bluuebunny/crossref_metadata_2025 huggingface dataset:
Methods used in the gross anatomy lab to facilitate learning by first‐year students in a new Physician Assistant Program (722.1)
Authors: Syed Mohsin , Akhter Noor , Hayes Helen , Davies David
Published: June 2021

The University of Arkansas for Medical Sciences opened a new Physician Assistant (PA) Program in its College of Health Professions in the summer of 2013 with 26 students. This small class size ensured more individualized faculty‐student interaction that greatly facilitated the learning. Unlike most gross anatomy courses that use a regional approach, the PA students were taught with a system‐based approach. During the weekly 3‐hour lab sessions, anatomical structures were demonstrated on four prosected cadavers. Four second‐year medical students were enlisted to prepare these prosections, each of which required about 100 hours of dissection time. During scheduled lab sessions the PA students identified relevant structures using the prescribed atlas and with the help of 3 faculty members. A dedicated lab area (740 sq. ft.) was designated for the study of anatomical models, bones, previously dissected regional specimens and cross sections. A lab incentive program (LIP) was introduced to keep the students focused. Four computers loaded with anatomical tables, charts and images were accessible in the lab for self‐directed study. At the end of each organ system unit, major concepts, important structures and their clinical relevance were reviewed in the lab before proceeding to another organ system. Briefly, the effectiveness of these lab‐teaching strategies is quantitatively evidenced by the strong performance of students on examinations.

Supported by: Dept. of Neurobiology and Developmental Sciences, UAMS
E‐Learning: effective or defective? The impact of commercial e‐learning tools on learner cognitive load and anatomy instruction (725.7)
Authors: Van Nuland Sonya , Rogers Kem
Published: June 2021

Traditionally, educational researchers have focused on Cognitive Load Theory (CLT) to guide the design of novel e‐learning tools. However, there is little evidence to suggest that the design of commercially available e‐learning tools has been guided by educational theories, including CLT. As e‐learning tools gain popularity as methods of instruction in anatomy education, more research is merited to gauge the impact of these tools on learner cognitive load. This study aims to examine three commercially available anatomical e‐learning tools to determine their effect on learner cognitive load. Anatomy students at Western University will be invited to participate in this study, and will be randomly assigned to view all three e‐learning tools; each featuring the anatomy of a selected joint. To quantify the cognitive load that each tool places on the learner, a dual task methodology (a paradigm wherein the learner performs two tasks simultaneously) will be utilized. Learning material from an e‐learning tool will be assigned as a primary task and visual observation will be assigned as a secondary task. Learners will be assessed using a baseline anatomy knowledge test, secondary task response times, and an anatomy knowledge post‐test. We hypothesize that longer reaction times on the secondary task indicate a high cognitive load imposed by the primary task, and will result in poor performance on the knowledge post‐test.
Narciclasine effects on myofibroblast clustering (734.8)
Authors: Mahoney Madeline , Ilikj Marko , Vaughan Melville
Published: June 2021

Myofibroblasts are wound‐healing cells that differentiate from fibroblasts. They are characterized by the expression of alpha‐smooth muscle actin stress fibers. Narciclasine is a plant growth modulator that was shown to promote stress fibers in cancer cells and growth arrest in normal cells through the Rho‐kinase pathway. These same characteristics are involved in myofibroblast differentiation. Our goal was to determine whether narciclasine treatment was sufficient to induce myofibroblasts. Adult human fibroblasts were grown on coverslips and treated +/‐ TGF‐beta1 for 24 hours, followed by narciclasine treatment. Myofibroblast differentiation increased from control with narciclasine treatment but no difference was seen when narciclasine was added to TGF‐beta1 treated normal human fibroblast cultures; however, cell clustering was observed in Dupuytren’s treated fibroblasts under these conditions. Work is currently underway to understand the basis for cell clustering in myofibroblasts mediated by narciclasine.
The mismatch repair pathway maintains genomic stability in human pluripotent stem cells (735.1)
Authors: Lin Bo , Heinen Christopher
Published: June 2021

The mismatch repair (MMR) pathway repairs polymerase errors to help maintain genome stability and prevent tumorigenesis. In addition to repair, MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain forms of DNA damage. The role of MMR in preserving genome integrity in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) has not been previously examined. Accumulation of mutation in pluripotent stem cells (PSCs) would be detrimental to the developing organism so we hypothesize that these cells have a robust MMR pathway. We find that ESCs and iPSCs express high levels of MMR proteins and demonstrate strong mismatch repair activity. PSCs are also hypersensitive to DNA damage, showing a high level of apoptosis after treatment with a DNA alkylating agent that is MMR dependent. However, the specific response observed differs from that seen in HeLa or other cancer or primary cells. In these other cells, a permanent G2/M cell cycle arrest is induced in the second cell cycle after damage, however, PSCs directly undergo apoptosis in the first cell cycle. Interestingly, there is no activation of cell cycle checkpoint kinases CHK1 or CHK2, although phosphorylation of H2AX is increased. This more immediate apoptotic response suggests a separate molecular mechanism by which the MMR pathway can induce a response to DNA damage in PSCs.

