bluuebunny/crossref_metadata_2025 huggingface dataset:
Catabolism of 4‐hydroxynonenal in the rat liver via omega‐ and omega‐1 oxidation (959.2)
Authors: Li Qingling , Berthiaume Jessica , Tochtrop Gregory , Zhang Guo‐Fang
Published: June 2021

In addition to our previous finding on the catabolism of 4‐hydroxynonenal (HNE), 3 novel catabolic pathways of HNE were identified in the perfused rat liver by using the combination of metabolomics studies and isotopic analysis. First pathway, omega oxidation of HNE forms 4,9‐dihydroxynonanoate that is further oxidized to 4‐hydroxynonanedioate. The catabolic degradation of 4‐hydroxynonanedioate started from C‐9 to 4‐hydroxyheptanedioyl‐CoA, 2‐hydroxyglutaryl‐CoA, 2‐hydroxyglutarate that enters citric acid cycle via 2‐ketoglutarate. The conversion of 2‐hydroxyglutarate to 2‐ketoglutarate is coupled by the reduction of succinic semialdehyde to 4‐hydroxybutyrate. This whole pathway was confirmed by perfused with (un)labeled 4‐ hydroxyheptanedioate and 2‐hydroxyglutarate, respectively. The second and third pathways are two parallel pathways of degradation of 4,8 ‐ dihydroxynonanoate that is omega‐1 hydroxylation product of HNE. Two parallel catabolic pathways of 4,8‐dihydroxynonanoate follow the same pathways that we reported earlier on HNE catabolism. Rat heart and brain have little activity of HNE omega‐ and omega‐1 oxidation by in vivo and in vitro experiment.
Effects of feeding Ricinus communis seed meal on liver enzymes makers of albino rats (959.8)
Authors: Omeh Ndukaku , Ejiofor Emmanuel
Published: June 2021

The effect of feeding seed meal on livers enzyme markers was evaluated using albino wister rats. Sixteen (16) male albino rats were randomly divided into 4 groups (1, 2, 3 and 4). Ricinus communis was incorporated into normal rat diet at 5%, 10% and 20% inclusion levels and fed to group 2, 3 and 4 rats respectively, while group 1 which served as control was fed with normal diet for 28 days. At the end of the feeding period, the level of the liver enzymes markers [(Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Alkalime Phosphate (ALP)] were determined using standard methods.

The serum bilirubin (conjugated and total) were also determined. The entire mean liver enzymes makers the conjugated and total bilirubin significantly decreased when compared with the control. The AST significantly increased when the test groups were compared with the control. The ALP also significantly reduced when compared with the control. The conjugated bilirubin concentrations significantly decreased when compared with the control while the total bilirubin concentration significantly increased when the test groups were compared with the control. The result of the experiment suggests that Ricinus communis meal may possess some hepatoprotective property.
Altered mitochondrial function in epididymal adipose tissue with advancing age (960.6)
Authors: Hart Corey , Layec Gwenael , Trinity Joel , Henson Grant , Morgan Richard , Walker Ashley , Donato Anthony , Lesniewski Lisa , Richardson Russell
Published: June 2021

We investigated the effect of aging on mitochondrial respiration in permeabilized epididymal white adipose tissue (eWAT) and skeletal muscle (SKM) in mice. Mitochondrial respiratory oxygen flux (JO2) was quantified in duplicate samples from eWAT and SKM (soleus) of young (n = 12) and old (n = 7) mice using a Clark respirometer (Hansatech, UK). Complex I and II respiration was measured using the sequential addition of glutamate and malate (GM), ADP (D), octanoyl carnitine (O), and succinate (S). Mitochondrial density, estimated from mtDNA concentration using qPCR technique, was not altered by age in either SKM or eWAT. Mitochondrial respiration was preserved with age in SKM, with no differences in respiratory or substrate control ratios (RCR and SCR). In contrast, eWAT JO2 was significantly increased (all P<0.05) with age after the addition of GM (young: 0.8 ± 0.1; old: 2.5 ± 1.0 pmol/sec/mg), ADP (young: 0.8 ± 0.1; old: 2.2 ± 0.5 pmol/sec/mg), and O (young: 1.3 ± 0.2; old: 2.6 ± 0.6 pmol/sec/mg). In addition, the SCR of GMOD/GMD (young: 1.7 ± 0.2; old: 1.2 ± 0.1) and GMOSD/GMD (young: 2.6 ± 0.4; old: 1.5 ± 0.1) was lower in eWAT from old mice (P<0.05), despite no difference in RCR. These data reveal that, while JO2 is unchanged in SKM, substrate control is decreased in the presence of a medium chain fatty acid in eWAT despite an increased JO2 in complexes I and II, suggesting altered lipid utilization with age.

