in global translational arrest of de novo synthesis of hepatic proteins including IκBα (>5‐fold) that was significantly reversed by heme repletion, but not the proteasome inhibitor MG132. This translational suppression of hepatic IκBα resulted in the nuclear activation of NF‐κB (p65/p50 heterodimer) by >7 fold, as detected by Electrophoretic Mobility Shift Assay (EMSA). To determine the precise role of HRI in IκBα translational suppression, we used HepG2 cells overexpressing HA‐tagged HRI protein. Upon acute heme depletion, this HRI protein was activated with consequently enhanced eIF2α phosphorylation as well as significantly decreased IκBα levels relative to controls. Collectively, our data suggest a plausible role for hepatic HRI in the activation of hepatic NF‐κB inflammatory pathway under heme‐deficiency stress. Supported by NIH grants DK26506, DK26743.
The Search for Novel Substrate Specificity in Mutants of L‐Alanine Dehydrogenase
Authors: Aldeborgh Hannah , Mundorff Emily
Published: June 2021

The enzyme L‐Alanine dehydrogenase from M. tuberculosis (AlaDH) catalyzes the conversion of L‐alanine to pyruvate by replacing the amino group with a carbonyl. AlaDH belongs to a superfamily of alcohol dehydrogenases that reversibly convert alcohols to carbonyls rather than the Glu/Phe/Leu amino acid dehydrogenase superfamily. Molecular modeling of the AlaDH binding pocket suggested that mutating the 94‐phenylalanine residue to alanine or serine would open up the binding pocket to accommodate larger, bulkier substrates. We predicted that these mutations would enable the enzyme to catalyze the same reaction on larger amino acids. An assay of the F94A and F94S mutants with different substrates showed novel activity on leucine and norleucine in comparison to the wild type, as well as greatly increased activity on norvaline and methionine. The Km values of both mutants are comparable to that of the wild type value, while the kcat values are <1% of the wild type value indicating that the mutant enzyme binds to the substrate well, but potentially in a less reactive conformation. We thank the HHMI for support for this project.
Glucuronides of antitumor agents C‐1311 and C‐1305 modulate cytotoxicity in cancer cells
Authors: Pawlowska Monika , Chu Rong , Bratton Stacie M , Augustin Ewa , Mazerska Zofia , Chambers Timothy C. , Radominska-Pandya Anna
Published: June 2021

These studies focus on two anti‐tumor compounds, C‐1311, an imidazoacridinone (IA), which has been selected for Phase II clinical trials, and C‐1305, a triazoloacridinone (TA), which is in extended preclinical studies. Our recent studies with human recombinant UDP‐glucuronosyltransferase (UGTs) revealed that these compounds are metabolized by the 1A10 isoform (UGT1A10), with the higher rate of glucuronidation C‐1305. Experiments with KB‐3 and MCF‐7 cells transiently transfected with UGT1A10 (50% efficiency) were carried out to investigate the effects of this isoform on cell cytotoxicity and cell cycle progression following C‐1311 and C‐1305 treatment. After transfection, cellular levels of C‐1311 and C‐1305 glucuronides were identified by HPLC‐ESI‐MS and their rate of production was time and concentration dependent. MTT assay revealed that sensitivity of cells with UGT1A10 overexpression compared to wild type increased by 31% in KB‐3 and by 40% in MCF‐7 cells only in the presence of C‐1305. Treatment of KB‐3 and MCF‐7 cells overexpressing UGT1A10 with C‐1305 led to the appearance of sub‐G1 population, suggesting apoptosis commitment. Collectively, these data indicate that the glucuronide of C‐1305 might be biologically active and its activity may contribute to overall cytotoxicity and pharmacology in these cancer cells.
Biochemical and mutational analysis of intracellular regions of the Plexin‐B1 guidance receptor as a R‐RasGAP
Authors: Kim SoonJeung , Hota Prasanta K , Buck Matthias
Published: June 2021

Plexins are single transmembrane receptors for semaphorin guidance cues, which regulate the cell motility through changes in the cells actin cytoskeleton. Since the actin cytoskeleton is regulated by small GTPases of the Rho family, interactions with small GTPases are of particular importance in the signaling mechanism of plexins. We mapped the interactions of small Rho GTPases, Rnd1 and active Rac1, with the plexin‐B1 RBD (Rho GTPase Binding Domain) to a common region of the structure [1]. In our recent study [2], the crystal structure of the intracellular region of human plexin‐B1 shows high homology to p120Ras GTPase‐activating proteins (GAPs). Down‐regulation of Ras GTPases appears to be a key mechanism for the plexin‐mediated regulation of cell motility. Molecular modeling and ITC measurements demonstrated that Ras GTPases can bind to the plexin GAP region. Furthermore, we showed that the intracellular plexin‐B1 binds R‐Ras but not H‐Ras. Recently we have for the first time been able to measure the GAP activity of plexin‐B1 with R‐Ras substrate using a biochemical assay. This assay now allows us to elucidate the role of the interactions between the intracellular regions and Rho GTPases for plexin function.
The uterine smooth muscle S‐nitrosylproteome in pregnancy and functional effects of S‐nitrosoglutathione
Authors: Ulrich Craig , Quilici David , Schegg Kathleen , Buxton Iain , Barnett Scott , Heyman Nathanael
Published: June 2021

The molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor are not known. Nitric oxide relaxes uterine smooth muscle in a cGMP independent manner. S‐Nitrosylation, the covalent addition of an NO‐group to a cysteine thiol is a likely mechanism to explain the ability of NO to relax myometrium. For the first time we describe the myometrial S‐nitrosylproteome in pregnant and non‐pregnant guinea pig and the human labor, not in labor, and pre term laboring comprehensive proteome and S‐nitrosylproteome using multidimensional protein identification technology. We show that S‐nitrosylation of the intermediate filament protein desmin is significantly increased (5.7 fold, p < 0.005) in pregnancy and that this increase cannot be attributed solely to the increase in protein expression (1.8 fold, p < 0.005) that accompanies pregnancy. We also show that both myosin light regulatory polypeptide 9 and myosin light chain kinase are disparately S‐nitrosylated in the different states of pregnancy. We further describe both the S‐nitrosylation and activation of the potassium channel TREK‐1 by the NO donor S‐nitroso‐glutathione. Elucidation of the myometrial proteome and S‐nitrosylproteome provides a list of mechanistically important proteins that can constitute the basis of hypotheses formed to explain the regulation of uterine contraction‐relaxation.
Trypanosoma brucei Tim50, a Protein Translocator of Mitochondrial Inner Membrane, Possesses Dual Specific Phosphatase Activity
Authors: Fullerton Marjorie S , Duncan Melanie R , Chaudhuri Minu
Published: June 2021

The single mitochondrion of Trypanosoma brucei is involved in multiple cellular functions. We recently identified a homolog of Tim50, a protein translocator of mitochondrial inner membrane, in T. brucei, which contains a unique CTD‐like phosphatase domain at the C‐terminal. To determine phosphatase activity, we expressed the C‐terminal 6X‐His‐tagged TbTim50 in Escherichia coli and the recombinant protein (rTbTim50) was purified to homogeneity from the bacterial lysate by Ni‐chelation chromatography. The Purified rTbTim50 displayed a phosphatase activity using standard substrate pNPP with a Km of 300 mM. The optimum activity of the TbTim50 phosphatase was observed at pH 5.0. The protein phosphatase activity of rTbTim50 was also measured against phosphotyrosyl and phosphothreonyl peptides. The phosphatase activity of rTbTim50 was two‐fold higher using phosphotyrosyl peptide compared to phosphoserine peptide with Km values 3.75[micro]M and 7.5 [micro]M, respectively. TbTim50 showed similar phosphatase activity to its human Tim50. The effect of site‐directed mutagenesis of two signature CTD phosphatase‐motifs in TbTim50 will be explored. Support by NIH Grant 2Sc1 GM 081146‐05.
The V‐ATPase Subunit d: New Roles for Functionally and Structurally Coupling of V1‐Vo
Authors: WEBER ELIJAH , Parra Karlett J
Published: June 2021

Vacuolar ATPase (V‐ATPase) proton pumps are complex proteins essential for pH regulation. V‐ATPases have fourteen different subunits which form a peripheral domain (V1) that hydrolyses ATP and a membrane domain (Vo) that is responsible for proton transport. In order to regulate pH in vivo, V1 and Vo reversibly disassemble, disassembly is linked to glucose availability but the mechanisms involved are unknown. Subunit d of Vo is essential for V‐ATPase activity, and we postulate that subunit d would also control reversible disassembly because subunit d connects V1 and Vo. Previous studies suggest that helix 5 of subunit d is important for functional coupling and assembly of V1 and Vo. Amino acid substitutions along the open reading frame of subunit d were introduced in order to address its role for V‐ATPase activity and assembly. We hypothesize that if helix 5 is essential for coupling of ATP hydrolysis and proton transport, then the mutations R118A, D117A, H116A, T114A, and M103A made within helix 5 will uncouple the enzyme and disturb V1‐Vo complex formation. The essential methods in the experiment include: site‐directed mutagenesis, whole cell lysis, western blots, vacuolar membrane purification by density gradient centrifugation, and measurement of ATPase hydrolysis and proton transport. The effects of these specific mutations on ATP hydrolysis, proton transport, and V1Vo assembly will be discussed.
Regulation of PI4KIIα distribution between the Golgi and endosomal compartments
Authors: Jovic Marko , Bojjireddy Naveen , Kean Michelle , Gingras Anne-Claude , Brill Julie , Balla Tamas
Published: June 2021

