bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the osteo‐inductive domain within the LMP‐3 protein (946.2)
Authors: McMichael Ashley , Zhou Liqiao , Li Xeusen , Robbins Paul
Published: June 2021

LIM Mineralization protein (LMP‐3), a transcript variant form the original LMP protein has proven to be a novel osteogenic molecule when overexpressed, inducing expression of osteogenic factors include BMP‐2, BMP‐6 and Osterix (Osx). Expression of LMP‐3 by gene transfer results in ectopic bone formation in vivo. The goal of this project is to identify the minimal osteogenic domain in LMP‐3, which then could be delivered intracellularly using a protein transduction domain to induce osteogenesis for bone healing applications. To identify the minimal osteogenic domain, we generated a series of deletion constructs in LMP in the context of a LMP‐3‐eGFP fusion. MC3T3 cells were transfected with the different deletion mutants of LMP‐3 and the activation of an Osterix promoter‐luciferase reporter plasmid determined. Fragment F3 initially was shown to contain the osteoinductive domain of LMP‐3. Further deletion analysis of the F3 fragment was then performed. Within the F3 sequence, fragments F4 and F8 activated the OSX promoter. These results suggest that a small domain in LMP‐3 is sufficient to induce expression of osteoinductive genes required for bone formation and healing. Future studies will assess the functionality of these specific fragments in activating other osteogenic genes that lead to bone formation. Moreover, we will assess the ability of F4 and F8 to induce osteogenesis when delivered using a protein transduction domain. These LMP‐3 derived osteogenic peptides could be used to for bone fractures, bone defects, spine fusions, and invertebral disc regeneration.
Carbohydrates downregulates sodium‐dependent neutral amino acid transporter‐2 expression (946.5)
Authors: Ortiz‐Ortega Vicor , Mendez‐Garcia Ana Luisa , Noriega Lilia Guadalupe , Torres Nimbe , Tovar Armando Roberto
Published: June 2021

SNAT has an important physiological role in the provision of amino acids that may enter the gluconeogenic pathway in liver. However, SNAT2 transcriptional regulation by carbohydrates has not been studied. The objective of this study was to determine the acute response to 70% sucrose diet (high sucrose diet, HSD) or 10% sucrose diet (low sucrose diet, LSD) on hepatic SNAT2 expression. Wistar rats fed HSD showed a decrease of hepatic SNAT2 mRNA and protein expression compared with rats fed LSD or fasted rats. Also, we found a decrease SNAT2 mRNA expression in rat hepatocytes primary culture or HepG2 cells incubated with 25 mM versus 5 mM glucose. SNAT2 promoter mapping showed a putative carbohydrate response element (ChoRE). A construct containing this site was ligated in a luciferase vector for functional studies in HepG2 cell line. Promoter activity was decreased when transfection was performed with 25 mM versus 5 mM glucose. Specific protein‐DNA complexes were detected by EMSA indicating that the carbohydrate response element‐binding protein (ChREBP) binds to ChoRE in the SNAT2 promoter. These results showed that sucrose‐fed rats decrease hepatic SNAT2 expression through ChREBP.

Grant Funding Source: Supported by CONACyT 183037
Modeling ToxT to explain how cholera toxicity can be regulated by fatty acids: The 2014 Pingry SMART Team project (946.7)
Authors: Abbott Kathryn , Chow Kevin , Hong Brad , Kwon Emily , Newman Matthew , Robinson Jacob , Sander Mariel , Sander Mariel , Wu Rachel , Kull F. , D'Ausilio Morgan
Published: June 2021

Infections of the enteric bacterium Vibrio cholerae cause the disease cholera and lead to severe dehydration and even death to those who ingest water contaminated with the organism. Two virulence factors are required for infection: the toxin‐corregulated pilus (TCP) and cholera toxin (CT). The production of both gene products is regulated by the transcription factor ToxT, a member of the AraC protein family. The structure of ToxT was solved by X‐ray crystallography and revealed a 16‐carbon fatty acid, cis‐palmitoleate, in the ligand binding pocket of the protein’s regulatory domain. Further analysis indicated that expression of both TCP and CT were reduced in the presence of cis‐palmitoleate and that binding of the fatty acid to ToxT prevented DNA binding and led to a reduction in virulence factor expression. Using the structure determined in this study, The Pingry School SMART (Students Modeling A Research Topic) Team, is using a 3D‐printer from the Milwaukee School of Engineering (MSOE) to model the interactions between ToxT and cis‐palmitoleate. Ligand binding by the regulatory domain of the ToxT leads to a change in conformation of the DNA‐binding domain, resulting in decreased interaction with DNA. Fatty acid regulation of ToxT‐DNA binding provides insight into the mechanism of controlling virulence gene expression in V. cholerae. Studies based on this research are currently investigating the structure and function of other AraC‐family members from a variety of infectious bacteria, seeking to determine if fatty acids may also be involved in regulation of virulence factor expression in other organisms.