Grant Funding Source: Supported by NIH CA 115783 and CT Stem Cell 13‐SCB‐UCHC‐06
Investigating the role of DNA repair enzymes in DNA replication and recombination (735.3)
Authors: Speed Clayton , Wiese Claudia , Parplys Ann , Hatherill J. Robert
Published: June 2021

Hypothesis: A mutant DNA repair protein (DSS1) will be unable to facilitate Homologous Recombination Repair (HRR) within osteosarcoma cell lines. Also depletions in the DNA repair enzyme RAD51AP1 will hinder the cells ability to rescue stalled replication forks and repair replication associated Double Strand Breaks (DSB), along with causing a S‐phase check point defect in cells. In this study DNA damage was induced via exogenous and endogenous stresses, to test the fitness of cells. To gain insight into the role DNA repair proteins play after DNA damage, mutant osteosarcoma cell lines were exposed to high intensity X‐rays. The ability for cells to faithfully repair double strand breaks was assessed by fluorescent activated cell sorting and immunocytochemistry. The levels of S phase check point activation by the phosphorylation of check point kinase1 was assessed by infrared Western blot analysis. We also depleted the DNA interacting enzymes and studied the effects on DNA damage, DNA replication, and the cell cycle. It was concluded that human RAD51AP1 expressed in chicken DT40 cells caused a checkpoint defect, leading to the cells inability to arrest after damage. This phenotype has been recently validated in human cell lines. The overall goal of this research was to determine the roles DSS1 and RAD51AP1 play in DNA repair and the interaction with other recombination mediators and effectors.

Grant Funding Source: NSF ATE DUE 1205059
Investigating the barrier that prevents penicillin catabolism genes from transferring from PE‐S1G‐1 to Acinetobacter baylyi strain ADP1 (735.5)
Authors: Bao Yiqiao , Lostroh Candi
Published: June 2021

Previous research discovered hundreds of phylogenetically diverse soil bacteria with the capacity to grow on a wide range of antibiotics as their sole carbon source. Some of them are closely related to human pathogens. The purpose of this study is to evaluate whether there is a barrier that might prevent the penicillin catabolism genes from transferring from PE‐S1G‐1 to Acinetobacter baylyi strain ADP1. Due to its natural competence and its close relation to human pathogen Acinetobacter baumannii, ADP1 is an ideal model for this study. Natural transformation with genomic DNA of PE‐S1G‐1, phi29 elongation with primers to add short homologous DNA to the genomic DNA, and Sau3A genomic plasmid libraries were used to test this barrier. None of these methods mentioned above transformed ADP1 to be a strain with penicillin catabolism phenotype. Surprisingly, a recent research challenges the concept of bacteria subsisting on antibiotics and argues that the selective media used in the original study (SCS) contains 15 mg/L EDTA which could be the carbon source that sustains the growth of these “antibiotic‐eaters.” The hypothesis that these bacteria could subsist on EDTA is currently being tested.
Poly‐Q aggregate trap interference with DNA repair genes in Huntington disease cells (736.10)
Authors: Hentosh Patricia , Sundin Tabetha , Mollica Pete
Published: June 2021

Huntington’s disease (HD)–an autosomal dominant neurodegenerative disease –is characterized by expansion of a (CAG)9‐34 trinucleotide sequence found in exon 1 of the human Huntingtin (HTT) gene. Posttranslational modifications of an expanded mutant Huntingtin protein (mHtt) renders polar poly‐glutamine (Poly‐Q) fragments that aggregate and interfere with cellular proteins. Poly‐Q aggregate traps (PQATs) hijack transcriptional regulators including cyclic AMP‐responsive element‐binding protein (CREB), CREB‐binding protein (CREB‐BP/CBP), transcription factor Sp1 and TATA‐box‐binding protein. CAG repeats form hairpin structures that typically trigger DNA replication fork stalling and are recognized by DNA repair enzymes. However, HD cells appear to have defective repair pathways in the absence of any known repair gene mutation. We hypothesized that PQATs are responsible for decreased transcriptional regulation of essential DNA repair genes, facilitating the characteristic trinucleotide expansion. Here we examined mRNA levels of cultured HD‐affected and unaffected fibroblasts from related individuals utilizing a RT2 Profiler PCR Array for human DNA repair genes. Our findings reveal down‐regulation of several DNA repair genes involved in base‐excision repair (BER), mismatch repair (MMR) and double‐strand break repair. Examination of their respective promoters revealed these genes have binding sites for the transcription factors Sp1, CREB and CREB‐BP/CBP. Thus, PQATs may contribute to decreased transcriptional regulation of multiple DNA repair genes. We suggest that diminished mRNA levels of an interconnected complex of repair genes involved in sensing a stalled replication fork as well as correcting a hairpin–rather than a single repair enzyme–contribute to repeat expansion.
HnRNP A2/B1 regulates alternative splicing of Tid1 isoforms (742.1)
Authors: Chen Chi‐Yuan , Chuang Yen‐Shu , Pi Wen‐Chieh , Wang Tzu‐Chien
Published: June 2021

Tid1, a DnaJ cochaperone, may promote degradation of oncogenic kinases. Tid1 is composed of 12 exons separated by 11 introns. Two alternatively spliced forms of Tid1 are expressed in humans, Tid1‐L and Tid1‐S. The Tid1‐S is generated by splicing of exon 10 to exon 12. Overexpression of Tid1‐S and Tid1‐L has been shown to produce opposing effects: cell death is repressed by Tid1‐S and enhanced by Tid1‐L. However, the mechanism of alternative splicing regulators to Tid1 isoforms development has been largely unknown. Heterogeneous nuclear ribonucleoproteins (hnRNPs) constitute a large family of proteins that associate with nascent pre‐mRNAs, packaging them into hnRNP particles. The A/B subfamily of hnRNPs (hnRNP A/Bs) are the most abundant hnRNPs in the nucleus. hnRNP A2/B1 is critical for precursor messenger RNA splicing. In this study, we tested the postulation that hnRNP A2/B1 may regulate alternative splicing of Tid1 isoforms. Our preliminary results indicated that knockdown of hnRNP A2/B1 in acute T cell leukemia‐derived Jurkat cells and lung cancer cells increased the expression of Tid1‐L gene and protein. Moreover, knockdown of hnRNP A2 produced a significant inhibition of cellular proliferation in Jurkat cells. These results suggest that specific knockdown for hnRNP A2/B1 decreases exclusion of exons 11 of Tid1 pre‐mRNA and influence cell proliferation of human cancer cells.
Understanding how two related RNA binding proteins determine different splicing outcomes (742.2)
Authors: Keppetipola Niroshika , Yeom Kyu‐Hyeon , Hernandez Adrian , Vashisht Ajay , Wohlschlegel James , Black Douglas
Published: June 2021