Phosphatidylinositol 4‐kinases (PI4Ks) are essential for trafficking along the secretory and degradative routes. The importance of PI4K localization is underscored by the role of PI4KIIα, an enzyme localized to trans‐Golgi and endosomes, in EGF receptor degradation and Wnt signaling, and its suspected role in the late‐onset neurodegeneration. Although membrane association of PI4KIIα through a palmitoyl moiety is suggestive of its cycling between endosomes and Golgi, little is known about the regulation of PI4KIIα subcellular localization. Here we performed a proteomic screen of potential binding partners of PI4KIIα and identified a vesicular fusion protein VAMP‐3, that was confirmed by co‐immunoprecipitation. Expression of Tetanus toxin (TeNT), which specifically cleaves VAMP‐3, resulted in accumulation of PI4KIIα on endosomes. Conversely, overexpression of TeNT with the TeNT‐resistant VAMP‐3 mutant recovered the Golgi localization of PI4KIIα. We also found that PI4KIIα rapidly trafficked back to the Golgi within 2–3 min following photobleaching of the Golgi subpopulation, whereas the rate of its reappearance was significantly decreased in the presence of TeNT. Collectively, these data suggest that VAMP‐3 regulates PI4KIIα retrograde transport, uncovering new aspects of the role of PI4KIIα in human health and disease.

Supported by the NICHD Intramural research program
The role of an ER‐localized metalloprotease in yeast cell wall integrity
Authors: Hecht Karen Alice , Wytiaz Victoria A. , Brodsky Jeffrey L.
Published: June 2021

The fungal cell wall protects against environmental stresses, and regulates morphogenesis. It is made up of a polysaccharide layer and a dense network of proteins necessary for cell wall function. Cell wall proteins are extensively glycan and lipid modified, and subject to endoplasmic reticulum (ER) quality control before secretion. When the cell wall is perturbed, stress signaling is essential to up‐regulate secretion of proteins required for cell wall repair. Understanding how yeast synthesize and maintain cell wall integrity is essential to combat fungal pathogens in human disease.

We have identified a gene, YBR074, displaying a distinct set of genetic interactions associated with lipid metabolism in the yeast Saccharomyces cerevisiae. Ybr074 is a putative ER‐localized metalloprotease of unknown function. In order to understand the role of Ybr074 in lipid metabolism, we have tested chemical‐genetic interactions of this gene under conditions of cell wall and ER stress. Interactions of YBR074 upon treatment with the cell wall perturbing agent calcofluor white have been observed with CDC48. Cdc48 belongs to the protein family of ATPases Associated with diverse cellular Activities (AAA). It is known to function both in ER quality control and cell wall maintenance. This genetic interaction suggests a role for Ybr074 in regulating components of cell wall maintenance under conditions of stress.

NIH grant GM75061
Impact of hydrophobic lipophilic molecules on worm burden in balb/c mice infected with heligmosomoides polygyrus
Authors: Foudjet Armand C
Published: June 2021

It was recently discovered that parasitic nematodes of plants, animals and humans secrete a structurally novel Fatty Acid and Retinol (FAR) Binding Protein into the tissues they occupy. The lipid‐binding characteristics of FAR proteins across multiple families of parasitic nematodes, and their presence at the host‐parasite interface, lends support to the idea that this nematode‐restricted family of proteins may play a crucial role in the life cycle of the roundworms. To investigate the possible role of these highly conserved nematode‐specific FAR proteins, our lab designed experiments to address the following hypotheses: 1) the FAR proteins are involved in lipid sequestration and delivery from the host to the nematode of a broad range of essential fatty acids, retinoids and other nutrients the parasites cannot synthesize themselves, 2) FAR proteins interfere with intercellular lipid signaling to manipulate the defense reactions of the host, and lastly, as these hypotheses are not mutually exclusive, 3) both mechanisms (hypotheses 1 and 2) are working together to allow for parasite survival and the establishment of chronic infection. To test these hypotheses, mice were dosed with acute, high levels of retinol and/or oleic acid prior to hookworm infection, to determine the impact on worm burden levels and circulating IL‐4. Our preliminary results demonstrate both a statistically significant increase in worm burdens and a significant decrease in circulating IL‐4, after treatments containing ether retinol or oleic acid, results which support hypothesis #3.
In vitro evidence that peptides derived from the candidate tumor suppressor gene Esophageal Cancer‐Related Gene 4 (Ecrg4) internalize into cells through the innate immunity receptor complex
Authors: Podvin Sonia , Dang Xitong , Kurabi Arwa , Coimbra Raul , Eliceiri Brian , Baird Andrew
Published: June 2021

