bluuebunny/crossref_metadata_2025 huggingface dataset:
New Applications in Anatomical Education Using Soft‐Embalmed Cadavers
Authors: Boaz Noel Thomas , Sikon Richard , Sikon Dana , Forest Lynette Nearn , Wells James R
Published: June 2021

Soft embalming (Thiel, W. Annals of Anatomy 174:185–195; 184:267–269) began at VSAP in June, 2012. The method shares with fresh bodies and a commercial no‐formaldehyde embalming procedure the highly desirable characteristics of absence of formaldehyde fumes, natural tissue color, and flexibility of structures, but it also offers an extended time of utilization because of its superior preservational and antimicrobial properties, without mold or deterioration for at least six months. Soft‐embalmed bodies require a “cure time” of 3 to 4 weeks and also cause a superficial epidermal desquamation with occasional fluid‐filled bullae, easily removed after embalming. Dissections on a soft‐embalmed female demonstrate the didactic advantages of the method: palmar aspect of the hand, especially the relationships of the deep and superficial flexors; infratemporal fossa and superolateral neck elucidating multi‐level relationships of the glossopharyngeal nerve and related structures; urogenital and anal triangles of the perineum with clear visualization of the urogenital diaphragm, pelvic diaphragm, and pudendal canal; and clear ultrasound imaging of knee and wrist joints, severely limited on formalized specimens. The relative ease of dissection, ready identification of structures, and formaldehyde‐free environment strongly recommend soft embalming for active learning in anatomical education.
Video lab guides: efficacy and student perception
Authors: Billings Heather J
Published: June 2021

In our anatomy laboratory course for nursing and dental hygiene majors, students use cadavers previously dissected in other courses. To facilitate locating structures more independently, video lab guides were produced. Previously, students performed similarly on laboratory practicals whether videos supplemented instruction or not. However, the previous cohort was introduced to the videos late in the course when overall scores improve due to experience with the testing format and may have masked an effect of the videos. Here we compared student performance on laboratory practical questions for two labs, one with (n=7 questions) and one without (n=9) video supplementation for the first quiz of the semester when the students were naive to the practical testing format. 123 students participated in the course. Numbers of students answering questions correctly did not significantly differ (p=0.6) for either instructional format. The previous cohort rated videos an average of 4.79 on a scale of 1 to 5 for benefit in studying. Student perception does not appear to reflect actual improvement in performance with the use of videos.
Refining the timeline of conjunctival papillae induction in the chick scleral ossicle system
Authors: Jourdeuil Karyn , Franz‐Odendaal Tamara
Published: June 2021

The conjunctival papillae, epithelial thickenings on the surface of the eye, are required for the induction of the underlying neural crest‐derived, intramembranous bones (the scleral ossicles). However, very little research has been conducted in the past 50 years on the mechanisms underlying the induction and patterning of these papillae. In order to further investigate this question, single papillae were surgically removed using fine tungsten needles at various time points during their development and the embryos were cultured for 1 to 5 days post‐surgery. Our results show that papilla removal had no effect on the patterning of the adjacent conjunctival papillae, and surprisingly, a normal patterned scleral ossicle develops under the site of surgery. This research demonstrates that the developmental timeline of the conjunctival papillae is much more flexible than previously noted. Our results also suggest that there is some compensatory mechanism taking place at the epithelial level for the induction of a complete ring of scleral ossicles. Thus, this study provides new insights into the mechanisms patterning the epithelium involved in epithelial‐mesenchymal induction of intramembranous bones. This research was primarily funded by the Natural Sciences and Engineering Research Council of Canada.

Grant Funding Source: Natural Sciences and Engineering Research Council of Canada
Multiscale modeling goes out on a limb: in silico simulations of developmental mechanisms shared between somitogenesis and the developing embryonic avian limb bud
Authors: Gens J. Scott , Srividhya Jeyaraman , Belmonte Julio M. , Hester Susan D. , Glazier James A.
Published: June 2021

Avian limb buds form primarily from a mixture of ectodermal and mesenchymal cells. The mesenchymal cells derive from two sources, the lateral plate mesoderm and the somites. Somite‐derived structures in mature limbs include muscles, tendons, and vascular endothelium. Limb development potentially shares many regulatory pathways and developmental mechanisms with somitogenesis, such as WNT and FGF8 regulation of cell proliferation, MET transitions, and differentiation. Although some mechanisms are likely to be conserved, tissue‐specific contextual cues are likely to introduce differences both significant and subtle. We present three‐dimensional, multi‐scale, multicell models using the open‐source CompuCell3D modeling environment to simulate these experimentally‐observed developmental mechanisms. We combine sub‐cellular (genetic and signaling networks), cellular (cell growth, death, division, differentiation and motility) and tissue (extracellular matrix and morphogen production) level scales to explore if combining WNT, FGF8 and Delta/Notch signaling (active during somitogenesis) with contact inhibition of growth in the ectoderm and the formation and maintenance of the Dorsal‐Ventral boundary at the apical ecotodermal ridge suffices to generate the unique patterning and growth dynamics of the limb bud. Supported by NIH 2R01GM076692–07and EPA R‐10–0049A3.