The process of alternative splicing is tightly regulated by RNA binding proteins that bind to corresponding cis‐acting elements and influence the assembly of spliceosomal components at adjacent splice sites. Many of these RNA binding proteins occur as gene families, with family members generally sharing high amino acid sequence identity and domain structure yet targeting overlapping but distinct sets of exons. PTBP1 (polypyrimidine tract binding protein 1) and PTBP2 (polypyrimidine tract binding protein 2) are paralogous RNA binding proteins. PTBP1 is well characterized as a splicing repressor. PTBP2, shares a similar domain structure and 74% primary structure identity to PTBP1, yet displays different splicing regulatory properties, with some exons affected by both proteins and other exons more strongly affected by PTBP1. The two proteins have non‐overlapping expression patterns, where PTBP1 is expressed in neuronal progenitor cells but is replaced by PTBP2 during neuronal differentiation. This mediates a switch to a neuronal splicing program with altered isoform expression and altered activity for many proteins. PTBP1 and PTBP2 have very similar RNA recognition properties and how they might exhibit different splicing regulatory properties is not understood. We created a series of hybrid proteins between PTBP1 and PTBP2. We assayed their in vitro RNA binding properties, their ability to repress neuronal exons in vitro and in vivo. We identified interacting partners for the two proteins via Flag‐IP and mass spectroscopy. We identified regions in PTBP1 that can confer splicing repression activity on PTBP2 for certain exons. Some of these regions were previously identified as sites of protein interaction on PTBP1. Together our results indicate that the differential targeting of the two proteins to different exons is largely determined by different protein‐protein interactions.
Mapping modified cytosines in the epitranscriptome of Trypanosoma brucei using sodium bisulfite sequencing (745.1)
Authors: Ackerman Sarah , Chen Leanne , Mandarano Alexandra , Valentine Erika , Militello Kevin
Published: June 2021

This study characterized the presence and location of modified cytosines in the tRNAs of Trypanosoma brucei, a parasitic protozoan. We are interested in these modifications, specifically 5‐methylcytosine (5mC), as a first step in characterizing the epitranscriptome. We used sodium bisulfite sequencing to determine the location of modified cytosines in procyclic form tRNAAsp(GUC), tRNAGly(GCC), tRNAVal(AAC), and tRNATyr(GUA). Our data show that all analyzed tRNAs have 1 to 4 modified cytosines. Each tRNA has a modification at C48 in the TψC stem and there is partial modification T C40 present in tRNAGly(GCC) in the anticodon stem. There are no modifications at C38, which is a common site in higher eukaryotes. tRNATyr(GUA), the only spliced tRNA in T. brucei, possess the same cytosine modification pattern in both unspliced and spliced molecules. In addition, the intron did not contain any cytosine modifications. Thus methylation can occur before splicing and it is unlikely that methylation is involved in splicing. We are now examining bloodstream form parasites for potential changes in methylation sites or levels. We are also determining if there is a correlation between the tRNA levels and modification levels. Elucidating the presence of these modifications has developed our understanding of the epitranscriptome in T. brucei and we hope to determine the global distribution and purpose of these modifications.

Grant Funding Source: NIH R15AI074035
The effect of cassette order on gene expression knockdown by triple tRNA‐shRNA constructs designed for anti‐HIV gene therapy (749.6)
Authors: May Mary , Lin Fang‐Chu , Haushalter Karl
Published: June 2021

Gene therapy utilizing RNA interference (RNAi) directed against HIV‐1 is being studied as an alternative to small‐molecule antiretroviral drugs. Because HIV‐1 is a rapidly mutating virus, it is advantageous to utilize multiple gene therapies at once to effectively prevent the virus from replicating and infecting new cells. Previous research has shown that it is possible to fuse a tRNA together with a short‐hairpin RNA (shRNA) to create a tRNA‐shRNA chimera capable of knocking down the expression of target genes. In this project, triple chimera constructs were created consisting of three different tRNA‐shRNA chimera cassettes on a single plasmid. The effect of differing the order of the tRNA‐shRNA chimeras on the plasmid was investigated using a dual‐luciferase assay with HCT116 cells and the relative knockdown efficiency was compared for each target. The long‐term goal of this project is to optimize a multiplexing strategy for anti‐HIV gene therapy.
Investigating the targeting and regulation of Ded1, a DEAD‐box ATPase, in returning repressed mRNAs to translation (749.9)
Authors: Hilliker Angie
Published: June 2021

Translation regulation is important, especially during stress, in neurons, and in early development, where many mRNAs are stored in translationally repressed states. These repressed mRNAs can be returned to translation, but this process is poorly understood. We have previously shown evidence that the RNA helicase, Ded1, performs two sequential functions to return a repressed mRNA back to translation in an ATP‐dependent manner. First, Ded1 promotes the formation of a “pre‐48S mRNP” that includes the initiation factors eIF4F and Pab1. Then, Ded1 hydrolyzes ATP, allowing the mRNA to become translated. We hypothesize that Ded1 acts as an ATP‐dependent switch to return mRNAs back to the translating pool.

