bluuebunny/crossref_metadata_2025 huggingface dataset:
Hydrazines as Potential Anti‐amyloidogenic Agents
Authors: Ghosh Sanjukta , Schifone Christine , Foster Michelle , Torok Bela , Levine Harry , Torok Marianna
Published: June 2021

Several neurodegenerative diseases are characterized by the formation of highly organized and extremely stable fibrillar aggregates as well as their soluble oligomers. One of the approaches to develop therapeutics for these diseases is to destabilize these aggregates and inhibit their formation. Our earlier work with hydrazones has resulted in strong inhibitors towards the self‐assembly of Alzheimer's disease's (AD) amyloid beta (Aβ) peptide. Due to the possible metabolic instability of the hydrazones, which may result in the reformation of their synthetic precursors, these studies were extended to the effect of the precursors, such as phenylhydrazines and benzaldehydes on the self‐assembly of amyloid beta. In our present work we describe the application of over 30 hydrazone precursor molecules in the inhibition of Aγ self‐assembly including fibril and oligomer formation, as well as, in the disassembly of preformed Aγ aggregates. As possible multifunctional scaffolds, the compounds were also assayed for antioxidant activity, which may result in potential drug candidates that can interfere with multiple pathways in the development of AD. The data indicate that phenylhydrazines are promising multifunctional agents for further drug development in AD.
Synthesis of Functional Proteins via Bioconjugation
Authors: Pirigyi Monica , Zondlo Neal Joseph
Published: June 2021

Large protein synthesis is a present day biochemical challenge. Proteins can efficiently be expressed from various sources; however, post‐translational modifications to introduce specific phosphorylation and spectroscopic labels can be challenging. One promising mechanism in creating these large, functional proteins is bioconjugation. This work is directed toward microtubule binding studies to elucidate the mechanism of tau aggregation in Alzheimer's Disease. Peptides from the tubulin‐binding domain of Tau were synthesized on an automated peptide synthesizer and modifications were conducted, on both solid and solution phase, to allow incorporation of specific phosphorylation events and multiple bioorthogonal functionalities. Peptides were modified and peptide conjugation reactions were conducted. To elucidate the efficiency of these bioconjugation reactions we have used model peptides and demonstrated clean, efficient conversions in aqueous solutions. Further, we are utilizing these tools to synthesize proteins with multiple tubulin‐binding domain repeats and to study their ability to bind, polymerize, and depolymerize microtubules in both their nonphosphorylated and phosphorylated states. Funding Sources: HHMI and NIH
Isoflurane preconditioning involves the upregulation of aquaporin genes
Authors: Schwamb Zackary , Seidler Norbert , Theisen Christopher
Published: June 2021

Anesthetic preconditioning is a phenomenon in which cells that have been previously exposed to volatile anesthetics exhibit protection from future cellular insult. The mechanism of isoflurane‐induced changes in gene expression is thought to involve the dehydration of proteins, which consequently leads to protein misfolding and an upregulation of protein chaperones including alphaA‐crystallin. In the present study we found that the neuronal cell line SH‐SY5Y exhibited increased expression of the genes for aquaporins 1, 4, 5, and 8, 48 hr after exposure to isoflurane. The levels of aquaporin 1 also remained elevated 72 hr after exposure which coincided with increases in alphaA‐crystallin expression. We also found that isoflurane exposure induced a preconditioning effect that was exhibited by an increase in SH‐SY5Y cell viability in the presence of ethanol, 72 hr after exposure to the anesthetic. These data indicate that isoflurane preconditioning is protective against dehydration stress in a cell culture model and may involve the regulation of water homeostasis via aquaporin channels.
Mass Spectrometric Immunoassay for Insulin‐Like Growth Factor 1
Authors: Nedelkov Dobrin , Niederkofler Eric , Phillips David , Krastins Bryan , Kiernan Urban , Tubbs Kemmons , Lopez Mary
Published: June 2021

Mass Spectrometric Immunoassays (MSIA) are protein quantification methods that utilize mass spectrometric detection for quantification of targeted protein analytes affinity retrieved by surface‐immobilized antibodies. This rather straightforward concept is realized through affinity pipette devices (MSIA‐Tips) that enable high‐throughput assaying of hundreds of samples per day. Described here is the development and validation of mass spectrometric immunoassay for insulin‐like growth factor 1 (IGF1).

MSIA‐Tips derivatized with anti‐IGF‐1 antibody were used for affinity isolation of IGF1 from human samples. Long R3 (LR3) IGF1, spiked into samples prior to the affinity isolation, served as internal reference standard for signal normalization and IGF1 quantification. Following capture and elution, both the intact proteins and their surrogate tryptic peptides were analyzed via LC/MS. Linear standard curves were built that spanned the range of 1–1,500 ng/mL. The assay exhibited intra‐ and inter‐assay precisions of <10%, and linearity and spiking‐recovery in the 90–110% range. Data was compared to that obtained with a commercially available ELISA, yielding good correlation. The data correlation between intact IGF1 and peptide‐based IGF1 MS analyses was good, demonstrating the applicability of the IGF1 MSIA across MS platforms.
The signaling pathways for compound DC072 induced apoptosis in acute myeloid leukemia
Authors: Li Kathy keqin , Chen Limin , Lu Juyan , Luo Liufei , Luo Cheng
Published: June 2021