Grant Funding Source: N/A
Testing hypotheses of growth in the developing chicken limb bud
Authors: Fowler Donald Alexander , Searle Eric B , Button Christine , Chan Edna K , Larsson Hans C. E.
Published: June 2021

Morphogenesis is not only a story of genes and phenotypes, but also one of growth and movement. To explain the large‐scale changes in shape and size that the developing vertebrate limb undergoes, a more complete understanding of mechanisms acting between gene expression and the developed phenotype is needed. Recent evidence has indicated that there is an anterior to posterior direction in the relative movement of cells compared to the contour of the chick wing bud at an early stage, HH16 (Hamburger and Hamilton, 1951, Wyngaarden et al., 2010; doi: 10.1242/dev.046987). Although older evidence has indicated that at later stages (HH20–27) there is an opposite posterior to anterior movement of cells relative to the margin of the limb bud (Vargesson et al., 1997; Development 124). Dii injections into the developing chicken wing at various stages were performed to establish the timing and directionality of movements of cells relative to the margin of the developing limb. We found these anterior to posterior movements of cells relative to the movement of the wing margin occur only at a restricted early phase of limb development and posterior to anterior movements are present generally throughout the remainder of limb bud outgrowth.

Grant Funding Source: by NSERC, McGill University, and a Canada Research Chair to HCEL
The chick embryo neural crest model system reveals a unique role for EphB6 in melanoma metastasis
Authors: Bailey Caleb Michael , Kulesa Paul M
Published: June 2021

We hypothesize that melanoma metastasis mimics aspects of the emigration program of its ancestral cell type, the embryonic neural crest. To test our hypothesis, we performed a molecular comparison between melanoma and the neural crest. Our study revealed that melanoma cells exploit portions of the embryonic neural crest development program to facilitate invasion. In particular, the purported metastasis suppressor EphB6 was reduced in invasive melanoma cells compared to poorly invasive melanoma cells and primary human melanocytes. Thus, we utilized the chick embryo transplant model system to assay the effects of EphB6 re‐expression in melanoma cells. We report that EphB6 over‐expression alters cell directionality of migrating melanoma cells, while also reducing invasive ability. We confirmed the anti‐metastatic effect of EphB6 in melanoma cells using a chick CAM metastasis assay, while tumor formation appeared unaffected. These results suggest that EphB6 acts specifically as a metastasis suppressor, likely at the metastatic step of intravasation. Our studies also demonstrate the chick embryo as a powerful tool with which to compare and dissect the complex mechanisms of metastasis in vivo that are similar or distinct from normal developmental processes. This work was supported by NRSA 1F32CA144297 (cmb) from the NIH, and by the Stowers Institute for Medical Research.

Grant Funding Source: NIH 1R01HD057922
Role of Ku in the DNA damage response activated from telomeres
Authors: Fell Victoria , Schild‐Poulter Caroline
Published: June 2021

The Ku heterodimer, composed of two subunits, Ku70 and Ku80, is the main DNA binding component of the non homologous end joining (NHEJ) DNA repair pathway. Intriguingly, Ku is also present at telomeres and is required for telomere protection and regulation, but its role in telomere maintenance is not fully understood. We previously showed that Ku70 S155 is involved in regulation of the DNA damage response leading to apoptosis in response to ionizing radiation (IR). Here we show that expression of Ku70 bearing a phosphomimetic substitution (Ku70 S155D) produces a robust DNA damage response, marked by an upregulation of phospho‐H2AX and ATM activation, even in the absence of any DNA damage treatment. Telomere FISH analysis showed colocalization of γ‐H2AX foci with telomere ends. Cells expressing Ku70 S155D displayed cell cycle arrest, in particular in the G2 phase and upregulation of several factors involved in cell cycle arrest. Conversely, cells expressing Ku70 with a S155 alanine substitution (S155A) showed an increased cell growth rate and downregulation of genes involved in cell cycle checkpoints. This suggests that the phosphorylation status of Ku70 S155 regulates the DNA damage response from telomeres to control cell cycle progression, providing a novel role for Ku as a signaling factor at mammalian telomeres.
DNA Damage Repaired by the Base Excision Repair Pathway Is Epigenetic
Authors: Strauss Phyllis R , Toomire Kim , Moore Stephen P.G.
Published: June 2021

Many of the enzymes involved in BER are embryonic lethals and at least one (AP endonuclease1) controls protein levels of the critical transcription factor Creb1. We examined whether alterations in the Creb1 consensus sequence repaired by BER affect Creb binding to DNA, using purified Creb1 and a 39‐mer oligonucleotide containing the Creb consensus sequence. Kd values, measured by electrophoretic mobility shift assay, were obtained for unmodified ds oligonucleotide and for one where one or more residues in the consensus sequence were replaced by U or T, 8‐oxoguanine (oxoG) or intermediates in the BER pathway on either upper or lower strand. Conversion of one G residue to oxoG but not the other enhanced recognition by Creb. A U residue opposite one G residue markedly enhanced Creb binding even more than replacement with oxoG but insertion opposite the other markedly decreased binding. Replacement of selected residues with various intermediates in the BER pathway or a T residue interfered with Creb binding. Only one alteration completely prevented dimerization. Since Creb recruits its binding partners as a dimer, this alteration abolishes Creb function. We conclude that DNA modifications repaired by BER control transcription factor binding and constitute a hitherto unrecognized source of epigenetic modulation. *Supported by the G. Harold and Leila Y. Mathers Charitable Foundation and Northeastern University.
ATP‐Dependent Heterochromatin Remodeling
Authors: Manning Benjamin Jon , Peterson Craig
Published: June 2021