Grant Funding Source: R21 AG033755, K01 AG033196, R01 AG040297, P01 HL 091830, VA Merit Grant E6910R
ATM mediates adaptive epigenetic responses to mitochondrial stress (961.1)
Authors: Schroeder Elizabeth , Christian Brooke , Shadel Gerald
Published: June 2021

Reactive oxygen species (ROS) are important signaling molecules that regulate numerous cellular processes. In contrast to their historic association with disease and accelerated aging, evidence in humans and model organisms suggests that ROS signaling can promote health and extend lifespan, yet ROS signaling pathways remain relatively unknown. We recently demonstrated that a mitochondrial ROS (mtROS) signal extends lifespan of the budding yeast Saccharomyces cerevisiae by activating conserved DNA damage response kinases that inactivate a jumonji‐family histone demethylase specifically at subtelomeric regions and repress gene expression. Here, we present evidence that this pathway is conserved in mammals. The DNA damage response kinase ATM, which dimerizes following ROS‐induced disulfide bond formation, also senses mtROS. Formation of the ATM dimer correlates with increased levels of H3K9me3, a histone modification characteristic of heterochromatin formation. Increased H3K9me3 following mtROS signaling is ATM‐dependent, and is associated with repressed telomeric and subtelomeric gene expression. Experiments are in progress to examine the role of jumonji‐family histone demethylases in the mammalian response to mtROS signaling. These results indicate that mtROS signaling via DNA damage response kinases and mtROS‐dependent subtelomeric heterochromatin formation are conserved from yeast to human and expand on the important roles of ATM in redox homeostasis, telomere regulation, and the DNA damage response.

Grant Funding Source: NIH F31AG043242‐01
Clustering protease domains for use in directed evolution of specificity (967.6)
Authors: Selby Thomas
Published: June 2021

The objective of this study is to determine the most successful protease domains for use in directed evolution experiments against substrates with novel amino acid sequences. Building on our previous work that created an in vivo expressible fluorescence resonance energy transfer (FRET) system for protease specificity determination, we have initiated a study of protease domain clustering for in vivo protease evolution against a single recognition sequence. Using backbone root‐mean‐square deviation (RMSD), buried active‐site surface area, secondary structure analysis, and hydrophobicity scores, we have clustered protease domains into groups with a likelihood of potential success for in vivo evolution against specific amino acid sequences of maximum length. Our results demonstrate that many protease domains are not amenable to evolution studies due to energetic concerns related to instability of secondary structure interactions around the active site. Additionally, many proteases exhibit active site recognition regions that are relatively small, which eliminate the potential to target these domains against longer, and more specific, amino acid sequences. Encouraging results were obtained for two types of protease domains that have the maximum likelihood for successful evolution studies. These protease domains will be used in fluorescence‐activated cell sorting (FACS) experiments and evolved against loop regions of signal transduction receptors using our FRET protease specificity substrates. Antibody conjugates of these domains will then be constructed to target these evolved proteases to specific cell types based on antigen availability on the cell surface.

Grant Funding Source: Supported by NIH GM080691
Harnessing sulfinic acid reactivity to report protein S‐nitrosylation (968.2)
Authors: Martin Brent
Published: June 2021

Oxidative stress describes the accumulation of reactive oxygen (ROS) and reactive nitrogen species (RNS), and is a hallmark of neurodegeneration and cancer. Cysteine residues can be oxidized by nitric oxide to nitrosocysteine (RSNO) or by peroxide to a sulfinic acid (RSO2H). We find that sulfinic acids react with nitrosothiols to form a thiosulfonate linkage. Using sulfinic acid‐linked probes, we demonstrate selective enrichment and mass spectrometry annotation of more than 1500 endogenous S‐nitrosylated proteins. This list includes nearly all previously annotated S‐nitrosylated proteins, including ion channels, chaperones, peroxiredoxins, p53, HDACs, hundreds of metabolic enzymes, as well as a rich set of novel proteins. Interestingly, the familial PD gene DJ‐1 protects cells from oxidative stress, and requires oxidation of an evolutionarily conserved cysteine to a sulfinic acid. Furthermore, high‐resolution mass spectrometry analysis and chemiluminescence NO detection confirms that S‐nitroso‐cysteine reacts with oxidized DJ‐1, causing elimination of nitric oxide. Furthermore, we discuss novel strategies to annotate and profile protein sulfinylation using orthogonal reactive probes. Based on these findings, we propose that sulfinic acids, such as in DJ‐1, function to reduce nitrosative stress, establishing a novel mechanism of redox regulation. Support is provided from the University of Michigan.
Evolution of telomeres and telomerase: new partners spice up the dance (97.1)
Authors: Shippen Dorothy
Published: June 2021

Telomere maintenance occurs through dynamic engagement of telomerase by telomere binding proteins. In Arabidopsis thaliana, duplication and diversification of core telomere components has the potential to introduce new modes of regulation. Here I discuss the consequences of two duplication events: telomerase RNA (TER) and the Protection of Telomeres (POT1). A. thaliana harbors three distinct TER subunits. TER1 is a canonical telomerase RNA template, while TER2 is a negative regulator of TER1. TER2 is rapidly induced in response to double‐strand DNA breaks (DSBs), triggering telomerase inhibition and thereby favoring faithful DNA repair. The third isoform, TER2s is generated by splicing and 3’ end cleavage of TER2. Notably, the TER2 intron is a transposable element, required for TER2 induction and telomerase inhibition. Thus, telomerase inhibition is not the ancestral function of TER2; it appears to be derived from transposon insertion.

A. thaliana encodes three isoforms of POT1. Unlike orthologs in vertebrates and yeast, A. thaliana POT1 proteins do not bind telomeric DNA, and instead bind TER. POT1a is a processivity factor that assembles with TER1 in the canonical RNP and docks the enzyme on the telomere. POT1b, by contrast, binds the inhibitory TER2 RNP. POT1c is the newest member of the POT1 gene family, and more closely resembles POT1 proteins in other eukaryotes. Mutations in POT1c grossly perturb telomere architecture, indicating that this isoform functions in chromosome end protection.

