bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development and testing of affinity tag purification vectors for a metal‐dependent regulator protein from Thermobifida fusca (946.14)
Authors: Sticher Zachary , Fenton Jordan , Novak Walter 
Published: June 2021

Metal‐dependent regulators are transcriptional regulator proteins that are used by a variety of pathogenic bacterial cells to maintain appropriate iron levels in the cell. The overarching goal of our research is to identify the function of the N‐terminus in DNA and metal‐binding in a metal‐dependent regulator from Thermobifida fusca. Previously, we demonstrated the metal‐dependent regulator protein from T. fusca could be overexpressed and purified to greater than 93% purity using nickel affinity and size‐exclusion chromatography. Although this technique works well for the wild‐type protein, we found that some mutations severely reduce the affinity of the protein for the nickel affinity column. Here we describe the creation of cleavable and non‐cleavable C‐terminal 6×His tagged fusion proteins. Initial results using the non‐cleavable tag suggest that the protein aggregates and also exhibits reduced affinity for the DNA operator.Grant Funding Source: Supported by the Wabash College Haines Biochemistry Fund.
Febrile temperature decreases the cell‐surface expression of the human ether‐a‐go‐go‐related gene (hERG) channel by facilitating channel degradation (949.3)
Authors: Zhao Yan , Koichopolos Jennifer , Wang Tingzhong , Zhang Shetuan 
Published: June 2021

The human‐ether‐a‐go‐go‐related gene (hERG) encoded IKr and KvLQT1+minK encoded IKs are primary K+ currents responsible for cardiac repolarization in many species including humans. Dysfunction of either IKr or IKs can lead to long QT syndrome (LQTS), which predisposes the affected individuals to arrhythmias, syncope, or sudden death. Recent studies have reported that fever may serve as a trigger for QT interval prolongation and arrhythmias in patients. In the present study, we investigated the effect of febrile temperature on hERG and KvLQT1+minK channels using Western blot, patch clamp, and immunocytochemistry. Our data show that culturing cells in febrile temperature (40°C) reduced the expression and current amplitude of hERG channels stably expressed in HEK cells as well as IKr in neonatal rat cardiomyocytes. However, this manipulation did not reduce KvLQT1+minK current. Our data further show that accelerated protein degradation is responsible for the reduced plasma abundance of hERG channels. We have previously shown that hypokalemia accelerates hERG degradation and reduces hERG expression at the plasma membrane. Our data revealed that a reduction in K+ greatly exaggerated, and K+ insensitive mutation S624T completely abolished febrile temperature ‐induced hERG degradation. We conclude that febrile temperature may facilitate the development of LQTS via accelerating hERG degradation.Supported by the Canadian Institutes of Health Research (CIHR).Grant Funding Source: Supported by Canadian Institutes of Health Research
Mutation of serine 32 to threonine in peroxiredoxin 6 preserves enzymatic activity but prevents trafficking to lamellar bodies in vivo (950.1)
Authors: Sorokina Elena , Dodia Chandra , Tao Jian‐Qin , Feinstein Sheldon , Fisher Aron 
Published: June 2021

Peroxiredoxin 6 (Prdx6), a peroxidase that also expresses phospholipase A2 (aiPLA2) activity, protects lungs from oxidative stress and participates in lung surfactant phospholipid (PL) turnover. Prdx6 has been localized to both cytosolic and acidic (lamellar body [LB] and lysosomal) compartments in lung epithelium. We propose that Prdx6 LB targeting facilitates its role in the degradation and remodeling of lung surfactant phosphatidylcholine. Although, we have previously shown that mutation of serine 32 (S32) to alanine abolishes Prdx6 aiPLA2 activity, we now show that serine to threonine mutation has no effect on either Prdx6 peroxidase or aiPLA2 activity. Since S32 is a necessary component of the amino acid motif that mediates Prdx6 LB targeting, we studied the effect of the S32T mutation on Prdx6 LB localization. In a “knock‐in” mouse model carrying an S32T mutation in the Prdx6 gene, there was phospholipid (PL) accumulation in the lung and a marked decrease in aiPLA2 activity in LB. Prdx6 was absent from LB of mutant mice by both western blot and immunofluorescence analysis despite normal protein expression in lung epithelial cells. Thus, the S32T mutation preserves the PLA2 activity of Prdx6 but does not support its targeting to LB, which results in PL accumulation, despite the normal Prdx6 PLA2 activity in lung tissue.Grant Funding Source: HL102016 and HL105509
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
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.
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
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: <http://waterdata.usgs.gov/nwis>: 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