Heterochromatin maintains genomic stability by inhibiting aberrant recombination. DNA lesions, however, still occur at heterochromatic loci. Therefore, a cell must have ways to circumvent heterochromatin structure during repair of DNA damage. Prior work by our lab identified a role for the chromatin remodeling enzyme SWI/SNF in heterochromatic DNA repair. Here, we present biochemical studies that suggest two distinct mechanisms by which SWI/SNF remodels heterochromatin in an ATP‐dependent manner. The first remodeling mechanism uses a unique physical interaction, between the BAH domain of Sir3p and the ATPase domain of the SWI/SNF catalytic subunit Swi2p, to evict Sir3p from nucleosomal arrays. The second remodeling mechanism occurs between SWI/SNF complex and Sir2/3/4 complex; this reaction results in the eviction of both the histone octamer and bound Sir proteins. These findings establish SWI/SNF complex as a unique regulator of heterochromatin structure.
Regulation of Glucocorticoid Production by Nuclear Lipid Metabolism
Authors: Sewer Marion B
Published: June 2021

Glucocorticoids are produced in response to activation of a cAMP signaling cascade that increases the interaction of the nuclear receptor steroidogenic factor 1 (SF1) with the promoters of multiple target genes. We have previously identified phosphatidic acid (PA) and sphingosine (SPH) as an agonist and antagonist for SF1, respectively. Based on the nuclear localization of SF1, we hypothesized that cAMP signaling regulates the ability of SF1 to activate target genes by modulating the nuclear concentrations of PA and SPH. We show that cAMP rapidly activates nuclear phospholipid and sphingolipid metabolism, where PA production is catalyzed by diacylglycerol kinase theta (DGKQ) and SPH biosynthesis by acid ceramidase (ASAH1). Ligand binding is facilitated by the ability of these lipid metabolizing enzymes to bind directly to the receptor. Silencing ASAH1 increases the capacity of adrenocortical cells to secrete cortisol, primarily by increasing the expression of genes required for steroid hormone production. In contrast, suppression of DGKQ expression attenuates the ability of SF1 to activate target gene transcription and impairs hormone biosynthesis. Collectively, we show that ASAH1 and DGKQ are novel coregulatory proteins that regulate SF‐1 function by directly binding to the receptor on SF‐1‐target gene promoters and identify a key role for nuclear lipid metabolism in regulating gene transcription.
Pch2 is a meiotic hexameric ATPase that binds to and alters Hop1 functions
Authors: Chen Cheng , Jomaa Ahmad , Ortega Joaquin , Alani Eric
Published: June 2021

In most organisms, the segregation of chromosomes during the first meiotic division requires at least one crossover between pairs of homologous chromosomes. Crossovers come from programmed double‐strand breaks (DSBs) that are preferentially repaired using the homologous chromosome as a template (this preference is called interhomolog bias). In budding yeast, PCH2 is required to establish proper meiotic chromosome structure, regulate timing of recombination progression and promote interhomolog bias. How Pch2 functions at the molecular level is unknown despite its wide conservation. PCH2 open reading frame contains regions homologous to AAA ATPase proteins, and here we show that: 1. Pch2 is an ATPase that forms a hexamer in the presence of nucleotides. 2. Both ATP binding and hydrolysis by Pch2 are critical for its in vivo functions. 3. Pch2 binds to Hop1, an axial component of the synaptonemal complex, and alters its DNA binding activity. Based on these and previous cell biological observations, we propose that Pch2 acts as a stringency factor in meiosis to facilitate Hop1 binding to specific chromosomal regions, thus licensing steps that monitor recombination progression and lead to interhomolog bias.

C.C. and E.A. are supported by NIH GM53085. J.O. is supported by Canadian Institutes of Health Research (MOP‐82930). A.J. is supported by a CIHR Doctoral Research Award.
Molecular delineation of BubR1 kinase function in kinetochore microtubule attachments
Authors: Jiang Hao , Huang Yuejia , Ding Xia , Yao Xuebiao
Published: June 2021

Accurate segregation of chromosomes into two daughter cells is essential for cell health, which is governed by spindle checkpoint surveillance. The spindle checkpoint is a signaling cascade ensures the correct kinetochore‐microtubule attachment and prevents aberrant chromosome segregation. It has been shown that checkpoint kinase BubR1 stabilizes kinetochore‐microtubule attachment and facilitates correcting aberrant microtubule attachments. To dissect spatiotemporal property of BubR1 during cell division, a combination of a phenotype‐based screens judging kinetochore microtubule attachment with enzymatic assay was employed to identify compounds that affect mitotic checkpoint kinase BubR1. One compound, here named bubrestin, prevents chromosome alignment and stable kinetochore attachment without alteration of cell cycle progression through S and G2. The bubrestin‐treated cells exhibit aberrant kinetochore attachments such as syntelic and merotelic. A combination of bubrestin with proteomic analyses has uncovered a list of BubR1 substrates implicated in faithful kinetochore microtubule attachment. One candidate is TIP150, a newly characterized microtubule plus‐end tracking protein. Importantly, inhibition of BubR1 kinase activity perturbs the localization of TIP150 and plus‐end microtubule dynamics. Consistent with this finding, non‐phosphorylatable TIP150 failed to track plus‐end and exhibited a dominant negative phenotype. We propose that BubR1 links regulation of chromosome‐spindle attachment to microtubule plus‐end dynamics.
Site‐directed mutagenesis, expression, purification and translesion synthesis analysis of human DNA polymerase η mutations found in xeroderma pigmentosum variant and melanoma patients
Authors: Kumar Mukesh , Jiang Xiaohua
Published: June 2021