We are investigating how Ded1 forms this pre‐48S intermediate by investigating the importance of its interaction with translation initiation factors and with the mRNA. Additionally, we are exploring the role of post‐translational modifications on the function of Ded1.
Discovery of an asymmetric ATPase with two catalytic centers that are separately activated by two different misacylated‐tRNAs (751.2)
Authors: Hendrickson Tamara
Published: June 2021

A majority of microorganisms lack a complete set of aminoacyl‐tRNA synthetases but still utilize the twenty standard amino acids in their proteomes. The pathogenic bacterium Helicobacter pylori is missing both the glutaminyl‐ and asparaginyl‐tRNA synthetases and instead uses indirect tRNA aminoacylation to generate Gln‐tRNAGln and Asn‐tRNAAsn. This process proceeds via misacylation of these tRNAs to produce Glu‐tRNAGln and Asp‐tRNAAsn, followed by transamidative conversion of the two amino acid side chains to produce Gln‐tRNAGln and Asn‐tRNAAsn. Both misacylated tRNA conversions rely on an amidotransferase called GatCAB. We have identified a new enzyme from H. pylori that induces the formation of a macromolecular complex that also contains this organism’s misacylating aspartyl‐tRNA synthetase (ND‐AspRS) and GatCAB. This enzyme contains two different ATPase active sites, one based on a classic P‐loop ATPase domain and the second based on an [[Unsupported Character ‐ Symbol Font □]]‐adenosine nucleotide hydrolase (AANH) domain. Remarkably, the P‐Loop ATPase is activated by binding of Asp‐tRNAAsn, whereas the AANH ATPase is activated by Glu‐tRNAGln. These ATPase activities modulate GatCAB activity to drive conversion of both misacylated tRNAs into their cognate products. The asymmetric nature of this enzyme and its activation by different misacylated tRNAs make it an unusual new participant in tRNA aminoacylation and protein translation.
Structure and dynamics of ribosome‐bound nascent chains: insights from NMR spectroscopy (752.2)
Authors: Christodoulou John
Published: June 2021

The folding processes of nascent chains are intricately linked to their chain elongation, which occurs in a vectorial manner as the N‐terminal part of the nascent chain emerges from the ribosome. Our use of NMR spectroscopy on ribosomes and ribosome nascent‐chain complexes (RNCs) is providing detailed structural insights of the conformations of protein chains while they are being created on the ribosome. By producing in‐vivo derived RNCs in which the nascent polypeptide is selectively labelled, our recent work has allowed us to use NMR follow, at a residue‐specific level, the co‐translational folding processes of proteins of several topologies, specifically, an immunoglobulin‐domain and of the intrinsically disordered protein, alpha‐synuclein. Recent developments in our use of NMR data (including the chemical shift alone) to produce the first experimentally derived structures of ribosome nascent chain folding and also in‐cell NMR studies to probe proteins and nascent chains within their physiological environment will also be discussed.
Function analysis of the different S6Ks isoforms (752.8)
Authors: Pavan Isadora , Ferreira Flávia , de Freitas Lidia , Tavares Mariana , Simabuco Fernando
Published: June 2021

mTOR signaling pathway has been related to several human diseases and disorders, including obesity, diabetes and many types of cancer. In the cell, mTOR signaling is responsible to induce cell growth and metabolism upon activation by signals from nutrients processing, such as insulin, ATP and amino acids. S6Ks, the known kinases of ribosomal small subunit S6 protein, are effectors of mTOR response. S6K family is composed of two genes (encoding S6K1 and S6K2) and different isoforms from alternative translation initiation (p70‐S6K1, p85‐S6K1, p54‐S6K2, p56‐S6K2) and alternative splicing (p31‐S6K1). The main function of S6Ks, upon activation, is to lead to increased protein synthesis. Nevertheless, one of the main questions in this field is to address the specific roles of the different S6Ks isoforms. In this study, we overexpressed p70‐S6K1, p85‐S6K1, p54‐S6K2, p31‐S6K1 in human cells and analyzed by immunoprecipitation followed by mass spectrometry identification the possible and different interacting partners of those S6Ks isoforms. The different roles of S6Ks isoforms was then evaluated in an adipocyte model and in cancer cell lines. Our study may provide new functions to the different S6Ks isoforms which may help the understanding of their roles in the mTOR signaling pathway.

Grant Funding Source: Suported by FAPESP #2012/13558‐7
A Tale of Two Distinct Mechanisms for IRE1 RNase (752.9)
Authors: Niwa Maho
Published: June 2021

IRE1, an ER transmembrane receptor kinase, is a component of the Unfolded Protein Response (UPR) pathway, and acts as an endoribonuclease (RNase) to cleave the intron from XBP1/HAC1 mRNA coding an UPR‐specific transcription factor. Interestingly, recent reports have revealed that UPR also induces a decrease in levels of ER‐associated mRNAs, including INSULIN mRNA in an IRE1‐dependent manner1, which has been termed Regulated IRE1 Dependent Decay (RIDD). While XBP1/HAC1 splicing and RIDD are proposed to be carried out by IRE1, the mechanism by which IRE1 differentiates between XBP1/HAC1 and RIDD substrate mRNAs has not been investigated. Major differences in the cleavage of the HAC1/XBP1 intron and of RIDD RNA include RNA sequences at the cleavage sites, and that HAC1/XBP1 mRNA cleavage occurs at the ends of the intron whereas certain RIDD substrate RNA are cleaved at multiple sites. In addition, HAC1/XBP1 exon cleavage must be coordinated with tRNA ligase activity while RIDD RNA fragments should be shielded from tRNA ligase to promote their degradation.