These findings underscore the extraordinary malleability of telomere components, and the potential to evolve novel modes of regulation and new molecular interfaces to promote genome integrity.
Role of the breast cancer suppressor BRCA1 in the maintenance of genomic integrity (97.3)
Authors: Jasin Maria , Kass Elizabeth , Zhang Yu , Sung Patricia , Chen Chun‐Chin
Published: June 2021

Homology‐directed repair (HDR) is a critical pathway for the repair of DNA double‐strand breaks (DSBs) in mammalian cells. Most mammalian HDR studies have focused on transformed and immortalized cell lines. We now report the generation of a DR‐GFP reporter‐based mouse model to study HDR in primary cell types from diverse lineages. Embryonic and adult fibroblasts as well as cells derived from mammary epithelium, ovary and neonatal brain were observed to undergo HDR at I‐SceI endonuclease‐induced DSBs at similar frequencies. When the DR‐GFP reporter was crossed into mice carrying a hypomorphic mutation in the BRCA1 tumor suppressor gene, a significant reduction in HDR was detected, demonstrating that BRCA1 is critical for HDR in somatic cell types, as previously reported in embryonic and tumor cell lines. These results indicate that the DR‐GFP mouse provides a powerful tool for dissecting the genetic requirements of HDR in a diverse array of somatic cell types in a normal, non‐transformed cellular milieu. Further studies are underway to examine the role of BRCA1 specifically in mammary epithelial cells.

We previously showed that BRCA1 works upstream of the other major breast tumor suppressor BRCA2 in HDR. Recent work from other labs is consistent with this result, and suggests that BRCA1 functions in an early step of HDR to control DSB repair pathway choice. We discuss our recent results in this regard.
Synthesis of muramic acid derivatives to investigate the promiscuity of bacterial cell wall biosynthetic enzymes (970.2)
Authors: Kenny Douglas , Grimes Catherine
Published: June 2021

Nod2 is an innate immune receptor found in mammalian cells that plays a role in recognizing bacteria. Specifically, Nod2 detects bacterial cell wall or peptidoglycan. Recently in vitro studies have shown that Nod2 can bind directly to a small fragment of peptidoglycan called muramyl‐dipeptide (MDP). Yet, it is still unclear how the bacterial cell wall fragments are generated and gain access into the cytosol to activate the receptor. In order to observe the cell wall and its interactions with the receptor, a method to label the cell wall of bacteria using fluorescent probes can be used. This method will allow us to track the uptake of the cell wall and observe its interaction with Nod2 in a cellular context. In order to label the cell wall, two derivatives of peptidoglycan precursors, UDP‐N‐Acetyl‐muramic acid, are being synthesized. These derivatives contain an azide on either the 2 or 6 positions, as they are the only positions not used in building the peptidoglycan framework. The azide functionality can act as a chemical handle and undergo quick, bioorthogonal coupling with a fluorescent probe for visualization. The derivative with the azide on the 6 position has been successfully synthesized, and the derivate with the azide on the 2 position is currently being synthesized. Once finished, the ability of the bacteria to incorporate these molecules into their cell wall will be tested through both in vitro, using the purified biosynthetic enzymes, and in vivo studies. With the fluorescently labeled bacterial cell wall, the mode of Nod2 activation can be studied with living cells.
Investigations of the specificity and promiscuity of receptors within the BCL‐2 protein family (971.2)
Authors: Carlson Kevin
Published: June 2021

The BCL‐2 protein homology plays a central role in regulating the intrinsic pathway of apoptosis. These proteins often hold the key to a cells decision to live or die following factors such as irreversible DNA damage. Not surprisingly, the anti‐apoptotic members of this family are over expressed in many cancers where they contribute to the formation, progression and chemo‐resistance of tumor cells. The promiscuous recognition of helical BH3 domains by the BCL‐2 class of proteins makes their exact roles in apoptosis difficult to determine and thus contribute to challenges in designing effective therapies. We seek to identify the molecular determinants of binding between the BCL‐2 homologs and their BH3 targets. Using in‐vitro translation and a luciferase based florescence assay we have examined the effects of mutations to the receptor pocket of BCL‐2 and MCL‐1 on interactions with 9 BH3 only proteins. Results from these experiments should formulate a picture of the role different members of this homology play in cell death. Moreover, these results will allow for the design of orthogonal receptor/ligand pairs, helpful for further interrogating this pathway.

Grant Funding Source: Arnold and Mabel Beckman Foundation
Lipophilic natural compounds (n‐3 polyunsaturated fatty acids) modulate plasma membrane organization in mouse CD4+ T cells (975.1)
Authors: Hou Tim , Barhoumi Rola , Rivera Gonzalo , McMurray David , Chapkin Robert
Published: June 2021