Sphingosine‐1‐phosphate (S1P) signaling are attractive targets for development of new therapeutics for cancers. By using computer‐aided drug design, we found a series of chemical analogues for Sphingosine or S1P, and then we tested their biological activities in enzymatic and cellular level. One of the analogues named DC072 shows very good anti‐cancer biological effects in some leukemia cells. DC072 could dramatically induce apoptosis of acute myeloid leukemia (AML) cell line kasumi‐1, and markedly reduce the growth of AML xenograft tumors in nude mice. We studied the genome‐wide gene expression profile by microarray analysis. There are group genes are associated with lipid metabolism. We further explored the molecular networking of how DC072 induced AML cell apoptosis. It was demonstrated that DC072 inhibited the activity of sphingosine kinase 1 (SPHK1) to make ceramide accumulation within the AML cells, and ceramide down regulated nutrition channel proteins and activated PP2A. PP2A activation triggered phosphorylations of c‐kit and Akt proteins, and ultimately resulted in the changes of downstream apoptotic cascade proteins such as Bcl‐2, Bcl‐XL, caspase 3 and PARP. Our data suggested that DC072 could be a potential addition to the therapeutic armamentarium in leukemia.
Carbon monoxide induces chromatin remodeling to facilitate endothelial cell migration
Authors: Li Mailin , Gallo David , Csizmadia Eva , Otterbein Leo , Wegiel Barbara
Published: June 2021

Objectives

Vascular injury to the endothelial monolayer (EC), either by mechanical removal or by chronic inflammation, is still awaiting effective therapies. The homeostatic enzyme, heme oxgenase‐1 (HO‐1) is induced by various stimuli such as oxidizing agents and its substrate, heme. HO‐1 degradation of heme generates the vasomodulatory gas carbon monoxide (CO), in addition to biliverdin and iron.

Results

In this study, we show that administration of exogenous CO enhanced acetylation of Histone H3 (Lysine 9) via the GSK3β‐dependent stabilization of Rev‐ERBα protein to increase migration of EC in response to mechanical injury. We found that CO acting via chromatin remodeling pathways inhibits PAI‐1 and thrombospondin genes, while concomitantly upregulating transcription of proangiogenic molecules such as VCAM‐1.

Conclusions

In summary, CO modifies chromatin structure through enhanced acetylation of histone H3 through the GSK3β‐Rev‐erbα pathway to increase migration and proliferation in EC. This mechanism is demonstrated to be important for vascular homeostasis as well as treatment of vascular complications associated with dysregulated thrombosis, wound healing, and angiogenesis. This work was supported by AHA 10SDG2640091 grant (BW) as well as Department of Surgery BIDMC internal funds (BW) and NIH grant HL‐071797 & HL‐076167 (LEO).
Impaired COX‐2/PGE2‐mediated anti‐viral response in G6PDknockdown A549 cells through NOX/MAPK signaling
Authors: Lin Hsin Ru , Wu Yi Hsuan , Yang Chuen Mao , Chiu Daniel Tsun Yee
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD) provides reducing power to all cells in the form of NADPH to meet the cellular needs for reductive biosynthesis and maintenance of the cellular redox homeostasis. We have previously reported that G6PD‐deficient cells suffer high oxidative stress and these cells are more susceptible to viral infection than normal counterparts. However, how G6PD deficiency may affect cellular signaling remains elusive. Toward this end, we determined the effects of G6PD status on inflammatory responses with TNF‐α to stimulate cells (15 ng/ml TNF‐α). By comparing the level of TNF‐α induced inflammatory response, decreased expression pattern of induced Cox‐2 and downstream PGE2 production were observed in G6PD‐knockdown A549 cells. We further analyzed TNF‐α‐induced antiviral activity and found that decreased PGE2 production enhanced cells more susceptible to Coronavirus 229E infection. Further investigation revealed that these phenomena were due to decreased phosphorylation level of MAPK (p38, ERK and JNK) comparing with control cells. By applying DPI pretreatment, the impaired inflammatory response in G6PD‐knockdown cells was through NOX signaling. All in all, these data suggest that G6PD status affects cellular inflammatory responses and decreased TNF‐α‐mediated anti‐viral response in G6PD‐knockdown A549 cells is due to dysregulated NOX/MAPK signaling.
Metformin relieves oxidative stress and leads to differential modulation of function in key metabolic tissues
Authors: Simmons Amber , Meshulam Tova , Labad Laura Nocito , Corkey Barbara
Published: June 2021

Metformin reduces hyperglycemia and hyperlipidemia in people with diabetes. We hypothesized that metformin alters a circulating redox state, thereby ameliorating dysregulation of metabolism in multiple tissues. The acute effects of metformin on redox state and tissue function were assessed in primary mouse hepatocytes, human adipocytes, and INS‐1 β‐cells. Metformin increased the NAD+ to NADH ratio in hepatocytes, implying an alteration in redox state. In neither adipocytes nor hepatocytes did metformin have an effect on basal production of reactive oxygen species (ROS), but metformin tended to decrease tert‐butyl‐hydroperoxide‐induced ROS production. A similar pattern was observed on lipid peroxidation in adipocytes. Interestingly, metformin inhibited both basal and forskolin‐induced lipolysis. However, metformin had no effect on basal or glucose‐stimulated insulin secretion in INS‐1 cells. These results suggest that metformin acutely altered the cellular redox state, which may explain its ability to yield various tissue‐specific effects and deliver pleotropic benefits.
The predictive validity of formative anatomy assessments
Authors: Finn Gabrielle Maria , Hanson Elizabeth
Published: June 2021

This retrospective study aimed to establish whether formative practical laboratory exams, taken after minimal anatomy teaching, were predictive of performance on end of year summative anatomy exams.