Upon development of a robust in vitro RIDD RNase assay with purified recombinant IRE1 expressed in baculovirus or E. coli, we examined the molecular mechanisms by which IRE1 cleaves either HAC1/XBP1 or RIDD RNA substrates. We have found that IRE1 engaged in cleavage of HAC1/XBP1 RNA cannot be competed by addition of cold RIDD RNA, while HAC1/XBP1 RNA can be effectively competed by excess cold HAC1/XBP1. The converse was true for IRE1 engaged in RIDD cleavage. Further analyses revealed that IRE1 oligomer constitutes the site for RNA substrate binding and catalysis, while IRE1 that acts on RIDD RNA does not require oligomerization. Our study provides a molecular basis for designing strategies to differentially activate (inhibit) one of the Ire1 RNase activities without affecting the other which may lead to new strategies for treating diseases associated with IRE1 activation.

#1: Hollien, J., Science 313, 104 (2006), Han, D., Cell 138, 562 (2009)
Determining amino acid propensity for propagation of the prion Sup35 in Saccharomyces cerevisiae (754.2)
Authors: Davis Emily , Knox James , MacLea Kyle
Published: June 2021

Proteins are built based on a DNA sequence template and fold into distinctive shapes or conformations. Amyloids are beta sheet‐rich proteins that can fold into two stable states, a normal form and an abnormal form. Prions, the infectious amyloids, are present in many eukaryotes including humans, cows, sheep, and the baker’s yeast, Saccharomyces cerevisiae and can cause the conversion of native protein structure into amyloid form. Unusually, yeast prion proteins have been shown to form amyloid structure on the basis of amino acid composition, independent of each protein’s primary structure. The most well‐studied prion protein in yeast, Sup35, consists of a prion‐forming domain, a middle domain, and a functional domain. Within the prion‐forming domain there is a glutamine/asparagine‐rich (Q/N‐rich) tract and a region of 5 ½ degenerate oligopeptide repeats (the oligopeptide repeat domain or ORD). The Q/N tract has been previously implicated in prion nucleation and the ORD was shown to be necessary for prion propagation. In this study we generated a library of yeast cells carrying varying, randomly‐generated, mutations in the fourth repeat of the Sup35 ORD region and on this basis we have been able to assign propensity values for each amino acid to favor or disfavor prion propagation.
Effects of lysine mutations on the structure and function of hemolysin A from Proteus mirabilis (755.4)
Authors: Adamczak Kyle , Weaver Todd
Published: June 2021

Proteus mirabilis (HpmA), a Gram‐negative bacterium, implements the two‐partner secretion pathway to produce a hemolytically active exoprotein. The full‐length HpmA was cloned and truncated to contain the first 265 amino acids (HpmA265) in order analyze the structure and function more readily. In the native crystal structure, a dimer was observed to form via on‐edge stacking of carboxy‐terminal parallel beta‐strands. A series of carboxy‐terminal lysine mutants were investigated in this study. Specifically, Phe241, Met245 and Leu263 were altered with the polar charged lysine residue to disrupt the non‐polar carboxy‐terminal dimer and thereby, induce monomer formation. The resultant HpmA265 mutants were analyzed via template‐assisted hemolysis assay, dynamic light scattering and circular dichroism. The results indicate that the loss of hemolytic activity can be correlated with replacement of the most carboxy‐terminal amino acid, Leu263. Additionally, there appears to be less well‐defined beta‐sheet structure in each of the HpmA265 carboxy‐terminal mutants. Further study will aim to conclude whether the HpmA265 monomer or dimer acts as the functional template during hemolysis.

Grant Funding Source: Supported by NSF‐RUI 1050435
Protective role of the mitochondrial Lon protease in homeostasis, oxidative stress‐adaptation, disease, and aging (756.6)
Authors: Davies Kelvin
Published: June 2021

The targeted removal of damaged proteins by proteolysis is crucial for cell survival. We have shown that the product of the human lon gene, the Lon protease, selectively degrades oxidized mitochondrial proteins, thus preventing their aggregation and cross‐linking. Decreasing Lon levels, by knock‐down, causes accumulation of oxidized mitochondrial proteins, compromised mitochondrial function, and increased mitophagy. We also show that lon is a stress‐responsive gene, whose protease product is a stress‐responsive protein that is induced by multiple stressors, including heat shock, serum starvation, and oxidative stress. Lon induction, by pre‐treatment with low‐level stress, protects against oxidative protein damage, diminished mitochondrial function, and loss of cell proliferation, induced by toxic levels of hydrogen peroxide and other metabolic, environmental, or drug oxidants. Blocking oxidant‐induced Lon induction, with lon siRNA, also blocks this induced protection. Lon protein levels can increase as much as eight‐fold in response to stress‐stimulation, yet mRNA levels increase only about 50%. Our studies indicate that a translational activation mechanism may underlie Lon’s extensive and rapid stress‐inducibility. Lon All of these results were obtained in young, healthy cells. Older animals (mice) show a marked decline in Lon levels, particularly in skeletal muscles. Interestingly, in both senescent cells, and in older postmitotic cells, Lon levels and activity decline, and adaptational responses become sluggish or even ineffectual. Studies in animals and humans now suggest that declining Lon activity and, perhaps, declining responsiveness to stress, may contribute to the ageing process, and to various age‐associated diseases. We propose that Lon is a generalized stress‐protective enzyme whose decline may contribute to the increased levels of protein damage, mitochondrial dysfunction, and mitophagy observed in ageing and various age‐related diseases