Grant Funding Source: NIH‐CTSA
Biochemical and biophysical insights into the function of the bHLH transcription factor TWIST2 (946.8)
Authors: Orengo‐Mercado Carmelo , Casasnovas Jose , Cadilla Carmen
Published: June 2021

TWIST2 is a transcription factor of the basic Helix Loop Helix family, which binds DNA as homo or heterodimer to consensus sequences called E ‐ boxes. Mutations in TWIST2 cause a rare recessive disorder called Setleis Syndrome. To this date, no structural or functional assays have been published using recombinant TWIST2. First, we performed a bioinformatics analysis ranging from multiple sequence alignments to key structural features. These led us to perform a 3D structural prediction of TWIST2 using the Phyre2 server. In order to produce recombinant His tagged TWIST2, the TWIST2 cDNA was cloned into the pQET7 vector and expressed in BL21 pLysS cells. The protein was purified under native conditions by a two‐step purification via Ni‐NTA Affinity followed by Ion Exchange Chromatography. After purification, a band of the expected size of TWIST2 was observed in SDS‐PAGE. Western Blot analysis and Mass Spectrometry validated this finding. TWIST2 functionality was demonstrated by Electrophoretic Mobility Shift Assay (EMSA) with a known E‐box probe. Obtaining recombinant TWIST2 protein will allow future structural and functional assays. These assays will expand the knowledge of this transcription factor and its interactions with DNA to regulate its target genes.

Grant Funding Source: Supported by NIH grants (2G12‐RR003051), (8G12‐MD007600) and (R25GM061838)
Isoaspartate accumulation in creatine kinase B in vivo and in vitro is associated with loss of enzymatic activity (949.1)
Authors: Dimitrijevic Aleksandra , Qin Zhenxia , Aswad Dana
Published: June 2021

Formation of abnormal isoaspartyl (isoAsp) residues occurs frequently in proteins and peptides under physiological conditions, often resulting in the alteration of their structure and function. IsoAsp residues can be efficiently repaired by the action of protein L‐isoaspartyl O‐methyltransferase (PIMT), a cellular repair enzyme that catalyzes the conversion of isoAsp to normal Asp residues. The brain‐enriched isoform of creatine kinase (CK‐B) plays a key role in intracellular energy metabolism and is highly susceptible to isoAsp formation. We investigated the effect of isoAsp formation on CK‐B activity, both in vivo in PIMT‐deficient mice, and in vitro using aged recombinant CK‐B. Accumulation of isoAsp residues in vivo was associated with a significant decrease in CK‐B activity. In vitro aging of recombinant human CK‐B also resulted in isoAsp formation and loss of CK‐B activity. IsoAsp sites in aged, recombinant CK‐B were efficiently repaired in vitro by the action of PIMT. These results (1) corroborate previous finding that CK‐B is a major target of PIMT action in vivo, (2) demonstrate that isoAsp accumulation in CK‐B is associated with significant loss of function, and (3) suggest that loss of CK‐B function in vivo may contribute to the neuropathology and susceptibility to epileptic seizures that are characteristic of PIMT‐deficient mice.

Grant Funding Source: Supported by NIH grant NS‐17269
The N‐terminal region of the Trypanosoma brucei Tim17 is critical for mitochondrial protein import (950.3)
Authors: Weems Ebony
Published: June 2021

The protein translocases of the mitochondrial inner membrane in higher eukaryotes, TIM23‐17 and TIM22‐54, possess three homologous proteins, Tim17, Tim23, and Tim22. In contrast, Trypanosoma brucei, a parasitic protozoon that causes African trypanosomiasis, possesses a single homologue of this protein family, TbTim17. TbTim17 is a part of a large protein complex consisting of proteins unique to trypanosomes. In spite of a significant similarity in the secondary structure of TbTim17 with the Tim17/23/22 family proteins, we report here that the growth defect caused by TbTim17 knockdown in T. brucei could not be complemented by the expression of either Tim17, or Tim23, or Tim22 from Saccharomyces cerevisiae and vice versa, suggesting TbTim17 is significantly divergent in order to full‐fill the species‐specific functions. Bioinformatics analysis revealed that TbTim17 N‐terminus possesses a longer stretch of hydrophilic amino acids at this domain (~30 amino acids) relative to Tim17 proteins expressed in other eukaryotes. In order to investigate the function of this region we expressed a number of N‐terminal deletion mutants of TbTim17 in T. brucei, where endogenous TbTim17 was knockdown by RNA interference targeted to the 3’‐untranslated region of its transcript. We found that the growth defect caused by TbTim17 RNAi was completely rescued by ectopic expression of the full‐length TbTim17 (FL‐TbTim17) but not by the N‐terminal deletion mutants, 20TbTim17, and 30TbTim17. All of these mutants are expressed and properly targeted to mitochondrial membrane in T. brucei. However, the import of nucleus‐encoded mitochondrial proteins into the mitochondrion is significantly inhibited in the N‐terminal deletion mutants. Suggesting a critical role for this region in the import of mitochondrial proteins in T. brucei.

Grant Funding Source: 2SC1GM081146, 5T32HL007737, 5T32AI007281, and 2R25GM059994
Carbonates are a new class of deubiquitinating enzyme inhibitors (952.5)
Authors: Long Marcus , Lawson Ann , Rozhansky Lior , Hedstrom Lizbeth
Published: June 2021

Deubiquitinating enzymes (DUBs) have emerged as promising drug targets, but few small molecule DUB inhibitors have been identified. Herein we introduce carbonates as a novel class of mechanism‐based DUB inhibitors. Activity profiling was used to determine inhibitor potency and specificity in cell lysates. The most potent inhibitor, C17, has a preference for USP9x and USP7. In K562 cells, C17 (50 micromolar) causes the degradation of Bcr‐Abl kinase, as expected when USP9x is inhibited. In MCF7 cells, C17 (25 micromolar) causes the degradation of Mdm2 and upregulation of P53, as expected when USP7 is inhibited. We believe that carbonates will be useful tools for studying the ubiquitination pathways and provide a new strategy for the design of cysteine protease inhibitors.