Exam (1 × formative, 2 × summative) results were collected for 8 cohorts of medical students (2004–12). Parametric tests were used as all data were normally distributed. Correlations were determined using a Pearson's rank test.

Consistently highly significant positive correlation (P<0.0010) between short, formative examinations taken and final summative examinations were demonstrated within all 8 cohorts. Students’ formative performance also correlated with their mid‐year summative examination performance (P<0.0010). There was also a significant correlation between mid‐year and final examination performance for the same 6 cohorts (P<0.0010).

Formative exams introduce students to practical anatomy exams. This is the first time that students have completed a timed exam where they can't look ahead review questions. We have demonstrated that students who do well in the formative exam do well in the subsequent exam, and conversely, students who perform poorly continue to do so. This raises debate as to whether anatomy practical exams test knowledge or instead are tapping into other unknown constructs as other formative written exams are not predictive of eventual performance.
Putting a Handle on the Pump: Translational Fusion of an Nterminal TAP tag to the Plasma Membrane Proton Pump AHA1
Authors: Rodrigues Rachel B , Sabat Grzegorz , Sussman Michael R
Published: June 2021

The Arabidopsis plasma membrane proton P‐type ATPases AHA1 and AHA2 are responsible for generating a proton motive force across the plasma membrane and are essential for successful embryo development. We report the translational fusion of AHA1 to a tandem affinity purification tag and stable expression of the tagged protein in transgenic double aha1/aha2 knockout plants. TAP tagged AHA1 complements the double knockout lethality, Western blots show the protein is targeted to the plasma membrane, and plants grow normally under standard conditions. An affinity purification mass spectrometry (APMS) workflow was used to identify proteins that co‐purify with TAP tagged AHA1 from plasma membrane enriched fractions. We identified two proteins co‐enriched with AHA1 by label free MS and an additional 17 using a metabolically labeled approach. Several of the proteins identified are consistent with previous literature on AHA1 protein interactions, while others are novel. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP tagged AHA1 has complemented all endogenous AHA1 and AHA2 function and have shown that these plants can be used to specifically purify AHA1 and identify in planta interacting proteins by mass spectrometry.
16 strains of Debaryomyces hansenii tested against Penicillium digitatum, a post harvest fungus, affecting citrus crops in Puerto Rico
Authors: Rodriguez‐Gonzalez Robert , Baez‐Saez Ernesto , Torres Stephanie , Colon‐Mercado Luis A. , Arroyo‐Gonzalez Nancy
Published: June 2021

Citrus crops contribute 11 million dollars to the Puerto Rican economy. Unfortunately, Penicillium spp. infects these crops causing a great economic loss. Debaryomyces hansenii, a marine halotolerant yeast, has the ability to produce a killer toxin which seems to be homologous to the KHR (killer toxin) gene of Saccharomyces cerevisiae whose activity in the reduction of the decay of citrus fruit caused by Penicillium spp. has been reported. Microbiology and molecular biology techniques have been used to characterize sixteen D. hansenii strains and isolate their killer toxin. Different assays of the killer toxin activity in vivo were realized to determine its potential as a biological control. Two strains, D. hansenii BCS 006 and YB‐17914, were more efficient against Penicillium digitatum.
Physicochemical and Thermal Properties of Wheat Gluten Fractions from Hard Red Winter Wheat Cultivar
Authors: Alamri Mohammed , Mohamed Abdellatif
Published: June 2021

Enriched gliadins, HMW‐GS and LMW‐GS were obtained by differential solvent fractionation from flour using a modification of the method described by Verbruggen et al (1998). Briefly, flour was defatted with chloroform (CHCl3) followed by gliadins extraction with 50% (v/v) n‐Propanol (n‐PrOH), glutenin subunits were extracted from the residue at 60°C by adding 50% n‐PrOH containing 1% (w/v) dithiothreitol (DTT), and the low molecular weight glutenins were precipitated by 85% n‐PrOH. The surface hydrophobicity, charge‐to‐mass ratio, and thermal properties of the three fractions were investigated using reverse‐phaseHPLC, capillary electrophoresis (CE), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Gliadin and LMW‐GS fractions resolved in peaks with different surface hydrophobicity and a relatively wide range of charge‐to‐mass ratio. Samples were pre‐heated to remove residual solvent using TGA. The DSC profile of the pre‐heated gliadin and LMW‐GS‐rich fractions revealed similar glass transition onset, midpoint and conclusion temperatures, averaging 134.9, 143.0 and 151.3°C, respectively, and 0.255 J/g/°C. While the glass transition for the HMW‐GS rich fraction showed higher trend averaging 137.5, 144.3 and 153.1°C for onset, midpoint and conclusion temperature, respectively, and 0.318 J/g/°C.
Identifying small molecule regulators of the Proteostasis Network that enhance CFTR protein function
Authors: Drew Lawrence , Miller John , Hutt Darren , Dobbs Wesley , Cullen Matthew , Zhu Yitan , Wachi Shinichiro , Andersen Jennifer , Khanna Shilpi , Giuliano Kenneth , Ge Hui , Garza Dan , Balch William , Reinhart Peter , Tait Bradley
Published: June 2021