We have previously shown that n‐3 polyunsaturated fatty acids (PUFA) increase plasma membrane lipid order in mouse CD4+ T cells, blocking T cell activation. Since lipid rafts are important for T cell activation, we hypothesized that n‐3 PUFA perturb CD4+ T cell plasma membrane lipid rafts. To probe the mechanism for n‐3 PUFA action, CD4+ T cells from Fat‐1 transgenic mice (enriched in n‐3 PUFA), were transduced with fluorescence energy resonance transfer (FRET) lipid raft probes Lck(N10) and LAT(ΔCP), and non‐raft probe Src(N15). Co‐clustering of Lck(N10) and LAT(ΔCP) in CD4+ T cells increased in the presence of n‐3 PUFA as assessed by FRET; conversely n‐3 PUFA did not alter the co‐clustering between Lck(N10) and Src(N15). These data demonstrate that n‐3 PUFA modulate membrane nano‐organization in CD4+ T cells. We have also previously demonstrated that n‐3 PUFA decrease the mass of phosphatidylinositol‐(4,5)‐bisphosphate [PI(4,5)P2] in CD4+ T cells. Since discrete pools of PI(4,5)P2 may exist in the plasma membrane, we also determined whether n‐3 PUFA modulate spatial organization of PI(4,5)P2 relative to raft and non‐raft domains. Co‐clustering of PH(PLC‐δ1), a PI(4,5)P2 probe, and Lck(N10) or LAT(∆CP) was not affected by n‐3 PUFA; however, co‐clustering of PH(PLC‐δ1) and Src(N15) showed a decrease in the presence of n‐3 PUFA, suggesting that n‐3 PUFA alter the spatial organization of PI(4,5)P2.

Grant Funding Source: Supported by NSERC and NIH
Bioactivity guided isolation of a novel protein from Moringa oleifera with antimicrobial activity (975.9)
Authors: Ahmed Sibtain , Jamil Amer
Published: June 2021

Natural products discovered from medicinal plants have provided numerous clinically used medicines. Moringa oleifera, a multipurpose plant first discovered in Asia and Africa, is now known worldwide for its nutritional and medicinal benefits as well as its industrial uses. This study describes the activity‐guided isolation and purification of a novel antimicrobial protein from Moringa oleifera. Purification was carried out by (NH4)2SO4 precipitation, gel filtration and ion‐exchange chromatography. Antifungal and antibacterial activities were determined after each purification step. SDS‐polyacrylamide gel electrophoresis revealed that the purified protein had a molecular mass of 15‐kDa. The protein possessed a strong and broad‐spectrum antimicrobial activity against pathogenic bacteria and fungi. It may be a future target for the development of drugs with strong antimicrobial activities.

Grant Funding Source: University of Agriculture Faisalabad, Pakistan
The multidrug resistance IncA/C transferable plasmid encodes a novel domain swapped dimeric disulfide isomerase (976.1)
Authors: Kurth Fabian , Lakshmanane Premkumar , Neyer Simon , Schembri Mark , Martin Jennifer
Published: June 2021

The multidrug resistance‐encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. DsbP, a plasmid encoded disulfide isomerase is crucial for conjugation. The crystal structure of DsbP revealed a unique V shaped homodimer that is inverted with respect to known disulfide isomerases from E. coli (DsbC and DsbG). Dimer formation is mediated by an N‐terminal domain that includes a novel domain‐swapped interaction. Overall, the structure, surface and catalytic properties of DsbP diverge from those of Escherichia coli DsbC and DsbG disulfide isomerases. Functional differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC‐like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural revealed conformational changes in the linker region, which contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid‐encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

Grant Funding Source: Supported by an Australian Research Council (ARC) Australian Laureate Fellowship (FL0992138) to JLM
High‐throughput screen for inhibitors of androgen receptor‐RUNX2 interaction in prostate cancer (977.2)
Authors: Vuong Winston , Tew Ben , Little Gillian , Frenkel Baruch , Jones Jeremy
Published: June 2021

Metastatic PC (mPC) is managed with AR‐targeted therapies; however, progression is inevitable. AR normally regulates prostate homeostasis but is compromised in PC to promote proliferation. RUNX2, a regulator of osteoblast differentiation, is expressed in many PCs and is associated with progression and metastasis. We found that RUNX2 modulates DNA‐binding and gene regulation by AR. Notably, AR/RUNX2 synergistically activates a subset of genes (type II) that includes the pro‐oncogenes Snai2 and Sox9. Thus, inhibition of AR/RUNX2 may provide a novel target for management of mPC. We devised a robust cell‐based assay (z‐score >0.7) for screening potential inhibitors of the AR/RUNX2 interaction in the C4‐2B PC cell line that expresses doxycycline (dox) inducible RUNX2. These cells were transfected with a type II/YFP reporter and a drug‐independent mCherry control plasmid. The cells were treated with dox to induce RUNX2 expression and DHT to activate AR then analyzed in a 96‐well plate format on a Tecan fluorescent plate reader. Each plate was assayed in duplicate with no drug, 0.25 mg/mL dox, 0.3 nM DHT, and dox+DHT controls. Using dox+DHT treated cells, we screened ~1800 compounds at 1 µM from the NCI Developmental Therapeutics Program library. Hits were identified with a custom algorithm, validated by performing a dose curve in the same assay, and then tested for activity against synergistic activation of endogenous genes by qPCR. Thus far, we have identified one inhibitor that is specific to the AR/RUNX2 synergism and are continuing to test validated hits in the qPCR assay. In the future, we will examine the AR‐RUNX2 inhibitors for efficacy against PC invasiveness in vitro and growth inhibition in PC xenograft mouse models.
Genesis of antibiotic resistance X: cross border procurement and disposal practices of antibiotics in Rio Grande Valley (986.10)
Authors: Kannan Subburaj , Subburaj Prabavathy
Published: June 2021