Grant Funding Source: Supported by the NIH R01 GM100921(LH), HHMI International Student Fellowship (MJCL).
Characterization of the mitochondrial function and genetics in the pathogenesis of collagen VI related disorders (958.1)
Authors: Angelin Alessia , Lin Chun Shi , Sharpley Mark , Derbeneva Olga , Potluri Prasanth , Bonaldo Paolo , Bernardi Paolo , Wallace Douglas
Published: June 2021

Background: Alteration in the collagen VI protein causes Bethlem Myopathy and Ullrich Congenital Muscular Dystrophy. Mitochondrial dysfunction, ultrastructural defects, increased apoptosis and autophagy impairment are associated to muscle of young Col6a1‐/‐ mice and patients.

Results: To clarify the pathophysiology of this disorder we are characterizing Col6a1‐/‐ mice at different ages. Preliminary studies show reduced cell growth, decreased respiratory capacity, increased ROS production and increased mitochondrial mass on myoblasts isolated from old Col6a1‐/‐ mice. Mutant cells show altered cytoskeleton structure that could explain why dysfunctional mitochondria accumulate and are not removed by autophagy. We are also determining if the progression of this myopathy is related to the accumulation of mtDNA mutations.

Methods: We study: cell growth capacity, mitochondrial membrane potential and mass, oxygen consumption capacity, ROS production, mitochondrial dynamics and cytoskeleton structure in primary muscle cells; and skeletal muscle tissue properties with histological methods. To assess mtDNA mutations levels, we had developed a next generation sequencing protocol that avoids any PCR step in preparing the DNA prior to sequencing. This innovative approach is now being applied to various tissues, with particular reference to the diaphragm the most affected muscle.
Simultaneous analysis of urinary fructose and sucrose by isotope dilution tandem LCMS (959.14)
Authors: Franke Adrian , Custer Laurie , Li Xingnan
Published: June 2021

Objective: Sugar intake has increased vastly in the US diet during past decades and has been associated with obesity, metabolic syndrome, type 2 diabetes, cardiovascular disease, and cancer. Measuring sugar intake by FFQ is not reliable and urinary fructose and sucrose excretion as a biomarker is recommended but existing methods for their analysis are complex, expensive or unreliable (GCMS, enzymatic kits, doubly labeled water). A simple liquid chromatography mass spectrometry (LCMS) method was therefore established for urinary fructose and sucrose.

Methods: This study evaluated many extraction, chemical derivatization and electrospray ionization (ESI) tandem MS variations in positive and negative mode using isotope dilution and reversed, normal or reversed reversed phase LC protocols.

Results: While sucrose was measurable reliably by many LCMS protocols myriads of interferences with fructose measurements could be avoided by derivatization of fructose to its dansyl hydrazone or by selective application of organic solvents during urine extraction.

Conclusions: A reliable, simple, fast and inexpensive isotope dilution tandem ESI LCMS assay for fructose and sucrose was established requiring 20 uL or less of urine.

Grant Funding Source: This research was supported by National Cancer Institute grants P30 CA71789.
Investigating effects of consuming different protein and/or oil sources on progression and severity of polycystic liver disease in young female PCK rats (959.9)
Authors: Nanda Nainika , Maditz Kaitlin , Oldaker Chris , Livengood Ryan , Benedito Vagner , Tou Janet
Published: June 2021

Polycystic Liver Disease (PLD) is characterized by multiple cyst formation increasing liver size, structural damage, and loss of function. There are no effective cures for PLD, but diet may be a potential therapy. The objective of this study was to investigate whether soy protein isolate (SPI) and/or dietary omega‐3 polyunsaturated fatty acid (n‐3 PUFA) supplementation attenuates PLD progression and severity. Young (age 28 d) female PCK rats, a genetic model of PLD, were fed diets (n=12/group) consisting of casein + corn oil (Casein + CO), casein + soybean oil (Casein + SO), SPI + soybean oil (SPI + SO), or SPI + 1:1 soybean/salmon oil blend (SPI + SB) for 12‐weeks. Liver weights were not significantly different among the diet treatment groups. Histological evaluation showed no significant differences in cyst effacement and liver fibrosis between diet groups. Rats fed SPI+SB diet had the highest (P=0.03) cyst obstruction and interstitial inflammation. Gene expression by real time quantitative polymerase chain reaction (RT‐qPCR) showed no significant differences (P>0.05) in fibrosis and inflammation factors: peroxisome proliferator‐activated receptor gamma (PPARγ), mammalian target of rapamycin (mTOR), mitogen‐activated protein kinase (MAPK1), transforming growth factor beta (TGFβ), and epidermal growth factor (EGF). Furthermore, serum liver function measurements showed no significant differences among diet groups. Based on the results, dietary supplementation of SPI and n‐3 PUFA had no effect on progression or severity of PLD.
Effects of avocado oil in brain mitochondrial ETC function and ROS generation in diabetic rats (960.5)
Authors: Ortiz‐Avila Omar , Hernandez‐De La Paz Lucio , Canizal‐Garcia Melina , Montoya‐Perez Rocio , Saavedra‐Molina Alfredo , Cortes‐Rojo Christian
Published: June 2021