Grant Funding Source: NIH/NIEHS ES003598
Quantitative analysis of mitochondrial dynamics illuminates links between mitochondrial dynamics and cell function, and allows single organelle localisation microscopy (759.2)
Authors: Saunter Christopher , Chalmers Susan , Girkin John , McCarron John
Published: June 2021

Mitochondrial structure is believed to be important to the normal function of cells. However, remarkably, in fully differentiated cells understanding of how mitochondrial structure controls cell function is preliminary. An ability to generate high resolution, high speed images of mitochondrial structure simultaneously with measurements of cell function is required. We present a suite of quantitative bio‐imaging tools developed for the measurement and analysis of mitochondrial dynamics from high speed fluorescence imaging of live cells, including the measurement and quantification of mitochondrial motion to sub‐diffraction limits, Single Organelle Localisation Microscopy, the use of transient depolarisation's of the mitochondrial membrane to segment physically and optically crowded clusters of mitochondria, and the co‐localization of mitochondria and Ca2+ signalling . We present the algorithmic basis of these tools and highlight their use in the context of changing mitochondrial dynamics in cellular proliferation and hypertension, with an emphasis on the generation of quantified measurements to yield insight into the different cellular states. These tools present a powerful combination of experimental procedures that give an unobstructed open view of new, previously hidden, mechanisms that control mitochondrial dynamics and cell function.

Grant Funding Source: Wellcome Trust; British Heart Foundation; EPSRC
Mitochondrial fission is necessary for vascular smooth muscle cell migration (761.1)
Authors: Wang Li , Yoon Yisang
Published: June 2021

Intimal hyperplasia occurs in response to arterial injury and is also an important feature of atherosclerotic plaques. Vascular smooth muscle cell (VSMC) migration is one of the main processes contributing to intimal hyperplasia. Mitochondria are energy producing organelles that also generate reactive oxygen species as a byproduct of ATP synthesis. Mitochondria inside the cell redistribute frequently and undergo constant shape change mainly through fission and fusion, which is referred to as mitochondrial dynamics. Although the involvement of mitochondria in VSMC migration has been reported in the context of oxidative stress and mitochondrial dysfunction, the role of mitochondrial dynamics in VSMC migration has not been studied. In the present study, we found that platelet‐derived growth factor (PDGF), a strong migratory stimulant, induced mitochondrial shortening in VSMCs. This phenomenon was accompanied by the translocation of the fission protein Drp1/DLP1 from the cytosol to mitochondria, suggesting that PDGF activated mitochondrial fission to form short mitochondria. PDGF also increased the mitochondrial functional parameters including mitochondrial membrane potential, respiration, and ATP levels. Importantly, we found that inhibition of mitochondrial fission greatly decreased PDGF‐induced lamellipodia formation and VSMC migration. Fission inhibition blunted the PDGF‐induced ATP production, suggesting that reduced ATP production is an underlying mechanism for the diminished VSMC migration by fission inhibition. These data indicate that the PDGF‐induced activation of mitochondrial fission is required for augmenting ATP levels and VSMC migration, and suggest that mitochondrial dynamics is a controlling component regulating mitochondrial function.
HIV protease inhibitors sensitize human head and neck carcinoma cells to radiation by activating ER stress (762.2)
Authors: Liu Runping , Zhang Xiaoxuan , Wang Xiang , Mikkelsen Ross , Song Shiyu , Zhou Huiping
Published: June 2021

Head and neck cancers are the eighth most common cancer worldwide. Despite significant advances in the delivery of treatment and surgical reconstruction, the mortality rates for this disease have not improved in the past 4 decades. Our previous study has shown that HIV protease inhibitors (HIV PIs) induce cell apoptosis via activating ER stress. It also has been reported that a few HIV PIs are able to radiosensitize tumor cells. However, the underlying cellular mechanisms remain to be identified. The aim of this study was to examine whether HIV PIs activate the ER stress response and sensitize human head and neck carcinoma cells to radiation. Methods: Human SQ20B and Fadu cells and the most commonly used HIV PIs, lopinavir and ritonavir, were used in this study. The mRNA and protein levels of ER stress‐related genes (CHOP, ATF4, XBP‐1, and GRP78) were detected by real time RT‐PCR and Western blot, respectively. Cell viability and apoptosis were analyzed using Cellometer Vision CBA. After treatment with HIV PIs, cells were irradiated at a dose of 2G or 4G. Colonies were stained and counted 10 days after irradiation. Results: HIV PIs significantly induced activation of ER stress and apoptosis. Treatment of HIV PIs inhibited Akt phosphorylation and increased tumor cell sensitivity to irradiation‐induced cell death. Conclusion: HIV PIs sensitize human head and neck carcinoma cells to radiation by activating ER stress.