Protein homeostasis is established and preserved by a collection of pathways and cellular processes known as the Proteostasis Network (PN). Protein misfolding disorders can arise through a reduced capacity of the PN to appropriately correct the folding, trafficking and degradation of disease‐associated proteins. We target PN pathways using small molecule Proteostasis Regulators (PRs) to identify modulators that confer functional rescue of misfolded proteins. Specifically, we have applied our PN approach to the correction of mutant Cystic Fibrosis Transductance Regulator (CFTR), the causative defect in Cystic Fibrosis (CF). A single mutation, deletion of phenylalanine 508 (F508del), is present in ~87% of all CF patients. The F508del mutation causes defective folding and trafficking, as well as enhanced degradation of the resultant CFTR protein. We carried out a high‐throughput screen using a multiplex gene expression platform to identify PRs that modulate proprietary PN pathway reporters. PRs found to correct mutant CFTR F508del exert effects on one or more pathways within the PN, suggesting they may target different mechanisms important in the maturation of CFTR. We present a novel approach for identifying therapeutics whose mechanism of action involves modulation of the PN. Funded in part by the Cystic Fibrosis Foundation Therapeutics, Inc.
Investigating the Functional Role of Reactive Cysteine Residues on Glycolytic Enzymes
Authors: Shannon Alex , Weerapana Eranthie
Published: June 2021

Cancer‐cell metabolism relies heavily on glycolysis and the pentose phosphate pathway to generate the energy and reducing power required for sustaining proliferation. Cancer‐cell proliferation is also accompanied by an increase in intracellular reactive oxygen species (ROS) that proceed to oxidatively modify proteins, specifically through reactive cysteine residues. Recently, it was shown that a highly reactive cysteine residue on the glycolytic enzyme pyruvate kinase M2 is inhibited by cysteine oxidation. Although this oxidized cysteine is not involved in the catalytic mechanism of the enzyme, it is still important in regulating protein activity and supporting cancer‐cell proliferation. To identify similar reactive cysteine residues on other glycolytic enzymes, we overexpressed the ten enzymes in glycolysis and applied a cysteine‐reactive chemical probe together with quantitative mass spectrometry to identify and rank all reactive cysteines within these enzymes. Through mutagenesis, activity assays and metabolomic analyses, we will investigate the role of these highly reactive cysteines in regulating protein activity and flux through glycolysis. Finally, we hope to develop cysteine‐reactive small molecules to covalently modify these reactive cysteines so as to chemically regulate glycolytic flux within cancer cells.
The role and mechanisms of microRNA let‐7g in regulating lipid metabolism in macrophages
Authors: Juo Suh‐Hang H , Wang Yung‐Song , Liao Yi‐Chu , Cheng Hsin‐Yun , Lin Ruey‐Tay
Published: June 2021

The transformation of foam cells from macrophages can lead to atherosclerosis. A micorRNA can bind to one or multiple mRNAs to inhibit protein synthesis, and/or degrade mRNA. We previously reported that microRNA let‐7g can bind to LOX‐1 mRNA, an oxLDL scavenger receptor, to knock down LOX‐1 expression. The present study aims to investigate let‐7g function macrophages.

Let‐7g regulates macrophage functions:

During foam cell transformation, macrophage's let‐7g level was significantly down‐regulated. Transfecting let‐7g mimic to macrophages led to a significant decrease of intracellular lipid. In addition, transfection of let‐7g mimic to macrophages could increase cell survival and reduce the levels of proinflammatory substances including IL‐1β, IL‐6 and MCP‐1.

Let‐7g contributes to cholesterol homeostasis:

We investigated whether let‐7g regulates cholesterol by influencing ABCA1 and SERBP2 expression. ABCA1 could be decreased by oxLDL but let‐7g could restore ABCA1 expression. Furthermore, SREBP2 expression was simultaneously down‐regulated by let‐7g mimic.

Let‐7g inhibits NF‐kB pathway:

Bioinformatic databases predicted that IKKα in the NF‐kB pathway is one potential target of let‐7g. Using the luciferase reporter assay, we proved that let‐7g can bind to IKKα to inactivate the NF‐kB cascade pathway. Our data suggest that let‐7g may play an important role in cholesterol metabolism in macrophages.

This research is supported

by National Health Research Institutes, Taiwan (NHRI‐Ex101–10107PI), National Science Council, Taiwan (NSC 101–2628‐B‐037–003‐MY2 and NSC 99–2628‐B‐037–037 ‐MY3)
Docetaxel‐induced alterations in cellular RNA in ovarian cancer cells
Authors: Narendrula Rashmi , Guo Baoqing , Lanner Carita , Parissenti Amadeo M
Published: June 2021

A major obstacle to the ablation of tumors using chemotherapy is resistance to anti‐cancer agents. Recently, the relationship between tumor characteristics and treatment response was assessed in a phase I/II clinical trial (Breast Cancer Res Treat, 119: 347) for advanced breast cancer patients treated with epirubicin and docetaxel. This study reported significant, dose‐dependent reductions in tumor RNA integrity (RIN) values which correlated with response to treatment. Results from this study suggest the possible utility of RIN as a measure of clinical response to chemotherapy. The purpose of the present study is to assess chemotherapy‐dependent alterations in tumor RNA quantity and integrity in vitro. Cells were plated and treated with docetaxel concentrations from 0.001–40 μM for 24 hr, to determine the effect on RNA content and integrity, measured using an Agilent Bioanalyzer 2100. RNA content increased per cell (p<0.05) while RIN did not change significantly in this range. Interestingly, discrete bands consistently appeared in the rRNA banding pattern at 0.005 μM, peaking at 0.2 μM DXL, just below the 28s and 18s rRNA bands. In contrast, docetaxel‐resistant A2780 cells did not display similar changes upon treatment, indicating that changes in tumor cell RNA content and integrity could be used to monitor response to docetaxel. Research funded by Northern Ontario School of Medicine and RNA Diagnostics Inc.
Students’ perceptions of two different study guides: the case of Human Anatomy and Physiology classes
Authors: Sturges Diana , Maurer Trent W
Published: June 2021