Diabetes mellitus is associated with functional and structural changes in the brain leading to neurodegeneration, with oxidative stress playing a role in the development of diabetic brain complications. In some cases, it has been reported that impaired function of the electron transport chain (ETC) may be involved in the development of diabetic encephalopathy and oxidative stress. Recently, we have reported that avocado oil supplementation has beneficial effects in kidney mitochondria during diabetes by increasing the resistance of the ETC to oxidative stress and decreasing ROS production in the complex III. Thus, the main goals of this work were to identify alterations in ETC function of brain mitochondria leading to ROS production in rats with 90‐days of STZ‐induced diabetes and the evaluation of the effects of avocado oil intake in these alterations. No alterations were found in the activity of any of the ETC complexes of diabetic rats. Avocado oil supplementation increases 1.2‐fold the activity of the complex III from diabetic rats and 3.5 times the activity of the complex II of normoglycemic rats. Despite no net changes in ROS generation were detected, a decrease in this parameter was identified in diabetic rats when ROS generation was normalized against complex III activity, with avocado oil further decreasing ROS production in the same group. These data suggest that 90 days of diabetes do not exert important changes in mitochondrial function of brain. However, early avocado supplementation may prevent further development of mitochondrial dysfunction in late stages of diabetes by improving the electron flow in the complex III and decreasing ROS generation.

Grant Funding Source: Suuported by CONACYT (130638 to CCR) and CIC‐UMSNH (16.12 to CCR) grants
Mitochondrial proteomes of Drosophila melanogaster adapted to chronic hypoxic environment (960.7)
Authors: Xue Jin , Yin Songyue , Perkins Guy , Ellisman Mark , Haddad Gabriel , Liu Siqi , Zhou Dan
Published: June 2021

To investigate the mitochondrial mechanisms underlying adaptation to hypoxic conditions, we quantitatively analyzed the protein abundance of the mitochondrial proteomes of Drosophila strains that tolerate extremely low O2 environments using iTRAQ. A total of 55 mitochondrial proteins displayed significant differences in abundance. The majority of the differentially expressed proteins associated with calcium regulation, detoxification, glycolysis, chaperon, antioxidant and translational regulation exhibited reduced abundances. And the differentially expressed proteins involved in respiration or associated with the mitochondrial membrane displayed increased abundances. Functional analyses suggested that the crosstalk between the mitochondrial, calcium and Notch signaling pathways may play important roles in regulating hypoxia tolerance. We believe that these findings will not only enhance our understanding of the mechanisms underlying adaptation to low oxygen conditions in Drosophila but also provide a clue in studying human disease induced by altered oxygen tension in tissues and cells.
Urzymology: experimental access to the origins of catalytic activity and translation (967.8)
Authors: Carter Charles , Weinreb Violetta , Li Li , Collier Martha , Chandrasekaran Srinivas , Fried Howard
Published: June 2021

Urzymology is the enzymological characterization of invariant cores, containing ~100 ± 40 amino acids, identified in enzyme superfamilies (1,2). Urzymes prepared from Class I and Class II aminoacyl‐tRNA synthetases (aaRS) accelerate cognate amino acid activation and tRNA acylation more than 105 times faster than necessary to support ribosome‐independent assembly of polypeptides and are therefore themselves highly evolved (3). Their high catalytic activities afford an experimental basis for combinatorial analysis of the effects of modular enhancements thought to have been involved in generating modern enzymes (4).

Among the central questions now accessible via the study of Urzymes are the likely origin of Class I and II aaRS on opposite strands of the same ancestral gene (5); the origin of intramolecular communication (6,7); the modular construction of the Urzymes themselves, and hence their descent from even more primitive catalysts; and the suggestion that an operational RNA code preceded development of the canonical genetic code.

Grant Funding Source: NIGMS 78227 and 40906
Characterizing the spectral properties of (‐)‐epigallocatechin 3‐O‐gallate fluorescence in solvents of different polarity (973.5)
Authors: Young Michael , Petroff Elena , Snitsarev Vladislav
Published: June 2021

(‐)‐Epigallocatechin 3‐O‐gallate (EGCG) is the most common catechin found in green tea and is known to have a plethora of bioactive properties. Recently, EGCG has been identified as a novel inhibitor of heat shock protein 90 (HSP90)[1], a cytoplasmic chaperone protein, which has received much attention as a potential drug target in cancer treatment[2,3]. Characterizing the spectral properties of EGCG fluorescence in environments similar to those of protein binding pockets provides an important tool to facilitate the study of protein‐EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in aqueous buffer (AB) at pH 7.0, acetonitrile (AN) (polar aprotic), dimethylsulfoxide (DMSO) (polar aprotic), and ethanol (EtOH) (polar protic). We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima ranging from 350 to 400 nm in a solvent‐dependent manner. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI) of EGCG is significantly quenched in AB at pH 7.0, and that it is about 85 times higher in the aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert‐Mataga equation, its fluorescence in AN points to non‐specific solvent effects on EGCG fluorescence. We conclude that significant solvent‐dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.
Identification of an antifungal peptide from hemolymph of an insect, Manduca sexta (976.4)
Authors: Alsouhail Qasim , Hiromasa Yasuaki , Kanost Michael
Published: June 2021

Insects secrete antimicrobial peptides as part of the innate immunity response. These peptides are often cationic, low molecular peptides with diverse structures. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized a newly identified peptide, antifungal peptide‐1 (AFP1) from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). AFP1 was isolated by size exclusion chromatography from hemolymph plasma of larvae. Fractions containing activity against the yeast, Saccharomyces cerevisiae were analyzed my SDA‐PAGE and MALDI‐TOF MS/MS and found to contain a 41 residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome data bases. AFP1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to AFP1, each with 6 conserved Cys residues. A cDNA for AFP1 was cloned from cDNA prepared from fat body RNA. AFP1 was produced as a recombinant protein in Escherichia coli. Purified natural and recombinant AFP1 were active against S. cerevisiae, with IC50 of 15 micromolar, but they had no detectable activity against bacteria. Experiments to investigate activity of AFP1 against other fungi are ongoing. AFP1 mRNA level strongly increased after larvae were injected with yeast or with Micrococcus luteus. Our results indicate that synthesis of AFP1 is part of the innate immune response to infection in M. sexta.