The variant form of human syndrome Xeroderma Pigmentosum (XP‐V) is caused by mutations in DNA polymerase η (hpolη). XP‐V patients are deficient in repairing the DNA damage caused by ultraviolet (UV) light in the form of cyclobutane‐pyrimidine dimers (CPD). Many missense mutations in hpolη catalytic core (1–432aa) have been identified in XP‐V patients, and mutations have also been implicated in Melanoma. The aim of current research is to confirm that the catalytic core of hpolη is 1–432aa. Hpolη (1–432aa) has been constructed. It will be expressed in E.coli and tested in primer extension assays to assess its effectiveness during translesion synthesis (TLS). Several of the point mutations have been successfully introduced in to hpolη (1–432aa) by site‐directed mutagenesis. Those mutants are expressed in E.coli cells and will be examined for their activity during translesion synthesis (TLS). Source of research support: Student research grant to Mukesh Kumar, Department of Chemistry, TTU, Cookeville, TN, Faculty grant to Xiaohua Jiang, College of Arts and Sciences, TTU, Cookeville, TN
Water oxidation chemistry of photosystem II
Authors: Brudvig Gary
Published: June 2021

Photosystem II (PSII) uses light energy to split water into protons, electrons and oxygen. In this reaction, Nature has solved the difficult chemical problem of efficient four‐electron oxidation of water to yield O2 without significant side reactions. In order to use Nature's solution for the design of materials that split water for solar fuel production, it is important to understand the mechanism of the reaction. The recent 1.9 Å X‐ray crystal structure of cyanobacterial PSII provides information on the structure of the Mn and Ca ions, the redox‐active tyrosine called tyrosine‐Z, chloride and the surrounding amino acids that comprise the oxygen‐evolving complex (OEC). However, reduction of the high‐valent Mn ions in the OEC during X‐ray exposure makes the redox state of the OEC represented in the crystal structure uncertain. Computational modeling has been used to refine the structure of the OEC in specific redox states by using spectroscopic data as a benchmark. In addition, EPR and ENDOR spectroscopy has been used to probe the binding of the substrate waters to Mn in the OEC by using 17O‐labeled water. The structure of the OEC in the intermediate oxidation states of the catalytic cycle, the binding of substrate water molecules to the OEC and the water oxidation chemistry of PSII will be discussed in the light of biophysical and computational studies, inorganic chemistry and X‐ray crystallographic information.
IGF1 Growth Hormone Response Element Undergoes Developmentally Regulated Demethylation Independent of STA5b
Authors: McKnight Robert A , Wiedmeier Julia , Yu Xing , Callaway Christopher W , Lane Robert H
Published: June 2021

The insulin‐like growth factor 1 (IGF1)/growth hormone (GH) axis, a major factor in growth, development and metabolism, is significantly affected by intrauterine growth retardation. Some of these alterations may lead to permanent pathological programming of the IGF axis. Prenatally, IGF1 signaling is GH independent, while postnatally, it is partly or fully GH dependent. GH regulates IGF1 via binding of the intermediate transcription factor, STAT5b, to a GH response element (GHRE) in the hepatic IGF1 gene. During adolescence, the rodent hepatic GHRE converts from closed to open chromatin and remains open throughout life. However it is not known when epigenetic events first begin to occur that prepare the chromatin for opening. CpG methylation and demethylation is often associated with closed and open chromatin respectively. We therefore sought to follow changes in DNA methyaltion in the GHRE as a possible indicator of transition to open chromatin. The GHRE was subjected to Na‐bisulfite sequencing at various time points in mouse development in wild‐type and STA5b knockout mice. The GHRE was also fused to a CG‐free reporter to test the effect of methylation on enhancer activity. The GHRE undergoes 3 stages of gradual DNA demethylation declining from a high of 80% methylation in embryonic stem (ES) cells to <30% by 5 weeks of life and remains hypomethyated for 2 yrs. Demethylation of the mouse GHRE is independent of STAT5b and is required for GHRE function in vitro.
High Fat Diet Induced Genome‐wide Differential Methylation Affecting Hepatic Signaling Pathways In Rat
Authors: Zhang Yukun , Zhou Dan , Ma Jian , Pan Yuan‐Xiang , Chen Hong
Published: June 2021

High fat diet is associated with progression of liver diseases. We hypothesize that high fat diet changes genomic landscape of DNA methylation, leading to differential expression of genes in hepatic pathways involved in pathological development. Methyl‐DNA immunoprecipitation assay in combination with high‐throughput sequencing (MeDIP‐seq) and methylation‐sensitive restriction enzyme sequencing were applied to illustrate genomic landscape of DNA methylation that is modified by high fat diet in liver of control and high fat fed rats. Genome‐wide analysis of differentially methylated regions (DMR) showed that 8294 DMRs induced by high fat diet were hypomethylated and 4622 were hypermethylated. DMRs with gene annotations (DMG) were further analyzed to show gene‐specific methylation profile. There were 88, 2680, and 95 hypomethylated DMGs identified within promoter; intragenic and downstream regions, respectively, comparing to fewer hypermethylated DMGs (45, 1623, and 50 in the respective regions). Pathway analysis showed that DMGs were involved in inflammatory pathway, fibrosis development, insulin response, cell cycle control, and lipid metabolism. We for the first time analyzed the global impact of high fat diet on the DNA methylation profile in liver of rats. Our finding identified epigenetic mechanisms of the effects of high fat on critical signaling pathways related to liver functions.
The Detection of 5‐hydroxymethylcytosine in Trypanosoma brucei DNA
Authors: Valentine Erika , Smindak Richard , Militello Kevin T
Published: June 2021