In light of data implicating the secreted precursor protein and candidate tumor suppressor Ecrg4 in the inflammatory response, we explored the possibility that predicted ligand domains in its primary sequence can target the innate immunity receptor complex. Predicted fragments of Ecrg4 were displayed on M13 phage and tested for internalization into cultured human embryonic kidney (HEK) cells that normally express low levels of the innate immunity receptor complex and HEK cells that stably overexpress. The receptor‐mediated internalized phage was recovered and phage DNA quantified by PCR. Internalized Ecrg4 targeted phage was compared to untargeted phage or epidermal growth factor (EGF) targeted phage that internalizes through the EGF receptor. Three peptide domains that derive from the Ecrg4 precursor internalized into HEK cells. Two of these, one at the amino terminus and one at the carboxyl terminus internalized 13 to 16 fold higher in HEK cells that overexpress TLR4/MD2/CD14 compared to wild type HEK cells. Untargeted phage internalization was low and EGF‐targeted phage internalization was high, but neither was different between the two cell lines. These data suggest that predicted Ecrg4 peptides derived from the secreted precursor protein can internalize into target cells through the innate immunity receptor complex.

Supported by a Mentored Young Investigator award from the Hydrocephalus Association
A high‐throughput screen for inhibitors of xenobiotic detoxification in nematodes
Authors: Leung Chi Kwan , Deonarine Andrew Stevenson , Choe Keith Patrick
Published: June 2021

Multidrug resistance is a growing problem in parasitic nematodes. In pathogens and tumor cells, multidrug resistance is mediated by enzymes that detoxify xenobiotics. The transcription factor SKN‐1 activates detoxification genes and is essential for development in C. elegans. Pharmacological compounds that target SKN‐1 would provide new tools for studying multidrug resistance and have the potential to inhibit embryonic development and drug resistance in adults. We identified the WD40 repeat protein WDR‐23 as a potent regulator of SKN‐1. Importantly, the homologous mammalian transcription factor Nrf2 is regulated by a distinct pathway and binds DNA by a distinct mechanism. We propose WDR‐23/SKN‐1 as a promising target for drugs that inhibit embryonic development, xenobiotic detoxification, and drug resistance in nematodes without affecting mammalian hosts. The small size, simple culturing characteristics, and genetic tractability of C. elegans make it an ideal system to screen for SKN‐1 inhibitors. We have developed a fluorescent‐based 1536‐well plate assay to measure SKN‐1 activity in C. elegans. Pilot screens with the Spectrum library and Library of Pharmacologically Active Compounds (LOPAC1280) indicate that our assay is amenable for HTS in whole animals. Inhibitors would provide tools for studying the function of SKN‐1 in parasitic species. This work is funded by NIH grant R21NS067678‐01.
A novel, time resolved immunofluorescence screening assay to assess PAR2 ligand binding
Authors: Hoffman Justin , Flynn Andrea N. , Tillu Dipti V. , Vagner Josef , Price Theodore J. , Boitano Scott
Published: June 2021

We describe the synthesis and use of a modified PAR2 peptidomimetic agonist, 2‐furoyl‐LIGRLO‐DTPA‐NH2 (2‐f‐ DTPA), for lanthanide (Europium)‐based time resolved fluorescence screening. We compared 2‐f‐DTPA to its parent compound, the PAR2 peptidomimetic 2‐furoyl‐LIGRLO‐NH2, across a full spectrum of in vitro assays to demonstrate its potency and specificity as a PAR2 agonist. 2‐f‐DTPA induced full PAR2‐ specific in vitro physiological responses. Current PAR2 ligand screening methods utilize radiolabeled ligands and/or have low throughput screening capabilities. The advantage of our 2‐f‐DTPA ligand is that it can be used in a radioactivity‐free, high throughput screen without sacrificing sensitivity. We used 2‐f‐DTPA in a competitive binding assay to evaluate ligand‐receptor interactions at PAR2 with known high potency agonists (2‐aminothiazol‐4‐yl‐ LIGRL‐NH2 and; 6‐aminonicotinyl‐LIGRL‐NH2) and a negative control (3‐indolacetyl‐LIGRL‐NH2). Both agonists demonstrated competitive binding reflecting their in vitro potency, whereas 3‐ indolacetyl‐LIGRL‐NH2 displayed limited competition for PAR2 binding. In summary, we report a PAR2 agonist that can be used in a highly sensitive competitive binding assay to screen novel PAR2 ligands in a high‐throughput manner. Supported by NIH grant NS073664.
Anti‐angiogenic effects of zoledronic acid on osteotropic breast cancer cells
Authors: Contreras Cynthia , Morshedian Shaghayegh , Hamud Walid , Dhakal Bishal , Khanal Bijay , Bush Jason A
Published: June 2021