Grant Funding Source: Supported by NIH grant GM41247.
Microfludic separation and electrochemical detection of neurotransmitters using a portable Lab‐on‐a‐Chip device (985.1)
Authors: Maughan Nichole , Gamagedara Sanjeewa
Published: June 2021

The objectives of this study are to develop a new microchip electrophoresis method to simultaneously separate and detect serotonin and epinephrine using a microfluidic device in a given sample and to further evaluate and validate the methodology developed using limit of detection, limit of quantitation, linearity, recoveries at low, middle, high concentration levels, and robustness. Microchip electrophoresis uses capillary electrophoresis to separate the analytes and electrochemical detection to detect them. It is considered to be the stride towards a “true” Lab‐on‐a‐Chip (LOC), which integrates all the steps of an analytical process (separation and detection) on a single compact microfluidic device. These devices offer several advancements such as high speed, great versatility, low cost, performance of parallel assays, portability for on‐site detection, and negligible consumption of reagents/sample and waste generation. Electrochemical detection in conjunction with these capillary electrophoresis microchips has been proven to be effective due to characteristics such as inherent miniaturization, sensitivity, low cost, portability, short measurement time, and compatibility with other microfabrication technology. The precision and sensitivity of the proposed microfluidic platform is comparable to previous reports using other conventional methods based on capillary electrophoresis and high performance liquid chromatography. Detection limits cited with these devices have typically been in the nM to μM range.
Modeling the Alcohol Binding Site of the NMDA Receptor Using the GluA2‐receptor Structure (LB110)
Authors: Ali Moid , Gundamraj Shivani , Kaur Tejas , Morris Sarah , Puri Simar , Singh Ricky , Singh Vipul , Smith‐Feinburg Lee , Wang Leah , Tuinstra Robbyn , Peoples Robert
Published: June 2021

According to the National Institute of Alcohol Abuse and Alcoholism, about 18 million people have an alcohol abuse disorder. Alcohol binds to the N‐Methyl‐D‐aspartate Receptor (NMDA) receptor, inhibiting cognition, short‐term memory formation, motor coordination, and overall regular CNS function. The Brookfield Academy SMART (Students Modeling A Research Topic) Team used 3D printing technology to model the alcohol binding site on the NMDA receptor. This receptor is an ion channel in CNS neurons. Binding of the neurotransmitter, glutamate, allows the passage of calcium and sodium ions through the channel, thus controlling multiple intracellular signaling pathways. Alcohol inhibits the gating of the receptor, preventing the flow of ions, leading to the symptoms of intoxication. The NMDA receptor is a heterotetramer, containing two GluN1 and two GluN2A subunits. Alcohol binds to the transmembrane domain of the receptor, interacting with the amino acids Gly638, Phe639, Phe639, Leu819, and Met818 (of subunit GluN1) and Met 823, Phe636, Leu824 and Phe637 (on GluN2A). Site‐directed mutagenesis studies have identified the importance of these residues. Mutations in the same position on different subunits can drastically modulate the inhibition of the receptor by alcohol. Further understanding of the NMDA receptor mechanisms could lead to treatment for long‐term alcohol abuse.

Grant Funding Source: The SMART team program is supported by a grant from NIH‐CTSA.
Bioactive lipid metabolism by carboxylesterase 1 (CES1) in macrophages (LB154)
Authors: Mangum Lee , Borazjani Abdolsamad , Crow John , Ross Matthew
Published: June 2021

Human carboxylesterases (CES) have an emerging role in lipid metabolism. CES1 in macrophages is a hydrolase with broad endogenous substrate specificity, including cholesteryl ester, triacylglycerol, and 2‐arachidonoylglycerol (2‐AG), which may be relevant to cardiovascular disease and other pathologies related to lipid metabolism dysfunction. 2‐AG is an endogenous ligand of the cannabinoid receptors (CB1 and CB2) which are involved in the regulation of numerous processes, including lipogenesis, inflammation, and cholesterol flux in macrophages. To characterize the cholesterol homeostatic functions of CES1, its expression was stably knocked‐down in a THP‐1 monocyte/macrophage line (CES1KD/THP‐1) and the extent of in situ 2‐AG catabolism and cholesterol uptake and efflux from foam cells was evaluated. Expression levels of several genes involved in cholesterol and 2‐AG homeostasis were also determined. Perturbation of CES1 function altered in situ 2‐AG levels as well as the expression of multiple genes involved in cholesterol uptake and metabolism through both compensatory upregulation (CES3) and downregulation (ABCA1, CYP27A1, CD36, SRA) mechanisms. CES1KD/THP‐1 foam cells exhibited altered free and esterified cholesterol mass compared to control cells, whereas cholesterol efflux was unchanged. These finding suggest a complex central role for CES1 in macrophage cholesterol homeostasis.