Grant Funding Source: NIH/NIDDK, China natiional science foundation
Methylthioadenosine phosphorylase is required for prostate cancer growth in vitro and in vivo (766.2)
Authors: Bistulfi Gaia , Morrison Carl , Gillard Bryan , Foster Barbara , Smiraglia Dominic
Published: June 2021

Methylthioadenosine phosphorylase (MTAP) helps to sustain methionine metabolism, which, either directly or indirectly, plays a significant role in polyamine synthesis, DNA synthesis, and DNA methylation. MTAP is one of the most frequently deleted genes in human cancers, possibly contributing to genetic and epigenetic damage. Because prostate cells have an exceptionally high level of polyamine biosynthesis impinging on methionine metabolism, we investigated MTAP status in human prostate cancer (CaP). In contrast to many other types of cancer, MTAP was never deleted in CaP, as assessed in a sample of 13 cell lines by PCR and 75 CaP patients at different stages of the disease by tissue microarray analysis. In the context of folate deficiency, which stresses methionine metabolism, transcriptional up regulation of MTAP allowed CaP cells to adapt to the metabolic stress in order to provide sufficiency of key metabolites as assessed by HPLC analysis. We therefore hypothesized that MTAP may play an essential role in sustaining CaP growth. We treated a panel of CaP cell lines with a specific MTAP inhibitor. Consistent with MTAP’s importance in prostate cells’ metabolism, we found that the inhibitor was very effective in preventing CaP growth in vitro, with an IC50 between 0.7 and 1.4 µM. Finally, MTAP knock down in LNCaP cells prevented tumor establishment in nude mice. We conclude that MTAP plays a pivotal role in CaP growth and that, because of its importance specific to prostate cells, it could be a novel, ideal target for CaP therapy.

Grant Funding Source: AICR American Institute for Cancer Research
Response gene to complement 32 deficiency protects against high‐fat diet‐induced insulin resistance and obesity with decreased hepatic lipogenesis in mice (767.7)
Authors: Cui Xiaobing , Chen Shiyou
Published: June 2021

The objective of this study was to determine the effects of response gene to complement 32 (RGC‐32) on the development of obesity and insulin resistance in mice. Mice with RGC‐32 deficiency (RGC32‐/‐) gained less body weight with reduced epididymal fat and liver weight compared to the wild‐type animals after a 12‐week high‐fat diet. Biochemical analysis of blood, as well as glucose and insulin tolerance tests showed that RGC32‐/‐ improved insulin sensitivity and lowered serum triacylglycerol and low‐density lipoprotein/very‐low‐density lipoprotein cholesterol concentration. Hepatic lipid accumulation was also decreased in RGC32‐/‐ mice. Additional analysis revealed that lipogenic genes sterol regulatory element (SRE) binding protein (SREBP)‐1c, fatty acid synthase, and stearoyl‐CoA desaturase‐1 (SCD1) were decreased in the livers of RGC32‐/‐ mice. Mechanistic studies using mouse primary hepatocytes showed that SCD1 regulation by RGC‐32 was SREBP‐1c‐dependent since deletion or mutation of SRE abrogated SCD1 promoter activity mediated by RGC‐32. These data demonstrate that RGC‐32 induces SREBP‐1c‐dependent hepatic lipogenesis, which is essential for the high‐fat diet‐induced obesity and insulin resistance.
Oxidation of DNA by high‐valent metal‐oxo porphyrins Mn‐TMPyP and Fe‐TMPyP (768.12)
Authors: Tandjigora Kaba , Pratviel Genevieve
Published: June 2021

The goal of this project is to regulate the reactivity of high‐valent metal‐oxo porphyrins by the control of the axial ligand trans to the oxo group. This work will allow a better understanding of the mechanism of metalloporphyrins oxidation. The chosen substrate is a DNA model. The oxidation products are analyzed by HPLC and LC/ESI‐MS. Meso‐tetrakis(4‐N‐methylpyridiniumyl)porphyrinatomanganese(III) (Mn‐TMPyP) and its iron analogue (Fe‐TMPyP) mimic the reaction mechanism of natural heme enzymes namely peroxidases and cytochrome P450. Mn‐TMPyP and Fe‐TMPyP become high‐valent metal‐oxo species when activated by dissymmetric peroxide such as KHSO5 and are able to oxidize DNA. Previous work has shown that Mn‐TMPyP/ KHSO5 was sensitive to pH and produced variable oxidation products of the DNA duplex model 5’‐d(CAGCTG) by electron transfer (ET) and oxygen atom transfer (OAT). At pH 8, the main products observed were those of OAT, whereas at pH 6 the dominant products were obtained from ET. Fe‐TMPyP as Mn‐TMPyP is a powerful oxidizing agent; however, its reactivity towards DNA is unknown. What is known is that it exists as a µ‐oxo species in basic and neutral medium. The µ‐oxo characteristic of Fe‐TMPyP should make it insensitive to pH. We showed that its oxidation products did not vary between pH 6 and pH 8. The present work has shown that the oxidation reactivity of manganese and iron porphyrins can be directed based on their proximal axial ligand (L‐ or X‐type). The proximal ligand can be controlled by simple modulation of the pH of the reaction in the case of manganese porphyrin or by the nature of the metal (iron porphyrins form iron µ‐oxo dimers). The control of metalloporphyrins reactivity may also prove useful in fine chemistry where these compounds are used as oxidation catalysts.