The method of procurement, consumption (utilization), and disposal of antibiotics through a multitude of channels vary among the general public all over the world. An effort has been made to clarify the ability to purchase the life‐saving antibiotics from the border town(s) of United States ‐ Mexico. In essence, Consul DE MEXICO EN LA CIUDAD, DE PIEDRAS NEGRAS, COAHUILA, communicated that a prescription from a physician is required to dispense antibiotics. Information on sharing of medications (antibiotics) and “buy back of unused medication by state or any other agency in Mexico is unavailable at this time”. Both the Mexican counsulate in Eagle Pass, Texas and also Texas Region 8 Medical Director, indicated that there is no unused medicine (Antibiotics) buy back program in effect in the Mexico or Rio Grande Valley region of Texas. The annual water report from The City of Eagle Pass Water Works, indicate that currently there are no methods in effect to determine the Pharmaceuticals and Personal Care Products (PPCPs) listed as Pollutants by The United States Environmental Protection Agency (US‐EPA); and also The Texas Commission on Environmental Quality (TCEQ). Information regarding the detection of and/or determination of PPCPs in the Maverick County Solid Waste Landfill is has not been made available during an inquiry process. Specific details pertinent to this presentation shall be discussed in Experimental Biology 2014 meeting.

Grant Funding Source: PROFESSIONAL DEVELOPEMENT FUNDS ‐
Genesis of antibiotic resistance II: monitoring of antibiotics and their metabolic breakdown products in the Rio Grande River water by United States Geological Survey (986.2)
Authors: Kannan Subburaj , Villarreal Carmen , Atchico Monya , Bonilla Magaly , Dingler Margarita , Subburaj Prabavathy
Published: June 2021

An analysis of the data on the detection and concentration of antibiotics, its metabolic breakdown intermediates in Site number: 08450900, Site Name: Rio Grande bl Amistad Dam nr Del Rio, TX of USGS: : has been undertaken. We have obtained the Water‐Data Report TX‐2005 08450900 USGS Rio Grande below Amistad Dam near Del Rio, TX; Rio Grande River Basin; LOCATION.‐‐Lat 29°25′30″, long 101°02′27″ referenced to North American Datum of 1927, Val Verde County, Hydrologic Unit 13080001, and 2.2 mi downstream from Amistad Dam. Data analysis was performed on WATER‐QUALITY RECORDS CHEMICAL DATA: July 1968 to Aug. 01, 2005 (discontinued); BIOCHEMICAL DATA: Oct. 1995 to Aug. 01, 2005 (discontinued); PESTICIDE DATA: Oct. 1995 to Aug. 01, 2005 (discontinued); SEDIMENT DATA: Oct. 1995 to Aug. 01, 2005 (discontinued). The available data indicates that no antibiotics or its metabolic breakdown products are monitored in the Rio Grande Water. However, the following indirect metabolic derivatives were detected: Disulfoton; Simazine; Terbacil; Terbu‐fos. At present efforts are underway to obtain the water data from 2005 to present from THE USGS for the detection of antibiotics and its metabolic derivatives. The detailed database search on sifter phrases and data analysis are to be presented in Experimental Biology 2014.

Grant Funding Source: Professional Development Funds from SWTJC‐Eagle Pass 78852
New copper binding components of blood plasma (992.1)
Authors: Dalphin Matthew , Salazar Dianne , Paik Samantha , Kidane Theodros , Vulpe Christopher , Page Kathryn , Linder Maria
Published: June 2021

It has been assumed for some time that the main Cu‐containing proteins in blood plasma are ceruloplasmin (Cp), albumin and transcuprein/a2‐macroglobulin, Cp being the main ferroxidase (Fx) implicated in mediating efflux of iron from certain cells. Recent studies in our laboratory suggest the picture is much more complex, and that other known and unknown factors containing Cu are still to be identified. We had previously shown in mice that knocking out Cp only knocked down plasma Fx activity by half. We now have evidence that knocking out hephaestin (Hp) a membrane‐tethered Cp‐homologue reduces plasma Fx activity and a peak in the Cu profile obtained by size exclusion chromatography (SEC), indicating that solubilized Hp may be entering the circulation. Also, a patient with twice normal level of plasma Cu but Cp protein in the normal range had twice normal levels of Fx activity, suggesting that ratios of holo to apo‐Cp were elevated or that another Fx increased in response to the contraceptives. Use of the anticoagulant, EDTA, to collect plasma had a dramatic effect on SEC Cu profiles, moving a large proportion of Cu in larger components (but not Cp) to one of low molecular weight normally present in trace amounts, which is not Cu‐EDTA or metallothionein. We conclude that the proportions of plasma Cu associated with Cp and Fxs can vary widely and speculate that a copper peptide normally attached to other proteins is dislodged by EDTA.

Grant Funding Source: HHMI 52007532
Structural and functional characterization of a heavy metal detoxifying ABC transporter (997.2)
Authors: Lee Jonas , Yang Janet , Zhitnitsky Daniel , Lewinson Oded , Rees Douglas
Published: June 2021

The superfamily of ATP Binding Cassette (ABC) transporters includes importers and exporters that translocate a wide variety of substrates across the cell membrane. Although significant progress has been achieved in the structural analysis of ABC exporters, much less is known about how ABC exporters selectively recognize varying substrates, and couple its binding to ATP hydrolysis. We have addressed these questions by the crystallographic and functional characterization of a dimeric Atm1/ABCB7/HMT1/ABCB6 bacterial ortholog. Glutathione and it's derivatives are demonstrated to serve as substrates. In vivo, this transporter confers resistance to silver and mercury toxicity to E. coli, implicating a physiological role in cellular detoxification processes. The 2.4 Angstrom resolution structure allowed detailed mapping of the binding interactions highlighting the articulated design of ABC exporters. The key ligand interactions are positioned at the boundaries between structurally conserved elements, which links ligand binding to the conformational changes underlying ATPase‐coupled substrate translocation.