The mixed method study aimed to investigate the effectiveness of two different study guides (outline vs. completed versions) in undergraduate Human Anatomy and Physiology classes (HAP) and to analyze their relationship with demographic factors and academic achievement (based on final grades). The targeted population consisted of 750 HAP students taught by the same instructor in spring and fall semesters of 2012. In spring 2012, HAPI students received the study guide outline only and HAPII received both the study guide outline and the completed version. In fall 2012, the design was reversed. The survey (32 quantitative questions and 4 qualitative questions) was administered in class and 586 surveys were returned (78% response rate). Quantitative data was analyzed using SPSS, while qualitative data was organized into categories based on the themes emerged. The majority of the respondents were sophomores (50.9%), females (74.5%), and white (65.5%) and were nursing (46.5%) or allied health (41.7%) majors. The results indicated that students used the study guides to make their own study tools, organize the information required for the test or make their own study guide. The study guides were beneficial in clarifying the material on the test and allowed students to focus on “main” points, although it was long and time consuming to complete and students were not sure if their answers were correct. The provided completed study guides were perceived as providing a lot of information, but not in enough detail, although they saved time in preparation for the test. Students indicated that despite disadvantages, the study guides should be completed and provided further in advance of tests, so students can make their own study tools based on the information provided.
Influenza Hemagglutinin Binding and Interactions with a Host Cell
Authors: Ambessie Sallem , Desai Ashley , Keating Elena , Shire Sequoia , Simpson Nick , Yenew Mihret , Dix Randy , Wahome Newton
Published: June 2021

The CDC estimates that during the flu season 35 to 50 million people get the flu. Hemagglutinin (HA) is one of the main proteins required for influenza virus to infect host cells. Located on the surface of the virus, HA is responsible for binding the virus to the cell, and for releasing the viral genome into the host cytoplasm. There are 17 different HA subtypes (HA1 ‐ HA17), but HA1, 2, and 3 are the only strains specific to human influenza. Even though they appear in different animals, HA binds to sialic acid, which is present on the surface of epithelial cells in the upper respiratory tract. HA is a trimer protein made of a globular and stem domain. The sialic acid binding region resides in the globular head. The binding depression surrounds the sialic acid domain with three identical primary regions. These regions are comprised of a loop‐helix‐ loop structure, which surrounds the sialic acid, forming the triangular opening into the beta‐sheet depression. The exact binding geometry between hemagglutinin and cell surface proteins differs within species, thereby accounting for the species specificity of HA. The Olathe North SMART team (Students Modeling a Research Topic) modeled HA using 3D printing technology. Program supported by a grant from NIH‐SEPA.
One Indole Ring to Rule Them All: How Modeling of Naltrindole Bound to the Delta Opioid Receptor Can Aid the Development of Novel Analgesics
Authors: Arnold E. , Bell N. , Boesen T. , Boldt N. , Borden A. , Bro J. , Douglas M. , Fuller J. , Furumo Q. , Gummin C. , Keuler A. , Kim D. , Martinez A. , Moldenhauer D. , Mullooly I. , Nelsen‐Freund R. , Ogunkunle D. , Ortega L. , Palmersheim S. , Sabatino T. , Schwabe B. , Sung R. , Trzcinski K. , Ulschmid C. , Klestinski K. , Kaiser C. , Vogt D. , Cunningham C. W.
Published: June 2021

According to the Institute of Medicine, 116 million Americans suffer from chronic pain, costing over $500 billion annually. As such, the use of pain‐killing drugs like morphine and oxycodone has increased dramatically over the past decade. Analgesic effects are produced through agonism, or activation, of the body's mu (MOR) and delta (DOR) opioid receptors, which are G‐coupled protein receptors. Tolerance, the decreased analgesic effect of MOR agonists after prolonged use, is a major problem facing opioid pain management. A drug that antagonizes, or inhibits, DOR can greatly reduce the development of tolerance to MOR agonists, offering new pain therapy potentials. One example of a selective DOR antagonist is naltrindole (NTI), which has a similar structure as morphine, except for a cyclopropylmethyl group on its nitrogen substituent and a bulky indole group. The large indole ring negatively interacts with the W318 residue on MOR but is able to bond with W284 residue on the DOR, producing DOR‐selective antagonism. Co‐administration of NTI with morphine represents a potential new approach to producing analgesics with less tolerance. Understanding the structure of this ligand and enzyme may further structure‐based drug designs. The Marquette University High School SMART Team (Students Modeling A Research Topic) modeled naltrindole bound to DOR using 3‐D printing technology. Supported by a grant from the NIH‐CTSA.
Proton‐Dependence of the MitoNEET [2Fe‐2S] Cluster : An Electrochemical and Structural Investigation
Authors: Bak Daniel W. , Elliott Sean J.
Published: June 2021

MitoNEET is a novel 3‐Cys, 1‐His ligated redox active [2Fe‐2S] cluster binding protein, whose biological function remains unclear. MitoNEET is of interest due to its interaction with the anti‐diabetes drug pioglitazone, a member of the TZD drug family, and its unique cluster ligation. Here, a characterization of the clusters unique ligand environment is presented. Using protein film voltammetry, the potential of the mitoNEET cluster is found to be 0 mV at pH 7, where upon binding of pioglitazone reduces the potential to −100 mV. This is one of the first reports of PFV being used to monitor drug binding. Additionally, the mitoNEET cluster demonstrates unusual proton coupled‐electron transfer as well as marked proton‐dependent cluster instability. Through a series of site‐directed mutations of conserved residues around the [2Fe‐2S] cluster, both of these phenomena have been examined, demonstrating the presence of multiple sites of protonation that affect both cluster midpoint potential and cluster stabilization. The high conservation of these residues, which reduce cluster stability, suggest a physiological role for cluster loss and lead to a putative model, in which mitoNEET may sense cellular oxidation state, proton concentration, or iron levels. Changes in MitoNEET cluster binding could potentially mediate either cluster transfer or additional signaling events within the cell.