Grant Funding Source: Supported by NIH 1R15ES015348‐02
Photoprotective effects of reinforced galactooligosaccharides supplementation against skin damage in hairless mice (LB357)
Authors: Hong Ki‐Bae , Jeong Min Geum , Kim Jae Hwan , Park Yooheon , Suh Hyung Joo
Published: June 2021

Ultraviolet B (UVB) radiation has been proposed as a main environmental stressor of skin damage because of its mutagenic and immunosuppressive effects as well the generation of reactive oxygen species (ROS). Photoprotective effects of probiotics have a potential to prevent UV‐induced skin damage and have drawn much attention recently. Galactooligosaccharides (GOS) are an important dietary prebiotics and generally defined as non‐digestible food ingredients that promote the function or viability of probiotics via their fermentation, but the effects of reinforced GOS between the immunomodulatory and anti‐inflammatory activities are still unclear. The aim of the present study was to investigate whether the oral administration of high content GOS by using enzyme reaction process to modulate the growth of beneficial bacteria and the immune‐suppressive effect of UVB and skin wrinkle development in male hairless mice. The oral administration of GOS was effective to alleviate skin damage by reducing the expression of primary matrix degrading enzyme as compared to control group, and modulate the water holing capacity and trans‐epidermal water loss. These result supports that a potential protective effect of high content GOS on skin damage induced by UVB exposure, and it may be mediated by the inhibition of ROS and modulation of MMP expression.

Grant Funding Source: Supported by Leading Industry Development for Economic Region, MOTIE, Republic of Korea
Trans fatty acids content of vegetable oil spreads and margarines in the USA (LB385)
Authors: Balentine Douglas , Garsetti Marcella
Published: June 2021

Historically vegetable oils spreads and margarines (“spreads”) contained significant amounts of trans fatty acids (TFAs) from partially hydrogenated vegetable oils (PHVOs). Our goal was to survey the US marketplace and determine the amount of TFAs and the use of PHVOs in spreads.

We sampled 43 spreads in 2011 and 46 spreads in 2013 and we measured TFAs by capillary gas chromatography. We searched for PHVOs in the ingredient declaration.

From 2011 to 2013, the mean TFAs content of spreads per 14g serving decreased from 0.51 g to 0.30 g (p=0.11). As a comparison, it was 1.7 g per 14 g serving in 2002 [1]. TFAs per 14 g serving decreased both in soft spreads (from 0.29 g to 0.13 g; p<0.05) and in stick products (from 1.62 g to 0.98 g; p=0.16). In 2013, 6 out of 11 manufacturers were no longer using PHVOs. Out of the 9 spreads found with PHVOs in 2013, 6 were private label and 4 were stick products.

Compared to 2002, the average TFAs content of spreads was markedly reduced by 70% by 2011 and 82% by 2013 to an average of 0.30g per 14g serving. Most manufactures of branded products, notably Unilever and GAF, provide products without PHVOs. Some private label spreads and some stick products still contain significant amounts of PHVOs. At the end of 2013, 80% of the national volume of branded spreads was free of industrial trans fats. [1] Satchithanandam et al. Lipids 2004;39(1):11‐18.

Grant Funding Source: Support for the analytical work was provided by Unilever
Enhanced production of ornithine in rice bran fermentation culture by using lactic acid bacteria isolated from the rice bran (LB398)
Authors: Oh Suk‐Heung , Kim Su‐Gon , Kim Eun‐Sun , Kim Ji‐Sun , Yu Ji‐Hyun , Kwon Mi‐Ra , Lee Jo‐Seph , Park Ki‐Bum
Published: June 2021

The worldwide production of rice bran is above 27.3 million tons per year. Rice bran contains various nutrients such as protein, fat, carbohydrate, cellulose, minerals, and vitamins, etc. making it a very useful food source. Lactic acid bacteria (LAB) involved in the fermentation of plant materials attracted many researchers because of its expected ability to ferment rice bran, barley bran etc. In this study, LAB strains isolated from rice bran powder were screened to determine the strains with ornithine‐producing capacity and to use such LAB for the preparation of products based on fermented rice bran. One strain, RBO1‐49 identified as Pediococcus sp. RBO1‐49 fermented D‐ribose, D‐glucose, D‐galactose, D‐fructose, D‐mannose, L‐rhamnose, L‐arabinose, D‐xylose but not D‐arabinose, L‐xylose, inositol and L‐sorbose. Culturing the Pediococcus sp. RBO1‐49 in 20%(w/v) rice bran solution (pH 6.5) containing 1%(w/v) arginine at 30℃ for 3 days by static culture method increased ornithine content in the culture up to 950mg/100g. These results suggest that rice bran has specific LAB for the production of useful amino acids such as ornithine and the RBO1‐49, which was isolated from rice bran, can be used for the production of even higher ornithine containing food materials including fermented rice bran and feeds. This research was supported by High Value‐added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs.
Characterizing and diminishing autofluorescence in formalin‐fixed paraffin‐embedded human respiratory tissue (LB482)
Authors: Davis Anne , Richter Anke , Becker Steven , Moyer Jenna , Sandouk Aline , Skinner Jeff , Taubenberger Jeffery
Published: June 2021

Tissue autofluorescence frequently hampers visualization of immunofluorescent markers in formalin‐fixed paraffin‐embedded respiratory tissues. We assessed nine treatments reported to have efficacy in reducing autofluorescence in other tissue types. The three most efficacious were eriochrome black T, Sudan black B and sodium borohydride. We then further characterized these changes in autofluorescence using white light laser confocal multi‐lambda analysis. Additionally, we assessed the impact of steam antigen retrieval and serum application on human tracheal tissue autofluorescence. Functionally fitting this multi‐lambda data to 2‐dimensional Gaussian surfaces revealed that steam antigen retrieval and serum application contribute minimally to autofluorescence and that the three treatments are disparately efficacious. Together these studies provide a set of guidelines for diminishing autofluorescence in formalin‐fixed paraffin‐embedded human respiratory tissue. Additionally, the characterization techniques are transferable to similar questions in other tissue types as demonstrated on frozen human liver tissue and paraffin embedded mouse lung fixed in different fixatives.
Identifying determinants of conformational stability in GTPases (LB607)
Authors: Sridharan Vishwajith , Isom Daniel , Dohlman Henrik
Published: June 2021