Emerging data suggest the bisphosphonate, zoledronic acid (ZOL), exerts both indirect and direct anti‐tumor effects by decreasing tumor cell proliferation, increasing apoptosis, and inhibiting angiogenesis. Recent data indicate that anti‐angiogenic factors exert cellular effects via suppression of mitochondrial oxidative phosphorylation/respiration and elevation of intracellular ROS levels. This project intended to define proteins involved in response to ZOL‐treatment of an osteotropic breast cancer cell line, MDA‐231BO. Results demonstrate that MDA‐231BO cells are more sensitive to ZOL cytotoxicity and salutary effects are both media‐ and calcium‐dependent. To investigate pro‐apoptotic mechanisms of ZOL, total RNA, whole cell protein lysates, or mitochondrial‐enriched fractions were isolated from MDA‐231BO cells and using a combination of RT‐PCR, 1D/2D‐PAGE separation and MALDI‐MS/MS, we identified several angiogenesis inhibitors that were upregulated and pro‐angiogenic factors that were downregulated after ZOL treatment. Functional studies demonstrate the improved efficacy of ZOL on MDA‐231BO compared to MDA‐MB‐231 controls. Taken together, these results further support a model that ZOL tips the ‘angiogenic switch’ in favor of anti‐angiogenic factors in cancer cells with a propensity for bone metastases.
Structural features for substrate recognition by bacterial 5′‐methylthioadenosine nucleosidase
Authors: Ronning Donald R , Mishra Vidhi
Published: June 2021

The bacterial enzyme 5′‐methylthioadenosine/S‐adenosylhomocysteine nucleosidase (MTAN) is a multifunctional enzyme that catalyzes the hydrolysis of the N‐ribosidic bond of at least 3 different adenosine‐based metabolites: S‐adenosylhomocysteine (SAH), 5′‐deoxyadenosine and 5′‐methylthioadenosine (MTA). These activities place the enzyme at the hub of 5 fundamental bacterial metabolic pathways: S‐adenosylmethionine (SAM) utilization, the purine salvage pathway, the methionine salvage pathway, the SAM radical pathways and autoinducer‐2 biosynthesis. The last pathway is important in synthesizing diffusible compounds used in bacterial quorum sensing, which affects biofilm formation in many pathogenic bacteria. Although structures of various bacterial and plant MTANs have been described, the interactions between the homocysteine moiety of SAH and the 5′‐alkylthiol binding site of MTAN have never been resolved. To gain information regarding these interactions, we have solved crystal structures of an inactive mutant form of Helicobacter pylori MTAN bound to MTA and SAH to 1.65 and 1.2 Å, respectively. These structures identify specific interactions between the homocysteine moiety and the 5′‐alkylthiol binding site of the enzyme that appear to be conserved in campylobacter but not across all bacterial MTANs. Comparison of the MTA and SAH structures also shows that the localized structure of 5′‐alkylthiol binding site is identical without regard for which substrate is bound. This suggests that HpMTAN binding of the homocysteine moiety contributes little or no entropic penalty to this binding interaction and that these new data may promote development of species‐specific inhibitors for MTAN that affect biofilm formation.
The Os Peroneum and Accessory Navicular: An integrated radiographic and cadaveric study
Authors: Delamarter Taylor , Benninger Brion
Published: June 2021

INTRODUCTION

Sesamoids and accessory bones commonly exist throughout the human body, especially in the foot. Pain from the foot and ankle is a common symptom, of which the Os Peroneum (OP) and Accessory Navicular (AN) are associated with pathologies that present to clinicians. The objective of this study was to investigate the use of CT as a paradigm in undiagnosed foot and ankle pain and to advocate the teaching the OP and AN in anatomy courses.

METHODS

Literature search was conducted on anatomical texts, atlases, websites, and journals regarding the OP and AN. Roentogram and CT scans were conducted on 15 embalmed cadaveric lower limbs, followed by palpation and deep dissection of the distal tibialis posterior and fibularis longus tendons.

RESULTS

Literature search revealed a 14 – 26% prevalence of the OP and 5–21% of the AN. Roentograms revealed OP in 2/22 and AN in 5/23 cadavers. CT scans revealed OP in 9/28 and AN in 13/28. Dissection revealed an OP in 9/30 and an AN in 10/28.

DISCUSSION

Despite the prevalence of the OP and AN in the literature, they are not addressed in anatomy courses. They are a known cause of pain and are associated with false negatives radiologically.