The goal of this project is to determine whether or not the modified base 5‐hydroxymethylcytosine is present in T. brucei nuclear DNA. T. brucei is the causative agent of African Sleeping Sickness, and has been previously shown to contain 5‐methylcytosine, 5‐hydroxymethyluracil, and base J. The T. brucei J‐base binding proteins (JBPs) synthesize 5‐hydroxymethyluracil, and are homologous to the mammalian TET (ten‐eleven translocation) proteins which synthesize 5‐hydroxymethylcytosine. Thus, we believe is it possible that the T. brucei JBPs synthesize 5‐ hydroxmethylcytosine in addition to 5‐hydroxymethyluracil. To test this model, an immunoblot assay was developed to detect 5‐hydroxymethylcytosine in DNAs spotted onto a nylon membrane. Our data indicate that both T. brucei bloodstream form and insect form DNA contain low but detectable levels of 5‐hydroxymethylcytosine. The same results were also generated using ELISA assays specific for 5‐hydroxymethylcytosine. We are currently using LC MS/MS to confirm the experiments above, and hydroxymethylated DNA immunoprecipitation experiments to determine the location of 5‐hydroxymethylcytosine in the T. brucei genome.
Large‐scale expression and purification of active pseudolysin in Escherichia coli
Authors: Tarpley Reid S. , Adekoya Olayiwola A , Sylte Ingebrigt , Odunuga Odutayo O
Published: June 2021

Pseudolysin, the extracellular elastase of Pseudomonas aeruginosa, is a member of the thermolysin‐like family of proteases. Pseudolysin has been identified as a key virulence factor in pathogenesis of P. aeruginosa infections due to its proteolytic activity on host tissue proteins causing severe hemorrhagic activity and weakening of the host defense mechanisms. The strong involvement of metalloendopeptidases in various diseases and infections makes them robust drug targets for therapeutic applications. Previous attempts to purify pseudolysin directly from P. aeruginosa or by heterologous expression in Escherichia coli and yeast yielded insufficient quantities of the enzyme for structural studies. We report the successful large‐scale heterologous expression of a correctly folded and active 33‐kDa pseudolysin in E. coli. The open reading frame coding for the secreted posttranslationally modified 33‐kDa pseudolysin was cloned into a plasmid vector and the protein product expressed in fusion with maltose‐binding protein (MBP). The MBP‐pseudolysin fusion protein expressed highly in E. coli and was found to be sufficiently soluble. Untagged pseudolysin obtained after cleaving the fusion protein with tobacco etch virus protease was soluble, stable and found to be active based on its ability to hydrolyze the Gly‐Leu bond in the substrate, 2‐aminobenzoyl‐Ala‐Gly‐Leu‐Ala‐4‐nitrobenzylamide.
The disassembly of post‐termination complex (PoTC) by ribosome recycling factor (RRF) and EF‐G occurs first with the releases of tRNA, mRNA, and splitting of the ribosome in its order ‐‐ IF3 does not participate in this reaction
Authors: Kaji Akira , Iwakura Nobuhiro , Kaji Hideko
Published: June 2021

Protein synthesis consists of four steps, initiation, elongation, termination, and ribosome recycling. The last step consists of three reactions; 1) tRNA release, 2) mRNA release from the PoTC, and 3) splitting of ribosomes into subunits. In E. coli, all three reactions are catalyzed by RRF and EF‐G/GTP. It has been suggested that IF3 participates in the splitting of ribosomes of PoTC (NAR 33, 5591–5601, 2005). Splitting has been measured with fluorescent labeled ribosomal subunits (Mol Cell 18, 403–412, 2005) which can detect the conformational change of ribosomes without splitting. The split subunits rapidly re‐associate. To prevent this, IF3 was added (RNA 11, 1317–1328, 2005). In other cases external 50S subunits were added and the split subunits (labeled) were observed as 70S ribosomes (Mol Cell 18, 675–686, 2005). These are indirect methods to measure splitting of ribosomes. In this paper, we haracterized PoTC as having one tRNA (P/E site) and mRNA. We further show direct evidence of splitting of PoTC without addition of IF3 by conducting the experiment in 4 mM Mg++. Using this system, kinetic analysis and measurement of subunits formation from the PoTC are consistent with the hypothesis that the release of tRNA takes place first followed by the mRNA release and the splitting of 70S ribosomes in this order. IF3 does not participate in any of these reactions. This research was supported by CBRI.
Actin Bundling Mutants in Translation Elongation Factor 1A Induce Altered Protein Synthesis at both the Initiation and Elongation Steps
Authors: Perez Winder B , Kinzy Terri G.
Published: June 2021

Apart from its canonical function in translation elongation, eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with the actin cytoskeleton. Amino acid substitutions in eEF1A that reduce its ability to bind and bundle actin in vitro, cause improper actin organization in vivo, as well as reduce total translation. Although initial analysis indicated the reduced translation was through initiation, in vitro analysis of the mutant proteins shows changes at the level of elongation. These strains exhibit increased levels of phosphorylated initiation factor 2α (eIF2α), suggesting the involvement of the Gcn2p protein kinase. Gcn2p is a key regulator of the general amino acid control (GAAC) pathway by responding to increases in uncharged tRNA levels in the cell and causing downregulation of total protein synthesis at the level of initiation. However, these eEF1A mutant strains do not exhibit increased levels of uncharged tRNAs, suggesting an alternate mechanism of Gcn2p activation. Thus, we hypothesize there is interplay between the translation initiation and elongation defects in eEF1A actin bundling mutant strains. The recent finding that Gcn2p and eEF1A directly interact sheds light on this new function. The interactions between Gcn2p and eEF1A actin bundling mutants as well as the specific functions of eEF1A within elongation are being assessed.