Grant Funding Source: National Institutes of Health
Binding affinity of epithelial sodium channel subunits and peptides measured through surface plasmon resonance (997.5)
Authors: Gonzalez Andres , Peltier Grant , Booth Rachell , David Wendi
Published: June 2021

Salt levels play a crucial role in regulating blood pressure, and cells are able to maintain salt levels via ion channels embedded within the plasma membrane. The Epithelial Sodium Channel (ENaC) is a heterotrimeric membrane protein that allows the passage of sodium, and this study’s aim is to further understand what structural aspects of ENaC are responsible for maintaining proper function of the channel. Potential residues critical for interaction between ENaC alpha, beta, and gamma subunits were identified and peptide segments containing the targeted residues were PCR amplified, ligated onto a pGEX‐4t‐2 vector, and transformed into Top10 E. coli. This vector contains a GST‐tag that was used to purify the peptides and immobilize them for further analysis by surface plasmon resonance (SPR). Binding between sets of peptides predicted to be important for stabilizing interactions in ENaC was investigated using SPR. One peptide from a potential interacting set of peptide regions was immobilized through the GST‐tag and binding with the corresponding partner (with GST‐tag removed) was quantified. Two interactions ‐one between the alpha‐palm and beta‐thumb, and the other between the beta‐palm and gamma‐thumb regions of ENaC ‐ were analyzed. The alpha‐palm and beta‐thumb interaction had a measured KD of 2.9 x 10‐8 M, whereas the interaction from the beta‐palm, gamma‐thumb region was in the micromolar range for KD. Additional sets of peptides have been expressed and will be analyzed for binding interactions. Results obtained from this initial study will be compared to those obtained from a mutation screen of ENaC function to confirm whether the predicted residues play a crucial structural role related to function.

Grant Funding Source: Supported by NIH R15 GM86798 (R. Booth & W. David) and Welch Foundation Departmental Grant AI‐0045.
An ancient, anatomical manikin of a pregnant woman in ivory by stefan ZIck (1639‐1715) (LB11)
Authors: LE FLOCH‐PRIGENT Patrice , GILLOT Jean‐Bernard
Published: June 2021

An ancient sample of a pregnant woman made in ivory belonged to the scientific library Alain Brieux/Gillot (Paris 6th). It was compared to a similar manikin from the National Academy of Medicine in Paris. It was smaller (12 cm long) and anatomically less precise than the Academy’s one with the absence of an intestinal shield and uterus and a lack of any vascular representation. The hair, the rendering of the upper limbs with a small horizontal axis of rotation, the lower limbs and the fœtus were very similar. A flat pillow with a finely worked border and a wooden basis with four ivory legs on which laid a lid in the shape of a sarcophagus were noticeable differences. It could be possibly attributed to Stefan Zick (1639‐1715) from Nuremberg or to his workshop. We acknowledge Mrs Domenica Scalisi and Mr Alain Piffault.
Assessment of medical student comprehension of basic biomedical and clinical science content within a unique integrated curriculum (LB112)
Authors: Peffley Dennis , Kahlil Mohammed , Hawkins Greg , Otto Ryan , Wright William , Crespo Lynn
Published: June 2021

The University Of South Carolina School Of Medicine Greenville has adopted curricular strategies that emphasize vertical and horizontal integration of basic biomedical with clinical sciences and active learning approaches. This study describes a comprehensive approach to student‐, learning module‐, and program‐level outcomes assessment. Assessment proceeds from the student‐level, which involves weekly formative exams mapped to learning module‐level objectives, primary and secondary biomedical sciences and clinical disciplines. Summative exam performance maps allow students to identify their strengths and weaknesses. Learning module‐level assessment includes establishment of student performance targets, correlation of student formative to summative exam outcomes, performance mapping to learning module objectives, and post‐module assessment reporting. Program‐level assessment utilizes online systems, i.e., ExamSoft™ and Weave™, that provide real‐time data demonstrating depth and breadth of student‐and module‐level assessments relative to program objectives. External measures of student progress and performance within the curriculum include National Board of Medical Examiner testing. Reporting of student‐ and learning module‐outcomes relative to program‐level objectives as described above has proven effective in guiding biomedical and clinical science content delivery.
Strategies for enhancing student understanding through active engagement (LB113)
Authors: Lutze‐Mann Louise
Published: June 2021

When teaching undergraduate students, especially in large classes, one of the most challenging aspects is providing students with an engaging experience that will foster their learning and increase their understanding of complex material. There are numerous studies in the literature which document the advantages for students of participating in “active learning” where interacting with other people (students, academic staff) in real or virtual worlds maintains their interest and hence enhances their learning, especially when they receive immediate feedback on their performance. However, it is not easy to design learning tasks that encourage student interaction, are academically rigorous and also able to engage the current generation of digitally native students. Three strategies for enhancing student understanding using active learning will be presented: the formative assessment lecture using mobile response devices; virtual laboratory activities using an adaptive feedback platform and student‐led game design. The value of each these strategies, apart from increasing student engagement, is that student misconceptions are frequently revealed allowing direct remediation, which further enhances student learning and provides instructors with feedback on troublesome areas of the curriculum.
Biotechnology: Science of the New Millennium ‐ Technology Based Programs at Thomas Jefferson University (LB115)
Authors: Bawazir Nada , Biswas‐Fiss Esther
Published: June 2021