CONCLUSION

This study suggests that anatomy courses include the OP and AN as clinically relevant structures. CT scans should follow roentograms as part of a diagnostic paradigm when treating unknown foot and ankle pain.
The Role of Sterol Carrier Protein 2 in the Endocannabinoid System
Authors: Osademe Anwuri , Sosa-Gonzalez Sonia , Miller Briana , Leachmon Gabriella , Osademe Ngozi , Cobb Michaun , Gonzalez David , Johnson Carol , Liedhegner Elizabeth Sabens , Hillard Cecilia J.
Published: June 2021

The endocannabinoid system (ECS) plays a role in diverse disorders such as anxiety, addiction, eating and memory disorders. The ECS is found throughout the body and consists of two lipid signaling molecules, N‐arachidonylethanolamine (AEA) and 2‐ arachidonoylglycerol (2‐AG), and their target receptor, CB1R. In the brain, these ligands bind to CB1R and modulate the release of neurotransmitters from nerve cells resulting in changes in synaptic transmission. Disorders result when levels of AEA and 2‐AG are either too high or too low. While 2‐AG is synthesized at the plasma membrane (PM), AEA is produced in the ER and may require an intracellular carrier protein to move through the cytoplasm to the PM where it is released. The lipid binding protein, sterol carrier protein 2 (SCP‐2) is hypothesized to transport AEA due to its ability to bind to membranes and its nonspecific hydrophobic binding pocket. This proposed binding pocket is composed of 2 alpha helices, 3 beta sheets and the core hydrophobic amino acid residues F13, F27, F35, F37 and F80. The Messmer SMART Team (Students Modeling A Research Topic) created a model of SCP‐2 using 3D printing technology. Understanding the structure of SCP2 and how this protein might regulate AEA and 2‐AG levels could lead to possible new treatments for debilitating mood, appetite, memory and anxiety disorders. Supported by grants from NIH‐SEPA and NIH‐CTSA
D‐Loop Facilitates Hydride Transfer in Transhydrogenase
Authors: Ramirez Robert , Kelly Conor , Tran Naomi , Hernandez Karen , Huettig Emma , Clay Addison , Mackey Joseph , Chowdhury Parag , Stout David
Published: June 2021

Transhydrogenase (TH) is located in the mitochondrial inner membrane and maintains NADPH levels to counteract oxidative stress resulting from oxidative respiration. Enzymes in the electron transport chain work to reduce oxygen to water and pump protons (H+) into the inter‐membranous space. The accumulation of H+ creates an electrochemical driving force, termed the proton motive force (pmf), which is used to power ATP synthase. However, the oxidative reactions also produce free radicals that are very damaging to cellular DNA and other molecules. To counteract this, the pmf powers TH to exchange hydride ions (H−) between two nicotinamide nucleotide cofactors, NAD(H) and NADP(H). TH is composed of three domains (DI, DII, and DIII). DII is membrane bound while DI and DIII are soluble, making them amenable to crystallization. DI and DIII are directly involved in hydride transfer between NAD(H) and NADP(H). The High Tech High SMART Team (Students Modeling A Research Topic) modeled TH (PDB: 1XLT, 1PNO) using 3D printing technology to visualize the hydride transfer. The models highlight the D‐loop in DIII in both open and closed conformations. The Dloop responds to changes in net charge when H− is transferred, causing DIII to move, which is linked to the inward transfer of H+ driven by the proton motive force. Hence, TH is able to make NADPH to counteract oxidative stress. Supported by a grant from the HHMI Pre‐College Program
E. coli's Multidrug Transporter Complex: A SMART Team Exploration of Drug Resistance Mechanisms
Authors: Carrel Elijah , Havlena Zoë , Hua Amy , Lou Thomas , Tan Xiangyi , Vorperian Sevahn , Bhattacharyya Basudeb , Nelson David L.
Published: June 2021

The treatment of bacterial diseases has become a serious problem in clinical medicine in large part because of antibiotic resistance. One way bacteria have increased resistance to drugs is the overexpression of multidrug efflux pumps, exemplified by AcrAB‐TolC, a group of three proteins that span both the inner and outer membranes of E. coli. AcrAB‐TolC transports a wide range of foreign compounds and a broad spectrum of antibiotics. AcrB forms a homo‐trimeric structure that uses cyclical conformational changes powered by ATP to transport foreign compounds into the TolC pore which eventually expels the compounds from the bacteria. The role of AcrA is less defined, perhaps providing structural support to AcrB. The Madison West High School SMART Team (Students Modeling A Research Topic) modeled the interactions between AcrB and associated proteins in the E. coli pump using 3D printing technology to further study the mechanisms of bacterial drug efflux, as well as gain a better understanding of other drug efflux transport mechanisms, such as that used by P‐glycoprotein, which is important in cancer biology and human resistance to chemotherapeutics. The study of the detailed structures of AcrAB‐TolC may be of paramount importance in the development of novel pharmaceuticals against bacterial infections. Supported by grants from NIH‐SEPA and NIH‐CTSA.
PBP2a: The Means for β‐lactam Resistance
Authors: Farooque Alma , Huettl Maggie , Islam Saiful , Kaiser Jennifer , Pathak Rahul , Shukla Neehal , Van Oudenhoven Quinn , Dopp Amy , Shukla Sanjay K.
Published: June 2021