Cellular functionalities are contingent upon the ability of proteins to maintain or transform their shape (conformation). The molecular forces that govern protein folding have long been known. These include hydrophobic interactions, hydrogen bonding, and disulfide bonds. However, how proteins maintain their conformation remains largely unanswered. In this study, twenty G proteins from Saccharomyces cerevisiae were cloned, purified from E. coli, and tested for conformational stability. The proteins vary in Tm from 35 to 60°C. This is paradoxical considering their highly analogous crystal structures and identical catalytic activity. We find that thermal stability correlates with percentage of Cys, Met, or Pro residues in the primary sequence. We also find that the stabilities group by their respective family members, with the following trend observed for least to most thermostable G proteins: Rho/Cdc42 < Ran < Large G‐proteins < Ras < Rab < Sar/Arf. Both these results suggest that thermal stability has a greater dependence on primary sequence than tertiary structure. Ultimately, a more detailed understanding of protein stability can help in engineering enzymes for new molecular therapeutics.

Grant Funding Source: Supported by National Science Foundation REU Site Award 1156840 to UNC‐Chapel Hill
Normoglycemia sensitizes MDA‐MB‐231 breast cancer cells to metformin through an AMPK‐dependent mechanism (LB610)
Authors: Zordoky Beshay , Bark Diana , Soltys Carrie , Sung Miranda , Dyck Jason
Published: June 2021

Background: Metformin has been shown to have strong anti‐proliferative effect in many breast cancer cell lines, mainly due to the activation of the energy sensing kinase, AMP‐activated protein kinase (AMPK). MDA‐MB‐231 cells are aggressive and invasive breast cancer cells that are known to be resistant to several anti‐cancer agents as well as to the anti‐proliferative effect of metformin. As metformin is a glucose lowering drug, we hypothesized that normoglycemia will sensitize MDA‐MB‐231 cells to the anti‐proliferative effect of metformin.

Methods: MDA‐MB‐231 cells were treated with increasing metformin concentrations in hyperglycemic or normoglycemic conditions. The growth inhibitory effect of metformin was assessed by MTT assay. The expression of several proteins involved in cell proliferation was measured by western blotting.

Results: In agreement with previous studies, treatment with metformin did not inhibit the growth of MDA‐MB‐231 cells cultured in hyperglycemic conditions. However, metformin significantly inhibited MDA‐MB‐231 growth when the cells were cultured in normoglycemic conditions. In addition, we show that metformin‐treatment of MDA‐MB‐231 cells cultured in normoglycemic conditions and not hyperglycemic conditions caused a striking activation of AMPK, and an AMPK‐dependent inhibition of multiple molecular signaling pathways known to control protein synthesis and cell proliferation.

Conclusion: Our data show that normoglycemia sensitizes the triple negative MDA‐MB‐231 breast cancer cells to the anti‐proliferative effect of metformin through an AMPK‐dependent mechanism. These findings suggest that tight normoglycemic control may enhance the anti‐proliferative effect of metformin in diabetic cancer patients.

Grant Funding Source: Supported by Canadian Institutes of Health Research and Alberta Innovates ‐ Health Solutions
Neonatal and adult cardiomyocytes differ in their susceptibility to the proapoptotic protein bnip3 (LB657)
Authors: Lee Youngil , Lee Hwa‐youn , Gustaffson Asa
Published: June 2021

Bnip3 is a BH3‐only protein that is upregulated during hypoxic condition and is known to cause cell death in many different cell types. Studies have found that overexpression of Bnip3 in neonatal cardiac myocytes (NCM) induces cell death in the absence of a hypoxic insult. Here, we compared the sensitivity of NCM and adult cardiac myocytes (ACM) to Bnip3‐mediated cell death under a normoxic condition. Freshly isolated NCM and ACM were infected with an adenovirus encoding either β‐galactosidase (β‐gal) or Bnip3 for 24 hours. TMRM and Yo‐Pro1 were used to detect the changes in mitochondrial membrane potential and apoptotic cell death, respectively. Interestingly, we found that overexpression of Bnip3 failed to induce significant cell death in ACM, whereas significant cell death was observed in NCM. Ultrastuctural analysis of ACM overexpressing Bnip3 revealed normal mitochondrial architecture. Instead, Bnip3 overexpression appeared to selectively activate mitochondrial autophagy under normoxic conditions. Moreover, overexpression of Bnip3 significantly augmented oxidative stress‐mediated cell death in ACM, suggesting that Bnip3 required the presence of additional stress to activate cell death in ACM. Bnip3 is known to induce ROS production and to investigate the mechanism underlying the difference in susceptibility to Bnip3‐mediated cell death between ACM and NCM, we examined levels of the antioxidant, manganese superoxide dismutase (MnSOD) in these cells. We found that MnSOD expression was significantly higher in ACM compared to NCM. Also, susceptibility to Bnip3‐mediated cell death was restored in ACM in the presence of 2‐ME (100μM), a pharmacological MnSOD inhibitor. Taken together, these results suggest that ACM are resistant to Bnip3 due to its enhanced antioxidant capacity and that additional stress is required to switch from Bnip3’s pro‐autophagy to pro‐death function. Supported by NIH R01 HL087023