Grant Funding Source: Supported by NSF‐CHM 1156907
Effects of kombucha tea on amylase (768.5)
Authors: Villeneuve Mallory , Mann Francis , Seehusen Katie
Published: June 2021

Kombucha is a fermented tea beverage produced using a culture of yeast and bacteria to create a bubbly beverage claiming many purported health benefits. The inhibitory effects of Kombucha were analyzed to assess its relevancy in digestive effects. The effects of Kombucha in relationship to breakdown of dietary starches in the mouth by amylase were examined. Results may be relevant to the health benefits of this beverage and others that can slow sugar absorption in the mouth. Preliminary data shows that this fermented drink inhibits the enzyme activity in the digestion of starch using a starch azure assay. Further analysis using a salicylate assay measuring freed maltose using a standard curve was conducted. In this method, the Vmax and Km of amylase were observed using varied sample preparations of Kombucha and Kombucha‐derived metabolites to assess the natural of amylase inhibition by Kombucha.
Probing the determinants of the metal binding specificity in HDAC8 (768.7)
Authors: Kim Byungchul
Published: June 2021

HDACs catalyze the hydrolysis of ε‐N‐acetyl lysine residues on proteins acetylated by histone acetyltransferases. Upon their discovery, this enzyme was identified as Zn2+‐dependent metalloenzymes. However, current activity and binding affinity studies suggest that Fe2+ could be the native metal cofactor in vivo. This has a number of biologically relevant implications, including 1) metal switching regulatory mechanism, 2) substrate specificity based on the identity of metal status. Therefore I am presenting the determinants of metal selectivity in HDAC8 in vitro to provide insight into the intracellular metal specificity in vivo.

In HDAC8, the second shell of amino acid surrounding the metal coordinating side chains is predominantly hydrophobic. This is contrary to many prototypical metalloenzymes, such as carbonic anhydrase II (CAII). The lack of second shell hydrogen bonds may be an important factor contributing to the weaker Zn2+ affinity of HDAC8 compared to CAII, allowing facile switching between Zn2+ and Fe2+ cofactors. To investigate these structural determinants, I have prepared HDAC8 mutants and I am measuring the metal ion affinity and dissociation rate constants of wild‐type and mutants. Surprisingly the dissociation rate constants for Zn2+ and Fe2+ from the HDAC8‐metal complex in mutants are comparable despite the large difference in binding affinity.

Previous studies indicate that binding to the 1st K+ site inhibits the activity, while binding to the second K+ site activates catalysis possibly through structural stabilization. This indicates that K+ binding to two different sites may affect the Zn2+ binding site. To study this, I am measuring the dissociation rate constant for zinc and iron binding to HDAC8 at varying concentrations of monovalent cation (K+ and Na+). The Zn2+ dissociation rate constant from metal‐bound HDAC8 decreases as the concentration of monovalent cation (K+/Na+) is increased.

Grant Funding Source: Supported by NIH's NIGMS 2RO1GM040602‐22
The minor role of Gln 183 in the NADH reductive half‐reaction of E. coli methylenetetrahydrofolate reductase (769.10)
Authors: Zuo Chong , Jolly Amber , Nikolova Andriana , Cao Sirui , Sanchez Jeremy , Trimmer Elizabeth
Published: June 2021

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10‐methylenetetrahydrofolate (CH2‐H4folate) to 5‐methyltetrahydrofolate (CH3‐H4folate) using FAD as a redox coenzyme. The reaction contains two different half‐reactions, the NADH reductive half‐reaction and the CH2‐H4folate oxidative half‐reaction. The focus of our work is the active‐site amino acid Glutamine 183 (Gln183). Structural studies have implicated the importance of Gln183 in both substrate binding and catalysis of the two half‐reactions, based on direct hydrogen bonds between Gln183 and the substrates NADH and folate. In order to investigate the function of Gln183 in MTHFR catalysis, two mutants with single amino acid changes, Gln183Ala (Q183A) and Gln183Glu (Q183E) have been constructed. Using the steady‐state NADH‐menadione oxidoreductase assay, we have examined the reductive half‐reactions of each mutant enzyme. For reasons of mutant stability, the temperature of the assay was optimized to 4°C. We expected that the change in hydrogen bonds and polarity of the Q183 mutant enzymes would affect their kinetic parameters kcat and Km. However, the mutant enzymes have similar kinetic parameters to the wild‐type enzyme at 4°C, consistent with our previous results at 25°C. Therefore, we conclude that glutamine 183 plays only a minor role in the reductive half‐reaction of MTHFR.

Grant Funding Source: Grinnell College
The mode of substrate entry into the 5‐lipoxygenase active site (769.3)
Authors: Mitra Sunayana , Newcomer Marcia
Published: June 2021

Leukotrienes are potent immune‐modulatory compounds involved in the inflammatory response. 5‐Lipoxygenase (5‐LOX) is the enzyme that catalyzes the production of Leukotriene A4 (LTA4), which is further metabolized to pro and anti‐inflammatory compounds from the substrate Arachidonic Acid (AA), a polyunsaturated fatty acid.

The crystal structure of a stabilized form of 5‐LOX has been recently reported (Gilbert et al, 2011). However, the structure does not reveal the mode of substrate recognition. The first step in the enzymatic reaction involves the removal of a hydrogen from the central carbon of a pentadiene present in the substrate. It is not known how 5‐LOX distinguishes among three such chemically equivalent pentadienes in AA to produce only 5‐ S‐HPETE, which is subsequently transformed to LTA4. The crystal structure suggests a somewhat crescent shaped active site lined with invariant Leucines and Isoleucines, with no clear access port visible for substrate entry.

A series of site directed mutants were produced and the activities of these mutants evaluated. Product analyses of these mutants with AA and substrate analogs suggest entry of the substrate by the opening of the so‐called Phenylalanine (F)‐Tyrosine(Y) ‘cork’. The data is also consistent with the orientation of AA as carboxyl innermost, as predicted from the stereochemistry of the product and mechanistic models of lipoxygenase function.

Grant Funding Source: Supported by NIH Grant # HL107887