Methicillin‐resistant Staphylococcus aureus (MRSA) is a scourge of modern hospitals causing significant morbidity and mortality. MRSA is a formidable threat due to its resistance to β‐lactam antibiotics. β‐lactams function in normal S. aureus strains by inhibiting the action of penicillin‐binding‐protein (PBP), which synthesizes the cell wall by forming peptide bonds between short segments of peptidoglycan. β‐lactams acylate the Ser 403 active site of PBP, causing the substrate to be unable to attach to it, thus inhibiting the enzyme and effectively initiating death. β‐lactam resistance in MRSA is due to mecA, which encodes a variant of PBP known as PBP2a. At normally lethal β‐lactam concentrations, PBP2a is resistant to the antibiotics due to its lower affinity to β‐lactams, which decreases the rate of formation of the acyl‐PBP2a intermediate. This reaction is also inefficient because the active site has to undergo conformational changes for acylation to happen; these changes do not occur in PBP2a. The Marshfield SMART Team (Students Modeling A Research Topic) designed a 3D model of the transpeptidase domain of PBP2a to understand the structural basis of β‐lactam resistance. Using this model, one can design newer antimicrobials with improved binding affinity between the drug molecule and the active site of PBP2a. Supported by grants from the Marshfield Clinic and School District of Marshfield and NIH‐SEPA
Improvement in Skin Carotenoid Levels Assessed by Reflectance Spectroscopy
Authors: Talbott Shawn , Quan Danyi , Xiong Wade
Published: June 2021

BACKGROUND

Carotenoids have been directly associated with health benefits, including lutein (eye health), lycopene (cardiovascular and prostate health), and beta‐carotene (ultraviolet skin protection). Various biophotonic spectroscopy techniques are accurate and noninvasive methods of detecting skin carotenoid levels and assessing change in antioxidant levels over time.

OBJECTIVE

We used Reflectance Spectroscopy to measure skin carotenoid levels following two supplementation regimens.

METHODS

Sixty‐six healthy volunteers were supplemented with: Group A = 15mg/day of mixed carotenoids (9mg beta‐carotene, 4mg lycopene, and 2mg lutein in a proprietary nano‐emulsion); Group B = 18mg/day of natural beta‐carotene; or Group C = placebo containing no carotenoids. Skin carotenoid levels were measured at Baseline and Weeks 8 & 16.

RESULTS

Skin carotenoid levels were increased 59% and 57% in Group A, and 37% and 36% in Group B at weeks 8 and 16, respectively, compared to Control.

CONCLUSION

These results show, via Reflectance Spectroscopy, that skin carotenoid levels are significantly increased by supplementation with natural beta‐carotene (18mg/d) or a proprietary nano‐emulsion (15mg/d). The magnitude of skin carotenoid increase was greater with the nano‐emulsion, suggesting a possible enhancement of absorption, delivery, deposition, or retention in the body.
Zinc depletion‐induced apoptosis is mediated through Ca2+‐ dependent depolarization of mitochondria membrane in human breast cancer MDA‐MB‐231 cells
Authors: Lin Alice , Xu Zhaoming
Published: June 2021

We have previously shown that zinc depletion induces a Ca2+‐ dependent apoptosis in MDA‐MB‐231 cells. This zinc depletioninduced apoptosis is associated with an increased Bax:Bcl‐2 ratio and an elevated caspase‐9 and −3 activities while caspase‐8 activity remains unchanged. We hypothesized that Ca2+‐dependent depolarization of the mitochondrial membrane is involved in zinc depletion‐induced apoptosis. To test this hypothesis, MDA‐MB‐ 231 cells were cultured in DMEM+10% FBS for 3 days followed by treating the cells with N,N,N′,N′‐tetrakis[2‐ pyridylmethyl]ethylenediamine (TPEN; 20μM) alone for 0–24h or in the presence of 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (BAPTA; 0–20μM) for 24h. Mitochondrial membrane potential was assessed by a DiOC6 flow cytometric assay. TPEN caused depolarization of the mitochondrial membrane in a treatment duration‐dependent manner (2–24h; R2=0.99). BAPTA reduced TPEN‐induced depolarization of the mitochondrial membrane by 62–79%. Collectively, these results indicated that zinc depletion‐induced apoptosis was mediated through a Ca2+‐dependent depolarization of the mitochondrial membrane in MDA‐MB‐231 cells. Supported by NSERC.
Effect of High‐Fat Meal and High‐Carbohydrate Meal on Daily Total Energy Metabolism in Adults Japanese Men
Authors: Takuya Taira , Hisao Fujii
Published: June 2021

Introduction

Diet‐induced Thermogenesis (DIT) is the increase in energy expenditure above resting associated with the consumption of a meal, accounts for approximately 10% of an individual fs total daily energy expenditure. The thermic effect of separate nutrients is highest for protein (20–30%), followed by CHO (5–10%), and fat (0–3%).