Support: NIH 5T32AI007403–18
Cloning and Characterization of Leucyl‐tRNA Synthetase from Pseudomonas aeruginosa
Authors: Medellin Eduardo , Salazar Humberto , Quach Nathalie , Chavero Pamela , Bullard James
Published: June 2021

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of nosocomial infections. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. We describe here the cloning and enzymatic characterization of the class I leucyl tRNA synthetase (LeuRS) from P. aeruginosa.

Results

LeuRS from P. aeruginosa was cloned and expressed in E. coli and purified to greater than 98% homogeneity. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl‐tRNA synthetases. The ability of LeuRS to function in aminoacylation of its cognate tRNA was determined in tRNA aminoacylation assays. The kinetic parameters for the interaction of P. aeruginosa LeuRS with its three substrates (tRNA, ATP, leucine) were determined. Initial velocities were determined for charging of tRNA using tRNA concentrations between 6.5 and 10 μM tRNALeu. The KM and Vmax for the interaction of LeuRS with the substrate tRNALeu was determined to be 7.14 μM and 0.36 μM min−1, respectively. The ATP:PPi exchange reaction was used to monitor interaction with ATP and leucine.

Conclusion

LeuRS identified in P. aeruginosa was cloned, expressed and purified and shown to be functional in assays suggesting that it is functional in protein synthesis. Research was partially supported by NIH grant 1SC3GM098173–01A1.
Cloning and characterization of EF‐Tu and EF‐Ts from Bacillus subtilis
Authors: Rivera Carlos , Garcia Velia , Bullard James
Published: June 2021

Introduction

Elongation factors (EF) facilitate many steps in the process of the biosynthesis of proteins. EF‐Tu in particular plays a central role in the GTP‐dependent placement of aminoacylated tRNA at the A site of the ribosome. EF‐Ts acts by catalyzing the change of EF‐Tu from a GDP‐bound inactive state to a GTP‐bound active state. Much work has been done with elongation factors from Gram − bacteria but little work has been carried out to analyze the activity of these proteins from Gram + organisms. We describe here analysis of EF‐Tu and EF‐Ts from B. subtilis.

Results

Analysis of both B. subtilis EF‐Tu and EF‐Ts showed they are 77% and 47% identical to their E. coli counterparts, respectively. Both B. subtilis EF‐Tu and EF‐Ts were cloned and over‐expressed in E. coli and purified to greater than 95% homogeneity. B. subtilis EF‐Tu was shown to be active when assayed for activity using the GDP binding assay. Kinetic parameters for the interaction of EF‐Tu with its substrate GDP were determined in the absence and presence of EF‐Ts. The dissociation constant (KGDP) for EF‐Tu•GDP was determined by forming the complex at 4 °C and diluting the complex 100 fold and measuring the amount of complex remaining at different times at various temperatures. EF‐Ts was shown to stimulated the exchange of GDP by EF‐Tu. EF‐Tu was also shown to be active in ternary complex formation as well as delivery of aminoacylated tRNA to the A site on B. subtilis ribosomes.

Conclusion

EF‐Tu and EF‐Ts identified in B. subtilis were cloned, expressed and purified and shown to be functional in assays suggesting that each is functional in protein synthesis.
Evolution of ribosomal RNA: universal and domain‐specific conserved sequences
Authors: Gerbi Susan A , Doris Stephen M , Smith Deborah , Raphael Benjamin , Beamesderfer Julia , Nathanson Judith
Published: June 2021

We present a new bioinformatics approach to identify Conserved Nucleotide Elements (CNEs) in the three domains of life (Eukarya, Bacteria, Archaea), that play important functional roles. Using ARB/SILVA, we developed a Full Length Organismal rRNA Alignment database (FLORA) that was filtered to include only complete rRNA sequences, each entered only once in the database. Our analysis identified 63, 49 and 48 CNEs that are ≥6 nt in 23S–28S rRNA of Eukarya, Bacteria and Archaea. Nine of these CNEs are universally conserved (uCNE) in all domains of life. They map to area of known function, but also to areas of undiscovered function in rRNA. In addition, structure‐based quantification of motifs across all domains of life identified domain‐specific CNEs: 12 uniquely conserved in all Eukarya, 3 only in Bacteria and 1 only in Archaea. Thus, domain specific CNEs are largely a eukaryotic phenomenon, and represent new structural features in eukaryotic ribosomes. We are completing a similar analysis to identify CNEs in the small ribosomal subunit, where we have identified 26, 24 and 24 CNEs in 16S–18S rRNA of Eukarya, Bacteria and Archaea.

