bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Hypoxia on Gene Expression in the Chick Embryonic Heart
Authors: Nanka Ondrej , Krejci Eliska , Barth Jeremy , Sedmera David
Published: June 2021

Our previous results showed that normal embryogenesis involves physiological tissue hypoxia. Hypoxic avian hearts show abnormal myoarchitecture, differences in the extent and distribution of hypoxic regions and increased HIF1α. Here we have used microarray analysis to identify genes differentially expressed between normoxic and hypoxic conditions

Chicken eggs were incubated at 38°C under normoxic (21% O2) conditions for the first 48 h and subsequently under hypoxic (16% O2) conditions for 24 h (acute hypoxia) or 144 h (chronic hypoxia). Total RNA was extracted from control and hypoxic chick embryonic hearts, converted into biotin‐labeled material, and then hybridized to Affymetrix Chicken Genome Arrays according to manufacturer's protocols. Transcripts differentially expressed in the short term and long term hypoxic states were identified by the criteria fold change >1.5 and p <0.05 (Student's t‐test). 79 transcripts were significantly up‐regulated and 75 down‐regulated in the hypoxic samples. Functional categorization of these genes detected significant enrichment of biological processes such as cell growth, differentiation, muscle contraction and signal transduction. Additionally, we performed a focused examination of genes previously linked to hypoxia adaption, identifying 10 transcripts that were significantly affected in our hypoxic samples (p<0.05). Given that important hypoxia‐regulated genes (e.g., HIF1α) are known to be regulated at the protein, rather than the mRNA level, our results add significant insight into the transcriptional response that occurs in response to hypoxia.

Supported by PRVOUK P35/LF1/5 and P302/11/1308.
TGFβ‐induced TAZ upregulation and the underlying mechanisms
Authors: Miranda Maria , Bialik Janne , Rozycki Matthew , Pedersen Stine , Kapus Andras
Published: June 2021

TGFβ is a prime inducer of myofibroblast (MF) transition from various precursors. TAZ, a Hippo pathway‐regulated transcriptional coactivator has been shown to crosstalk with TGFβ signaling, primarily as a Smad3 nuclear retention factor. During our studies aimed at assessing the role of TAZ in fibrogenic phenotype shifts, we observed that TGFβ caused a substantial (»5‐fold) and fast (6 h) rise in TAZ protein level in two MF precursors, pericyte‐like (10T1/2) cells and renal fibroblasts. TGFβ increased TAZ in tubular epithelial cells as well, an effect that was strongly potentiated by cell contact uncoupling, a prerequisite for epithelial‐MF transition. Interrogating the underlying signaling in 10T1/2 cells, we found that the Smad3 inhibitor SIS3 failed to abolish TGFβ‐induced TAZ upregulation, whereas inhibition of Akt or p38 abrogated this effect. These findings imply that non‐canonical TGFβ pathways are central to the control of TAZ. While interference with TAZ‐degrading pathways (Casein Kinase δ/ε, GSK3β) increased TAZ expression with similar magnitude and kinetics as TGFβ, cycloheximide‐provoked TAZ degradation was not prevented by TGFβ. Instead, TGFβ increased TAZ mRNA. Since myocardin‐related transcription factor (MRTF) is a TGFβ‐regulated key inducer of MF transition, we treated cells with its inhibitor, CCG‐1423. The drug strongly suppressed TGFβ‐triggered TAZ expression and the concomitant induction of a TAZ‐dependent luciferase reporter. Thus, TGFβ enhances TAZ transcription in an Akt‐, p38‐ and MRTF‐dependent manner, signifying a hitherto under‐recognized TAZ regulatory mechanism with potentially key roles in the pathobiology of organ fibrosis.
Application of Laser Feedback Interference Microscopy to Localize the Ciliary Vector
Authors: Ovryn Ben , Satir Peter
Published: June 2021

In migrating fibroblasts with primary cilia, the primary cilium points in the direction of migration but why this is so is unknown. The objective of our work is to investigate whether directional signaling from the primary cilium (the ciliary vector) affects the direction of cell migration or vice versa and to determine what molecules and mechanisms are involved. Using a well defined cilium‐based RTK signaling pathway whose receptor (PDGFRα) specifically localizes and signals from the ciliary membrane, we use micropipettes to apply a variable concentration of directional stimulus to the cilium and phase shifted laser feedback interference microscopy to determine the transverse and axial position of the cilium in living fibroblasts. We have obtained data which demonstrate that we can correlate fluorescence imaging of GFP labeled cilia with our interference imaging. Although analogous to confocal imaging, our interferometer is a form of label free imaging that has high sensitivity to dense protein aggregations and can determine axial position with an accuracy and precision of better than 10nm in live cells. We have obtained data which indicates that we obtain a strong reflective signal from the primary cilium that we can use for localization. We plan to use this signal in a feedback loop so that we can introduce control perturbations to the direction of the cilium, with an AFM like probe, while simultaneously tracking signaling molecules. Assistence of Tabrez Alam is acknowledged.
Cyclophilin A, a Novel Component of Listeria monocytogenes Comet Tails
Authors: Dhanda Arandeep , Guttman Julian
Published: June 2021