NIH‐NIGMS R01‐GM072663
Akt is required for Rac1 mediated mitochondrial oxidative stress and pulmonary fibrosis
Authors: Casey Jennifer , Murthy Shubha , Ryan Alan J , Carter A. Brent
Published: June 2021

The production of reactive oxygen species, especially hydrogen peroxide (H2O2) from alveolar macrophages, has been linked to the development of pulmonary fibrosis. We have shown that Rac1 is imported into the mitochondria and modulates H2O2 production in macrophages. In this study upstream regulators of Rac1 mitochondria import were investigated. Rac1 and Akt have been shown to positively and negatively modulate each other. We hypothesized that Akt activity was required for Rac1 activation and mitochondrial import. We found that macrophages exposed to chrysotile asbestos had increased Akt activity. Rac1 mitochondrial import was enhanced by the over‐expression of constitutively active Akt and conversely the knockdown of Akt was shown to abrogate Rac1 mitochondrial translocation, as Rac1 remained in the cytoplasm of macrophages expressing Akt siRNA. In contrast to the cytoplasm, Rac1 activity was significantly upregulated in the mitochondria of macrophages expressing constitutively active Akt, which consequently resulted in increased H2O2 production in these cells. These observations suggest that Akt plays a vital role in the development and progression of pulmonary fibrosis, as it is necessary for mitochondrial Rac1 import and regulating mitochondrial oxidative stress.

(Supported by 2R01ES015981–06, R01ES014871, VA Merit Review 1BX001135 (to A.B.C.) and NIH T32 CA078586).
A new type of Bacillus subtilis thiol peroxidase acting as an antioxidant during the stationary growth
Authors: Cha Mee‐Kyung , Kim Il‐Han
Published: June 2021

As a laboratory model organism Bacillus subtilis is used as the Gram‐positive equivalent of Escherichia coli, an extensively studied Gram‐negative rod. We found two types of thiol peroxidase (TPx) by searching a database of Bacillus substilis, which are homologous form of E. coli AhpC. We named the two bacillus TPx as AhpC_H1 and AhpC_H2. First, we characterized structural features of the two AhpC homologous. The structure features of the two proteins, compared to that of E. coli AhpC, suggest that AhpC_H1 is a new type of bacillus thiol peroxidase (TPx), and AhpC_H2 is a count part form of E. coli AhpC. The oxidized form of AhpC_H1 is reduced by Trx system (Trx reductase, Trx1, and NADPH), suggesting AhpC_H1 as a thioredoxin‐supported thiol peroxidase. The protein level in Bacillus subtilis was estimated to be ¡‐2% of total soluble protein. The expression of AhpC_H1 is significantly induced by oxidative stress. The growth of the null mutant is severely retarded, especially at the stationary‐phased growth. Taken together, we conclude that the new type of bacillus TPx is a predominant thiol peroxidase acting as one of principlal antioxidants during the stationary growth of Bacillus subtilis..*This work was financially supported by NFR (2011–0008913).
Red Rover, Red Rover, Send BCCP Over!: Coordinating Catalysis in Pyruvate Carboxylase
Authors: Dix Monica , Lozier Emilie , Murali Ananya , Murali Anjana , Whittle Daniel , Whittle Kelly , Murali Lalitha , Lietzan Adam
Published: June 2021

Cells exist in a state of continuous metabolic flux. The Krebs cycle, a central metabolic hub in the cell, is responsible for supplying precursors for the synthesis of amino acids, nucleotides, and compounds required for energy transfer. During periods of increased metabolic flux, metabolites in the Krebs cycle become depleted and must be replenished. Pyruvate carboxylase (PC), a multifunctional enzyme, replenishes the Krebs cycle by catalyzing the conversion of pyruvate to oxaloacetate, a Krebs cycle intermediate. The Shorewood SMART Team (Students Modeling A Research Topic) created a model of PC using 3D printing technology. PC contains four distinct domains: biotin carboxylase (BC), central allosteric, carboxyltransferase (CT), and biotin carboxyl carrier protein (BCCP). The overall reaction is initiated by BCCP‐biotin carboxylation in the BC domain. BCCP‐carboxybiotin physically translocates to the CT domain to transfer its carboxyl group to pyruvate. The active site of the CT domain undergoes a reconfiguration upon pyruvate binding to accommodate the docking of BCCP‐carboxybiotin for pyruvate carboxylation. With the rise in antibiotic resistance, understanding how PC functions may provide a target in developing new antibiotics, whereby the new drug would eliminate critical metabolic activity, thus killing the bacteria. Supported by a grant from the NIH‐CTSA UL1RR031973.
FAM129B, a Protein that Promotes Cancer Cell Invasion, forms a Complex with KEAP1
Authors: Hachem Fatme A , Chen Song , Evans Hedeel Guy , Evans David R
Published: June 2021