A recent trend in molecular life science education at the associates, undergraduate and master’s levels has been the introduction of a variety of Biotechnology (BT) degree majors. Often these have evolved as offshoots of existing chemistry or biology curricula. At Thomas Jefferson University, we have designed novel, hands‐on, industry‐oriented programs in Biotechnology. This report details factors contributing to the successful development of these programs, whose goal is to prepare students to hit the ground running as “entry level scientists”. Components which were found to be significant to this endeavor included: (i) development of a highly‐hands on based curriculum; (ii) incorporation of practical rotations; (iii) design of instruments to assess technical competency; (iv) establishment of articulations with neighboring community colleges offering AS‐BT degrees; and (v) promotion of “biotechnology” as a major for students interested in science‐based careers. Outcomes analyses suggest that a competency based curriculum not only ensures that graduates are workplace ready, but also can provide a solid foundation for further study at the doctoral or professional level.
Novel Role for PTEN in Regulating Non Homologous End‐Joining Factor 53BP1 to Suppress Replication Fork Instability (LB120)
Authors: Schlacher Katharina , Guo Wen , Jasin Maria , Wu Hong
Published: June 2021

PTEN is one of the most frequently inactivated tumor suppressors with unresolved connections to DNA repair processes that prevent genomic instability. Using single molecule DNA spreading, we here show that PTEN suppresses replication fork instability by promoting efficient replication restart of stalled replication forks, a phenotype mirroring ATM and BLMs helicase functions. Moreover, we find that similar to BLMs, PTEN suppresses replication stress dependent DNA bridges. Previous studies show non‐homologous end‐joining (NHEJ) protein and ATM phosphorylation target 53BP1 in complex with BLMs at stalled fork, an interaction requiring phosphorylation of 53BP1. Yet, the role of 53BP1 at stalled forks remains ill‐defined. Strikingly, 53BP1 is highly phosphorylated in PTEN proficient cells upon replication stalling, but not in cells lacking PTEN. We further find 53BP1 to be readily recruited to nuclear foci upon replication stalling. Yet foci formation is compromised in PTEN defective cells, implying PTEN controls a function for 53BP1 when replication forks are stalling. Lastly, we reveal a direct requirement for 53BP1 in restarting stalled replication forks. These data reveal a surprising function for NHEJ factors controlled by PTEN to suppress replication fork stability and genomic instability.

Grant Funding Source: Supported by 1K22CA175262‐01 (K.S.) and UO1CA164188 (H.W.)
Perception and emotional impact of dissection on medical students (LB13)
Authors: Hajj Hussein Inaya , Hankin Mark , Dany Mohammed , Wasserman Jason , Jurjus Abdo
Published: June 2021

Dissection contributes significantly to anatomical knowledge and the development of professionalism for medical students. This study assessed student emotional stress and coping in the anatomy lab and the perception of dissection on learning. A survey of Medicine I students (n=100) assessed: demographics, emotions and stress of the first dissection, anxiety, coping, and learning. With a response rate of 40%, our findings include: positive attitude towards the first dissection (n=33) although some students still found it stressful (n=17); cultural views impacted the lab experience (n=24); some used spirituality (n=10) or humor (n=6) to cope; most agreed that dissection enhanced understanding of anatomy (n=31) and the connection of between theoretical and applied knowledge (n=37); promoted teamwork (n=35); and highlighted respect for the human body (n=31) and the spirit of organ donation (n=28). While some have assumed that students have a negative attitude towards human dissection, our study showed that the majority of students reported a positive impact on various aspects of professionalism and humanism. We conclude that dissection is useful for students to establish not only technical skills and knowledge of the body, but also qualities associated with inter‐professional teamwork and a humanistic attitude, without posing significant emotional stress.
Substitutions in the Ω‐Loop of TEM β‐Lactamase Change the Substrate Profile of the Enzyme (LB140)
Authors: Stojanoski Vlatko , Hu Liya , Chow Dar‐Chone , Prasad BV , Palzkill Timothy
Published: June 2021

Serine β‐lactamases are bacterial enzymes that hydrolyze β‐lactam antibiotics. Mechanistically, serine β‐lactamases are similar to serine proteases such as chymotrypsin. They both utilize acylation and deacylation of an active site serine in their mechanism of catalysis. TEM‐1, a common plasmid‐encoded serine β‐lactamase, catalyzes the hydrolysis of early penicillins and cephalosporins. Here we examine a previously identified triple mutant of TEM‐1, 165‐TyrTyrGly‐167 (wild type165‐TrpGluPro‐167) with substrate specificity altered from preferential hydrolysis of ampicillin to that of ceftazidime. When compared to chymotrypsin, the Glu166Tyr substitution in the TEM‐1 triple mutant is analogous to a substitution of the general base His57 in chymotrypsin that results in an enzyme that maintains function. Our findings agree with previous observations of altered substrate specificity of the triple mutant, which displays increased hydrolysis of ceftazidime. Additionally, enzyme kinetic analyses show that the hydrolysis of ceftazidime follows a branched pathway characteristic of substrate‐induced reversible inactivation. Structural analysis of the triple mutant reveals enlargement of the active site making it more accessible for larger substrates. Furthermore, in the crystal structure of the triple mutant, Tyr166 is within hydrogen bond distance to Ser70 suggesting involvement in the catalytic mechanism of the enzyme. These findings indicate that Glu166 general base residue can be substituted in the mechanism of serine β‐lactamases. More generally, this provides insights into alternate mechanisms for reactions catalyzed by serine hydrolases.