(NSF grants MCB‐0718714, MCB‐1120971)
Characterization of putative mitochondrial processing peptidase α‐subunit in Trypanosoma brucei
Authors: Mak Ivy Lynn , Lu Zhiquan , Mehta Vaibhav N , Salavati Reza
Published: June 2021

Trypanosoma brucei, the causative agent of African sleeping sickness, contains a single mitochondrion which imports proteins responsible for RNA editing process. Using RNA interference (RNAi), this study aims to characterize the gene Tb 11.02.1480, annotated as mitochondrial processing peptidase α‐subunit (MPP‐α) that is believed to cleave mitochondrial import signal to aid protein maturation. Severe growth inhibition resulted from RNAi. Analysis of transcript level by RT‐PCR showed a 2–8 fold accumulation in pre‐edited mRNAs that encode electron transport chain components apocytochrome b, ATP synthase subunit 6 and NADH‐ubiquinone oxidoreductase subunit 3 (ND3), along with relatively unchanged levels of never‐edited mRNAs ND4 and cytochrome oxidase 1. Mitochondrial extract prepared from glycerol gradient fractionation suggest disruption of editosome integrity, although maturation of editosome subunit KREL1 was unaffected. These observations suggest that the proposed MPP‐α in T brucei might not be involved in protein import as reported in other species; but impaired RNA editing activity suggest this protein contributes to editosome stability. This was supported by computational analysis where this protein appears to interact with editosome accessory proteins MRP. Ongoing work on identification of protein interacting partners would allow further elucidation of gene function.
Structural insights into RNase T in RNA maturation and DNA repair
Authors: Yuan Hanna S. , Hsiao Yu‐Yuan , Chu Woei‐Chyn
Published: June 2021

RNA maturation relies on various exonucleases to remove nucleotides successively from the 5′‐ or 3′‐end of nucleic acids. However, little is known regarding the molecular basis for substrate selection and cleavage preference of exonucleases. Here, we show how RNase T makes the final 3′‐end trimming to generate the mature ribosomal and transfer RNA with a precise 3′‐end by biochemical and structural approaches. Our crystal structural analyses on various RNase T‐DNA complexes show that RNase T dimer has an ideal architecture for binding a duplex with a short 3′ overhang to produce a digestion product of a duplex with 1 to 4 nucleotide overhangs depending on the sequence and structure of the substrates. Our results reveal the general principles for the final trimming step made by RNase T in the maturation of stable RNA. We also found that RNase T participates in trimming of structural intermediate DNA during DNA repair. The crystal structures of RNase T bound with a bubble and a Y‐structure DNA further demonstrate how RNase T binds and trims these structural DNA. These studies thus provide molecular insights into the nucleic acid 3′‐end trimming mechanism by RNase T during RNA maturation and DNA repair. These results can be extrapolated to the thousands of members of the RNase T superfamily of exonucleases, some of which are linked to human diseases.
Investigation of Functionally Critical Residues in the Pseudo‐ Helicase Domain of Brr2
Authors: Anguiano David , Brittsan Christine M , Titus James K , Boswell William , Guthrie Christine , Maeder Corina
Published: June 2021

Pre‐mRNA splicing is the removal of introns from mRNA to create mature mRNA. The conformational rearrangements that are required to remove introns are driven by a family of helicases called DExD/H box proteins. A member of this family of helicases, Brr2, is an RNA dependent ATPase that unwinds U4/U6 prior to catalytic activation of the spliceosome and unwinds U2/U6 during disassembly. Brr2 has a unique structure composed of an N‐terminal domain and two modules that resemble the DNA Helicase Hel308. Previous work identified the Hel308‐I domain of Brr2 contains the DEIH motif and is responsible for Brr2's helicase activity. The Hel308‐II domain of Brr2 lacks the canonical DExD/H motif and is not directly responsible for RNA unwinding. However, the domain is conserved, implying the domain's importance to the protein. Using Saccharomyces cerevisiae, we have identified cold‐sensitive and temperature‐sensitive mutations in the Hel308‐II domain, including several mutations that cluster around the putative ATP binding pocket. These findings support a functional importance of ATP binding to this region. We will present data on the ATP binding properties of these mutations and their effects on in vitro splicing.
The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)‐associated C9ORF72 r(GGGGCC)n hexanucleotide repeat forms extremely stable uni‐and multi‐molecular RNA G‐quadruplex structures
Authors: Reddy Kaalak , Zamiri Bita , Stanley Sabrina , Macgregor Robert , Pearson Christopher
Published: June 2021

G‐rich nucleic acids in promoters, telomere repeats, immunoglobulin class switch motifs, and fragile X repeats can form G‐quadruplex structures. Quadruplexes can affect promoter activity, splicing and neurite mRNA targeting. Amyotrophic lateral sclerosis (ALS), commonly referred to as Lou Gehrig's disease, and frontotemporal dementia (FTD) were recently shown to be caused by expansion of a G‐rich (GGGGCC)•(CCCCGG) hexanucleotide repeat in the C9ORF72 gene and these mutant transcripts can aggregate in nuclear foci possibly sequestering repeat‐binding proteins. Here we demonstrate that the (GGGGCC)n RNA forms extremely stable uni‐ and multi‐molecular parallel G‐quadruplex structures, whose formation is influenced by repeat length. Furthermore, MBNL1, a structure‐specific splicing factor that is sequestered in myotonic dystrophy type 1 patients by binding to expanded CUG RNA repeats, does not bind the ALS‐FTD RNA hexanucleotide repeat, while the ASF/SF2 splicing factor can bind the repeat. These observations may shed light on the normal (GGGGCC)•(CCCCGG) repeat function in C9ORF72, its interacting partners, and upon pathogenic roles the expanded repeat may have in ALS‐FTD.
The effect of HSF‐1 and HSP70 on autophagy regulation
Authors: Dokladny Karol , Deretic Vojo , Moseley Pope L
Published: June 2021