Objective

The objective of this study was to clarify the effect of high‐fat meal and high‐carbohydrate on daily energy metabolism in adult Japanese men using indirect human calorimeter.

Methods

The subjects were fourteen adults Japanese men (21.5 ± 0.3 years). We measured EE (energy expenditure), RQ (respiratory quotient), GOR (glucose oxide rate), LOR (lipid oxide rate), LF/HF (Low Frequency/High Frequency), HF (High Frequency). EE was measured using the IHC for 36 hours. The activity of sympathetic nervous system was assessed by real‐time analysis system of heart rate fluctuation.

Results& Conclusion

Twenty‐four‐hours total energy expenditure was no different among three trial. On the other, RQ and GOR at supper time were significantly lower in HF trial than HCHO trial during the 120 minutes after ingestion (p < 0.05). LOR at supper time was significantly higher in HF trial than HCHO trial during the 120 minutes after ingestion (p < 0.05). Those results showed that high‐fat meal and high‐carbohydrate meal changed energy substrate.
Trends in dietary intake among U.S. 2–6 year olds, 1989 to 2008
Authors: Ford Christopher Nicholaus , Slining Meghan M. , Popkin Barry M.
Published: June 2021

Objective

Describe changes in diet among 2–6 year olds from 1989 to 2008 related to sharp rises in obesity during this period.

Methods

This analysis included 10,647 children ages 2–6 from five nationally representative surveys of dietary intake in the U.S.: CSFII 1989–1991; CSFII 1994–1998; and NHANES surveys 2003–2004, 2005–2006 and 2007–2008. Diet data were categorized into groupings using the UNC‐CH approach. Analyses were carried out using a single 24‐h dietary recall with appropriate survey weighting. T‐tests were used to compare means across survey years with P<.05 considered significant.

Results

Over the 20‐year period, there were increases in per capita intake of savory snacks (+51 kcal, P<.01), pizza/calzones (+32 kcal, P<.01), sweet snacks and candy (+25 kcal, P<.01), corn‐based mixed Mexican dishes (+22 kcal, P<.01) and fruit juice (+18 kcal, P<.01), whereas total daily energy intake increased by 212 kcal (from 1833 to 2045) (P<.05). Fruit intake increased marginally (+24 kcal, P<.01).

Conclusions

Foods that tend to negatively impact diet quality, and contribute excess calories, such as savory snacks, pizza/calzones, mixed Mexican dishes, sweet snacks and candy, and fruit juice, predominated the top changes in per capita consumption between 1989 and 2008.
Immuno‐stimulating Activities of Polysaccharides from Cheonggukjang
Authors: HONG HEE-DO , CHO CHANG-WON , HAN CHUN-JI , RHEE YOUNG KYOUNG , NAM SO-HYUN , KIM YOUNG-CHAN
Published: June 2021

In this work, we examined in vitro, ex vivo and in vivo immuno‐stimulating activities of polysaccharides from Cheonggukjang. The crude polysaccharide (CHW‐0) was isolated from Cheonggukjang by hot water extraction, ethanol precipitation, and ultrafiltration. The major constituents in CHW‐0 were neutral sugar (51.4%), and the uronic acid (16.3%). The result of in vitro assay, CHW‐0 showed an anti‐complementary activity (ITCH50 at 1,000 μg/ml: 30.3%). The immuno‐stimulating effects of CHW‐0 were also analysed ex vivo by means of nitric oxide (NO) and cytokines in supernatants of mouse peritoneal macrophage. The mouse peritoneal macrophage stimulated with CHW‐0 produced NO and various cytokines such as IL‐6 and IL‐12 in a dose‐dependent manner. We also investigated the immuno‐stimulating actions of CHW‐0 in 30 male Chinese Kun Ming (KM) mice. The mice were divided into three groups, and treated by oral administration with CHW‐0 into two dosages (100 mg/kg and 200 mg/kg body weight) for 4 weeks. The results showed that there were no significant differences in body weight and thymus index, but spleen index was increased. In the splenocytes proliferation test, the T and B lymphocyte proliferation abilities of CHW‐0 were significantly increased than that of control group. Overall, Cheonggukjang crude polysaccharide may have immuno‐stimulating activities beneficial to human health.