FAM129B/Minerva, a protein implicated in melanoma cell invasion, has been shown to suppress TNFα induced apoptosis in cancer cells (Song, Evans & Evans (2010), JBC 286, 10201–09), thus contributing to the survival of metastatic cells. KEAP1, a substrate recognition molecule for the CUL3 E3 ubiquitin ligase, plays a central role in the activation of the TNFα pathway. We wish to test the hypothesis that the up‐regulation of FAM129B in cancer cells suppresses apoptosis by sequestering KEAP1, thus preventing its participation in the apoptotic pathway. The first step is to demonstrate that FAM129B and KEAP1 form a complex. We have cloned and expressed the proteins in E. coli and purified both to homogeneity. Pull downs and immunoprecipitation clearly showed that the two proteins form a high affinity, stoichiometric complex. Using individually cloned domains, as well as mutations of the ETGE motif in FAM129B, the binding sites on each protein have been located. The proline rich region of FAM129B was shown elsewhere (Old et. al. (2009) Mol Cell. 34,115) to be phosphorylated by MAP kinase (Erk1/2) at four sites. The effect of MAP kinase, which is upregulated in cancer cells, and PKC phosphorylation on the stability and formation of the complex has also been investigated.
Structural and functional analysis of Cas2 from T.onnurineus Archaeal type III CRISPR/Cas system
Authors: Jung Tae‐Yang , Woo Eui‐Jeon , Oh Byung‐Ha
Published: June 2021

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) is RNA based prokaryotic immune system against exogenous genetic elements such as plasmids and virus DNA/RNA. Cas2 is a conserved essential component for an adaptation stage in CAS (CRISPR Associated protein) complex. We present the crystal structure of Cas2 from Thermococcus onnurineus with the metal‐dependent activity against single stranded (ss‐) RNA. The dimeric arrangement exhibits an obvious RNA binding site at the interface lined with positive residues and aromatic side chains, shaping deep and wide groove. The flexible loops that reported as DNA and RNA recognizing region were missed in Ton_Cas2, implying that Ton_Cas2 may interact with different types of substrate. Cas2 showed endoribonuclease activity toward ss‐RNA, but no cleavage patterns for linear double stranded (ds‐) DNA, circular ds‐DNA, ss‐DNA and ds‐RNA. However, 18nt DNA‐RNA hybrid was hydrolyzed with magnesium cofactor. Therefore, Ton_Cas2 is thought to be a new subfamily of Cas2 that involved in phage or cellular mRNAs degradation in type III‐B CRISPR‐mediated immune system in Thermococcus onnurineus.
Lasalocid induces apoptosis via the cell cycle arrest and ROS‐mediated mitochondrial pathway in prostate cancer PC‐3 cells
Authors: Kim Kwang‐Youn , Kim Sang‐Hun , Yu Sun‐Nyoung , Park Seul‐Ki , Kong2 In‐Soo , Ahn Soon‐Cheol
Published: June 2021

Lasalocid is an antibiotic from the group of carboxylic ionophores and is produced by Streptomyces lasaliensis. But there is limited information on the effect of lasalocid on human prostate cancer cells. In the present studies, the lasalocid was examined to better understand its effect on apoptosis and associated signal pathways in vitro in human prostate cancer cells. Using PC‐3 cells, we demonstrate that lasalocid triggers apoptosis by elevating the intracellular ROS level, which was accompanied by increased mitochondrial membrane potential, prompted the levels of Bax and caspase‐3 and reduced the level of Bcl‐2, and cleavage of PARP‐1, a caspase‐3 substrate. These results are the first to link elevated oxidative stress and mitochondrial membrane hyperpolarization to caspase‐dependent apoptosis of lasalocid‐treated prostate cancer cells.
Identifying the Nature of Iron‐Sulfur Clusters in the CDGSH‐Family Protein AlVin0680
Authors: Kulkarni Rishikesh , Bak Daniel , Elliott Sean
Published: June 2021

The CDGSH domain is a small [2Fe‐2S] cluster binding domain typified by the presence of three ligating cysteines and one ligating histidine residue. The human protein mitoNEET, an example of a CDGSH domain protein, has attracted interest due to its interaction with the type II diabetes drug pioglitazone. A greater understanding of other CDGSH domain containing proteins may provide insight into the functional role of the unique [2Fe‐2S] cluster. The Allochromatium vinosum protein, AlVin0680, has been identified as a domain fusion protein consisting of two CDGSH domains and a putative [3Fe‐4S] cluster binding DUF1271 domain. Basic characterization of AlVin0680 via UV/Vis spectroscopy, EPR, protein film voltammetry, and colorimetric assays of the iron content indicate that both the mitoNEET‐like CDGSH clusters and the DUF1271 clusters are bound by the protein. Further work will involve the exploration of the potential interactions between the two [2Fe‐2S] clusters and the [3Fe‐4S] cluster, specifically determining whether electron or cluster exchange occurs between the three clusters. A greater understanding of the iron‐sulfur clusters in AlVin0680 may provide insight into the general function of CDGSH domain proteins and their iron‐sulfur clusters.
Association of I/D ACE polymorphisms with hormonal status and oxidative stress in Mexican Mestizo women
Authors: Lugo Gabriela , Hernandez‐Caballero Marta , Nicolás‐Franco Ricardo , Ocharan‐Hernández Esther , Jiménez‐Zamarripa Carlos , Calzada‐Frías Jesús , Linarez‐Pérez Luz , Calzada‐Mendoza Claudia
Published: June 2021

OBJECTIVE

Analyze the association between the presence of I/D ACE variants with hormonal status and oxidative stress in Mexican Mestizo women.