Grant Funding Source: Supported by NIH
Novel Transporters for Enzyme Replacement Therapy (LB148)
Authors: Tong Wenyong , Hamil Kristina , Tor Yitzhak , Esko Jeffrey
Published: June 2021

Mucopolysaccharidoses (MPS) are inherited metabolic diseases caused by a deficiency in degradative enzymes that reside in lysosomes. A few of these disorders can be treated by enzyme replacement therapy. However, significant challenges remain because key organs and tissues remain inaccessible to intravenously injected enzymes. We are exploring the utility of guanidinoglycosides as carriers in vivo in mice. The approach is based on our previous observation that guanidinylated neomycin (GNeo) can deliver high molecular weight proteins across cell membranes and into lysosomes via cell surface heparan sulfate proteoglycans. All cells highly express heparan sulfate proteoglycans, suggesting the potential utility of GNeo‐conjugates for delivery to multiple tissues. Our study demonstrates that conjugation of guanidinylated glycosides to lysosomal enzymes can facilitate entry into the brain after intranasal administration, thus bypassing the blood brain barrier. This novel delivery system might be applicable to a variety of lysosomal storage disorders as well as for the delivery of antibodies and growth factors to the brain.

Grant Funding Source: Supported by an NIH LDN post‐doctoral fellowship (W.T.) and grants from the MPS Society and from Improved Therapies for MPS I Program through the University of Pennsylvania (to J.D.E.).
Enteropancreatic Endocrine Cells Response to Streptozotocin Induced Hyperglycemia. (LB16)
Authors: Otobo Tarimobo , Tarimobo‐Otobo Rugina
Published: June 2021

The Enteroendocrine cells form the largest endocrine system in the body. They secrete multiple regulatory molecules which control physiological and homeostatic functions particularly postprandial regulation of glucose and GIT motility (Sternini et al. 2008).In Diabetic setting, it is speculated that enteroendocrine cells which secrete hormones involved in carbohydrate metabolism will undergo changes aimed at maintaining carbohydrate homeostasis (Stephanie et al. 2009).To determine how enteroendocrine cells respond to diabetes and their likely role in diabetic complications, Sixty albino Wister rats (male=30 and female =30), weighing between 200‐300g were divided into five experimental groups (n=10) per group and a control (n=10) . Diabetes mellitus was induced with a single intraperitoneal dose of 65mg per kg of streptozotocin. Blood was sample from tail vein 30 minutes after the application of EMLAR cream (2.5% lignocaine and 2.5% prilocaine) for glucose test and only those with blood glucose within the ranges of 250‐ 600mg/dl were anaesthetized with isofluran , cervical dislocation was done to sacrifice the animals. (group 1= 2weeks, group 2=4weeks, group3=6weeks, group4=8weeks, group5=12 weeks). Experimental animals without selective beta cells destruction of the pancreas, demonstrated by chrome alum hematoxylin‐phloxine staining technique were excluded from the study. The stomach, duodenum, jejunum, ileum and large intestine were collected fixed in boin’s fluid and paraffin sections, 5 microns thick were stained using lead hematoxylin, Toluidine blue metachromasia, and the Grimelius methods. There was an increase in enteroendocrine cell population in the small intestine, large intestine; however few enteroendocrine cells were stained in the stomach when compared to matched control. Toluidine blue metachromatic staining technique stained enteroendocrine cell intensely in the jejunum and ileum when compared to matched control. These findings suggest a proliferation of enteroendocrine cell and by implication an increase in enteroendocrine cell hormones in chronic diabetes. Therefor it is reasonable to conclude that, enteroendocrine cells and their hormones are likely involved in the complications and management of diabetes mellitus.
Ceramides, mitochondrial fission, and reactive oxygen species a novel mechanism of obesity‐induced insulin resistance (LB167)
Authors: Tucker Brady , Bikman Benjamin
Published: June 2021

Reactive oxidative species (ROS) is a known byproduct of mitochondrial dysfunction and is believed to contribute to inflammation signaling commonly found in insulin resistance. Ceramide is a sphingolipid that serves as an important second messenger in an increasing number of stress‐related pathways. Ceramide has long been known to have morphological and physiological effects on mitochondria, which could potentially alter levels of ROS. The focus of this study is to determine whether increased ceramide levels result in ROS generation and decreased mitochondrial respiration in myotubes via altered mitochondrial morphology. C2C12 Skeletal muscle cells were used to assess the effect of ceramide on ROS generation and electron microsopy was used to image the morphological changes. Ceramide synthesis can be stimulated by treatment with exogenous fatty acid, thus myotubes were simultaneously treated with exogenous fatty acid following SPT2 knockdown (SPT is the rate limiting enzyme in ceramide synthesis), ROS increased within the myotubes and cellular respiration dropped. To verify the disruptive effect of ceramide on respiration caused by a change of mitochondrial morphology, cells were treated with a fission inhibitor (Mdivi‐1) prior to the addition of exogenous fatty acid. The resulting inhibition of mitochondrial fission via Mdivi‐1 helped protect the myotubes from ceramide‐induced metabolic disruption, including maintained mitochondrial respiration, and reduced ROS levels. Images taken with the confocal microscope verify that cells with increased levels of ceramide undergo mitochondrial fission while cells treated with Mdivi‐1 are protected from morphological changes. Altogether, these data suggest a critical role for mitochondrial fission as a mediator of ceramide‐induced metabolic disruption.