Grant Funding Source: NIH R01 HL087023
A novel method to detect in vivo oxidant production reveals increased susceptibility to high‐fat diet induced penile oxidative stress in GSNOR knockout mice (LB688)
Authors: La Favor Justin , Burnett Arthur
Published: June 2021

Excessive production of reactive oxygen species (ROS) has consistently been described as a cause of erectile dysfunction. However, direct measurement of ROS in the penis has remained elusive due to the short half life of ROS and limited accessibility to appropriate tissue beds in humans or animals. Binding of nitric oxide to a cysteine thiol is termed S‐nitrosylation, which can mediate function of many proteins. S‐nitrosylated glutathione reductase (GSNOR) is a major mediator of de‐nitrosylation, which can impact reactive oxygen/nitrogen homeostasis. The objective of this study was to optimize a new technique to measure in vivo ROS production in the mouse penis, and to investigate the effect of a high‐fat diet on penile ROS production in wild type (WT) and GSNOR knockout (KO) mice. WT and GSNOR KO mice were fed a standard chow or high‐fat diet for 3 or 6 weeks (n=3/group). A linear microdialysis probe (3mm membrane, 20kDa cutoff) was inserted into the shaft of the penis of mice under ketamine/xylazine anesthesia, allowing sampling of extracellular fluid. To measure extracellular ROS, probes were perfused with saline containing 100 µM Amplex Ultrared and 1 U/ml horseradish peroxidase. Dialysate fluorescence was measured at the outlet with a Turner Designs (TD‐700) fluorometer at 510Ex/590Em. Mean hydrogen peroxide (H2O2) concentrations were: WT (chow = 0.664, 3wk HFD = 0.666, 6wk HFD = 1.202 µM), GSNOR KO (chow = 0.735, 3wk HFD = 1.349, 6wk = 1.389 µM). Following 3 weeks of HFD, mean penile H2O2 was increased 103% in GSNOR KO vs. WT mice. It can be concluded that lack of GSNOR accelerates the development of oxidative stress in the penis in response to a high‐fat diet.

Grant Funding Source: Supported by a trainee grant from the Sexual Medicine Society of North America
Virtual Care in an Outpatient Subspecialty Substance Use Disorder Treatment Program
Authors: Levy Sharon , Deister Diana , Fantegrossi John , Green Leslie , Lunstead Julie , Martinez Tamara , Riccardelli William , Schizer Miriam , Schram Jesse , Schram Patricia , Weitzman Elissa
Published: June 2021

Objectives:

This article reports on the experience of an outpatient adolescent substance use disorder treatment program transitioning to virtual care during the COVID-19 pandemic. We describe the processes used to make the transition, including development of a safety protocol for patients seen virtually, present clinical volume data before and after the transition, and we describe a range of patient experiences through 3 clinical vignettes.

Methods:

Using data from the electronic health record, we generated counts of the total number of scheduled and completed appointments between December 2019 and June 2020. We used simple proportions to calculate the completed visit rate.

Results:

Both the absolute number of scheduled appointment and the percent of appointments completed increased with the initiation of virtual care, supporting the acceptability of this modality. Several patients experienced clinical improvements in conjunction with greater engagement, though challenges were also noted.

Conclusions:

Virtual visits are a practical mode of treatment for adolescent substance use disorders, and more evidence is needed to understand the risks and benefits of this treatment modality.
The Use of Magnesium Sulfate and Peripheral Nerve Blocks
Authors: Zeng Jie , Chen Qi , Yu Cong , Zhou Ju , Yang Bin
Published: June 2021

Objectives:

With the popularization of ultrasound, nerve blocks have been widely implemented in current clinical practice. Although, they have seen limited success due to their shorter duration and suboptimal analgesia. Magnesium sulfate as a local anesthetic adjuvant for peripheral nerve blocks could enhance the effects of local anesthetics. However, previous investigations have not thoroughly investigated the analgesic efficacy of magnesium sulfate as an adjunct to local anesthetics for peripheral nerve blocks. Thus, we attempted to fill the gap in the existing literature by conducting a meta-analysis.

Materials and Methods:

We performed of a quantitative systematic review of randomized controlled trials published between May 30, 2011 and November 1, 2018. Inclusion criteria were: (1) magnesium sulfate used as adjuvant mixed with local anesthetic for nerve blockade, (2) complete articles and published abstracts of randomized controlled trials, (3) English language.

Primary and Secondary Outcome Measures:

The primary outcome measures were time of onset, total duration of the sensory blockade, and Visual Analog Scale pain scores. The secondary outcomes were postoperative oral and intravenous analgesics consumption and the incidence of nausea and vomiting.

Results:

The 21 trials analyzed in this study included 1323 patients. Magnesium sulfate effectively prolonged the total duration of sensory blockade (mean difference [MD]=114.59 min, 95% confidence interval [CI]: 89.31-139.88), reducing Visual Analog Scale pain scores at 6 hours (MD=1.36 points, 95% CI: −2.09 to −0.63) and 12 hours (MD=1.54 points, 95% CI: −2.56 to −0.53) postsurgery. Magnesium sulfate also effectively reduced postoperative analgesic use within 24 hours postsurgery (standard MD=−2.06, 95% CI: −2.67 to −1.35). Furthermore, adjuvant magnesium sulfate significantly reduced the incidence of nausea and vomiting after transversus abdominis plane blockade (odds ratio: 0.39, 95% CI: 0.18-0.81).

Conclusion:

Adjuvant magnesium sulfate enhanced the anesthetic effects of local anesthetics and improved postoperative analgesia following the perineural blockade.