METHODS

In this study were included 38 healthy women and divided into two groups (pre‐menopausal or postmenopausal), to which a) menopause was corroborated by clinical evaluation and hormonal profile; b) polymorphisms were identified by real‐time PCR, sense oligonucleotide (5′‐CTG GAG ACC ACT CCC ATC CTT TCT‐3′) and antisense oligonucleotide (5′‐GTG GCC ATC AT ACA TTC GTC AGA‐3′); c) malondialdehyde was identified as a marker of lipid damage oxidative stress.

RESULTS

The women included were classified according their estrogenic status (premenopausal or postmenopausal), which was corroborated by medical history and hormonal profile. With respect malondialdehyde and glucose levels we found that were significantly higher in post‐menopausal (Mann‐Whitney U Statistic, p <0.001). However, in each group, oxidative stress and blood glucose were not associated with genotypes.

CONCLUSIONS

The I/D ACE genotypes were not associated with damage to lipids or with hyperglycemia observed in postmenopausal but it could participate associated with other risk factors. Before might due to the analyzed women were healthy, right now we are studying the association between I/D ACE genotypes and response to tibolona therapy in hypertenses postmenopausal.
Modulation of Amphiregulin Gene Expression by Estrogen in Breast Cancer Cells
Authors: Luvizon Aline Carbonera , De Sibio Maria Teresa , Conde Sandro Jose , Luvizotto Renata Azevedo Melo , Olimpio Regiane Marques Castro , Moretto Fernanda Cristina Fontes , Fecchio Denise , Nogueira Celia Regina
Published: June 2021

This study aimed evaluate whether estrogen modulates amphiregulin (AREG) gene expression in breast tumor cells. For this, MCF‐7 cells were treated for 1 or 4 hours with estrogen (E2 10−7 M) in the absence or presence of estrogen receptor inhibitor (ICI 1μM), cyclohexamide (CHX 10μg/ml), actinomycin D (ACTD 5.0 pg/ml) or LY294002 (50μM). Gene expression was assessed using RT‐qPCR, at 5% significance. Estrogen treatment increased AREG gene expression at both moments evaluated. This effect was partially blocked by ICI in MCF‐7 cells, suggesting that a hormone effect requires estrogen receptor (ER). ACTD treatment does not affect the AREG gene expression, demonstrating the independence of mRNA synthesis in the AREG induction in E2‐treated cells treated. Treating cells with CHX significantly decreased gene expression compared to E2 treatment alone, indicating that protein synthesis is necessary for an estrogen effect. Estrogen induced AREG gene expression is partially blocked by LY, showing that the PI3K pathway is required for a portion of this expression. These data demonstrate that estrogen augments AREG gene expression by ER and by PI3K pathway in MCF‐7 breast cancer cells. This effect is dependent on protein synthesis and independent of mRNA synthesis, indicating extranuclear actions at both times evaluated.

Supported by: Fapesp and CNPq
Initial Experiences with Web‐Based Independent Self‐Study as a Major Component in a Medical Developmental Anatomy Course
Authors: Conway Marianne Leovey , Sinning Allan R
Published: June 2021

Based on evidence in the literature that pedagogical methods utilizing active learning modalities produce more favorable learning outcomes than traditional, teacher‐centered approaches, the traditional lecture‐based developmental anatomy course has been replaced by a first‐of‐its‐kind course at the University of Mississippi Medical Center that introduces independent self‐study as a major component. The new hybrid course is student‐centered, utilizing fifteen web‐based, self‐directed study modules complemented by formative on‐line quizzes, clinical correlation lectures and interactive review sessions that focus on application of basic science concepts to solve clinical problems. The content is closely integrated with the gross anatomy course. Final grades before and after the implementation of the self‐study component were compared and analyzed using a single factor ANOVA design. Results show that student performance has not been adversely affected by replacing much of the traditional lecture hours of the course by the web‐based active learning component. Student perception of the new course format, however, has been disappointing; course evaluations conducted by the Evers Society, a unique, student‐run honor society, indicate that although the web‐based component has been accepted as part of the new course, the desire for a larger, face‐to‐face lecture component remains prominent.
Protein Kinase C Isoforms are Expressed During Intestinal Regeneration in Sea Cucumber Holothuria glaberrima.
Authors: Rosado Edwin Antonio , García José E
Published: June 2021

Molecular and cellular studies on the intestinal regeneration process of the sea cucumber Holothuria glaberrima allows us to understand how cells are rearranged and assembled into a functional organ. Protein kinases c (Pkcs), a superfamily of serine/threonine kinases, have been implicated in a wide range of cellular processes and in the regulation of growth, apoptosis, and differentiation in a variety of cell types. We now report the characterization of at least four Pkc isoforms from regenerating intestine cDNA libraries of H. glaberrima. One of the Pkc isoform showed similarity to the PB1 domain required for the enzymatic activity of atypical Pkcs (identity >; 63%). Another three Pkc isoforms showed the highest similarity to the C1 activation domain of the classical Pkc isoforms (identity >; 46%). Two of the Pkcs, one classical and one atypical isoform, were significantly upregulated in the regenerating intestine from early to middle stages of regeneration (3–14 days post‐evisceration) compared to normal uneviscerated intestine where its expression was almost negligible. The increase in gene expression coincides with drastic dedifferentiation, intense cellular division, and apoptosis events that take place during early to middle stages of regeneration implicating Pkcs as being key modulators of these processes.

Research supported by NIH Grant Number 5T34GM007821–33.