Autophagy is an evolutionarily conserved mechanism that plays an important role in maintaining cellular homeostasis by recycling large protein aggregates, damaged proteins, entire organelles, and bulk of cytoplasm within double membrane vacuoles delivered for degradation to lysosomes. Heat stress response renders protection to macromolecules, cells, as well as whole organism by assisting in folding of newly synthesized proteins, in stabilizing of unfolded protein, and in degradation and reactivation of damaged proteins. Previously, we have reported that autophagy is regulated by heat stress response. The purpose of the study was to investigate the role of heat shock factor 1 (HSF‐1), the central regulator of heat shock response and heat shock protein 70 (HSP70), principal effector of heat shock response on starvation‐induced autophagy in A549 cells. We utilized HSF‐1 siRNA to inhibit HSF‐1 protein expression and adenovirus to overexpress inducible form of HSP70. Their effects on starvation‐ induced autophagy were tested. Exposure to HSF‐1 siRNA augmented the starvation‐induced increase in autophagy and HSP70 significantly blocked this increase. We conclude that heat stress response exert a regulatory role on autophagy.
Nuclear MMP‐2: presence and activity in cardiac myocytes
Authors: Kondo Marcia Yuri , Castro Michele Mazzaron , Fan Frank (Xiaohu) , Schulz Richard
Published: June 2021

MMP‐2 is expressed in almost all heart cells and is found in subcellular compartments including the nucleus. MMP‐2 can be directly activated by oxidative stress (as peroxynitrite). Its activity is further regulated by its phosphorylation. To better study nuclear MMP‐2 in the heart, we extracted the nuclear, cytosolic and membrane fractions from isolated rat hearts (briefly perfused to clear them of blood). Western Blots for lamin A/C (nucleus), GAPDH (cytosol) and VDAC/Porin (membrane) were used as specific markers to confirm fraction purity. Gelatin zymography of these fractions revealed MMP‐2 but not MMP‐9 activities and MMP‐2 activity was about 15 times higher in the cytoplasm than in the nucleus. Interestingly, Western blot analysis showed that MMP‐2 is present in similar amounts both in the nucleus and in the cytoplasm. In order to characterize possible regulators of MMP‐2 activity, we analyzed for the presence of tissue inhibitors of metalloproteases (TIMPs). Western blots revealed that all forms of TIMPs (1–4) are found in the cytoplasm, TIMP‐4 is also present in the membrane fraction, and no TIMP was detected in the nucleus. Our data suggest that myocardial MMP‐2 activity in the nucleus is regulated by a mechanism different from inhibition by TIMPs, possibly by its phosphorylation.

Funding: Canadian Institutes of Health Research.
Development of affinity purification systems for isolation and proteomic analysis of proteases from Tetrahymena thermophila
Authors: Lu Brian , Carreon Jay R. , Straus J. William
Published: June 2021

To better understand the functioning of proteases, we initiated a proteomic study to determine partial primary structures and to identify functional genes for intracellular cysteine proteases from Tetrahymena utilizing affinity chromatography and LCMS/MS (liquid chromatography‐tandem mass spectrometry). A peptidealdehyde tight‐binding protease inhibitor, antipain, was coupled to aminated agarose via carbodiimide condensation, and utilized to isolate proteases from clarified detergent extracts of late exponential phase cells. An immobilized lectin, concanavalin A (Con A), was used to isolate a subset of cellular glycoproteins from the detergent extracts. Protein isolates were subjected to trypsin digestion and the resulting peptides were further purified and sequenced by LCMS/MS. Cell extracts prepared with CHAPS detergent and purified against immobilized antipain, had fewer proteases and fewer contaminating proteins than Triton X‐100 extracts, but yielded higher quality spectra. Con A isolates from CHAPS extracts yielded few proteases. ESI Q‐TOF LC MS/MS confidently identified seven and tentatively identified four proteases from the extracts. Candidate genes for cysteine proteases in the Tetrahymena genome were subjected to phylogenetic analysis and the evolutionary relationships of the identified enzymes were examined. This work was funded by Vassar College and an HHMI‐USEP grant.
Contribution of an inner core hydrogen‐bonding network to β‐helix stability within the two‐partner secretion exotoxin family
Authors: Weaver Todd Michael
Published: June 2021

TPS pathways, harboring both a membrane bound β‐barrel B‐component (TpsB) and a β‐helical A‐component (TpsA), can be found within bacterial, fungal and animal kingdoms. Bacterial TpsA components, signified by adhesions, proteases, and hemolysins, adopt a biogenic beta‐helical fold concomitant with TpsB‐dependent secretion. β‐helix proteins (βHPs) have become a prevalent structural fold being associated with antifreeze proteins, β‐amyloid, and prion proteins. The research implemented HpmA265 to investigate the βHP structure‐function (SF) paradigm. Specifically, a series of site‐directed mutants (SDMs) were constructed to target the inner core hydrogen‐bonding network shared between Q125 and Y134 at β‐arc 2 within first β‐circuit. Each of these two residues was replaced singly or doubly with either alanine, serine or phenylalanine residues. Additionally, these were combined with two other SDMs aimed at disrupting a carboxyamide ladder forming a bridge between non‐consecutive parallel β‐strands. The SF relationship attributed to these various site‐directed alterations was analyzed functionally within a template‐assisted assay and structurally using circular dichroism. The phenylalanine SDMs provided the greatest loss in hemolytic activity coupled to a weakened β‐signal and 10 °C decrease in Tm. These studies illustrate that the hemolytic activity of HpmA265 is linked directly to structural integrity of the β‐helix fold. In addition, the intricate inner core hydrogen‐bonding network within β‐arc 2 of β‐circuit 1 appears to be an important factor leading to successful TpsA activation.