The intracellular bacterium Listeria monocytogenes hijacks the actin polymerization machinery within human cells for its entry, intracellular movement and subsequent spreading from cell‐to‐cell. Intracellular motility of Listeria involves Arp2/3‐mediated actin polymerization at one pole of the bacteria, resulting in the generation of structures known as comet tails. During cell‐to‐cell spreading, comet tails protrude from the host cell membrane from structures referred to as listeriopods. Although the minimal protein constituents required for these actin‐based structures were identified years ago, a complete understanding of the molecular constituents and their specific functions at these sites remains to be investigated. Cyclophilin A (CypA) is a cytosolic protein involved in protein folding and potentially actin nucleation. Thus, because Listeria depends on actin‐based structures for crucial events in its disease process, we tested the hypothesis that CypA is recruited to Listeria comet tails and sites of cell‐to‐cell bacterial spreading. Through the use of fluorescently‐tagged CypA DNA constructs and time‐lapse imaging, we demonstrate that CypA is present at 1) the surface of Listeria prior to comet tail formation, 2) comet tails themselves and 3) listeriopods. Our data reveals a new component in the actin‐based motility of Listeria and suggests that CypA may play a crucial role in the cell‐to‐cell spreading of Listeria.

Grant Funding Source: NSERC and departmental funds
A PRACTICAL APPROACH TO CAREER ADVISING FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY MAJORS.
Authors: Kennelly Peter
Published: June 2021

The career landscape for biochemistry and molecular biology students has changed dramatically over the past two decades. While to some extent these changes reflect declining demand within the academic sphere, the predominant drivers have been the progressive expansion and diversification of the “bio‐based” sectors of the economy and a substantial growth in the number of undergraduate students choosing to major in biochemistry and molecular biology. These trends have generated a demand for more intentional and comprehensive career education and advising for undergraduate biochemistry and molecular biology; a demand which many programs are struggling to meet. The majority of current college / university career service offices are ill‐equipped to offer informed and insightful career support services for biochemistry and molecular biology majors. While faculty may feel that their lack of personal work experience outside the halls of academia leaves them ill‐prepared to aid students considering careers beyond the familiar spheres of academia, medicine, and research & development; in point of fact their scientific training renders them far better equipped than the typical university career advising specialist. So how can faculty better serve student needs for career information and advising? This poster outlines practical strategies and readily accessible resources by which educators can assist students in planning and preparing for the next, post‐graduation, steps in their career development. It also describes the syllabus for a simple course — “Careers in Biochemistry” — for proactively addressing student career information needs.
Flipped Classroom Activities in Introductory Chemistry and Biochemistry Courses for Nursing and Nutrition Majors
Authors: Anderson Ellen
Published: June 2021

There is a growing trend in the university classroom to invert the common teaching pedagogy. Instead of lecturing during class time and assigning homework to be completed outside of the classroom, the flipped classroom is gaining popularity. Several such activities have been developed for the two USJ required nursing and nutrition chemistry courses. Flipped classroom activities on stoichiometry and on dehydration and oxidation reactions of alcohols have been employed in Principles of Inorganic and Organic Chemistry while an activity on enzyme classifications has been utilized in Biochemistry of the Human Body. These topics require students to be able to solve problems entailing numerical calculations, writing reaction products or deciphering enzyme reactions. The most effective means to reach these ends are for the students themselves to actively engage in sufficient practice of these skills rather than passively watching the professor solve these type of problems. Initial findings show measurable increases in student performance on assessments of the information taught by this method. Thus flipping the classroom for challenging assignments supports the learning very effectively.
An Expanded Rubric for Assessing Molecular Visualization Literacy
Authors: Franzen Margaret , Craig Paul , Dean Diane , Dries Daniel , Listenberger Laura , Novak Walter
Published: June 2021

A discussion of molecular visualization skills initiated at the 2013 ASBMB Education Symposium has led to an expanded rubric for biomacromolecular 3D literacy. The rubric is divided into twelve areas, including alternate renderings, molecular dynamics and structure‐function relationships. We are developing broad learning goals, learning objectives (what students will know or do), sample assessments (questions to be asked or problems to be resolved), and expected competencies for each area. The competencies also take levels of expertise into account. Novices are first year life science students; amateurs are upper division undergraduates; experts are advanced practitioners who may be graduate students, post‐doctoral fellows or faculty members. We will present our suggestions with the goal of building a learning community for face‐to‐face discussions at future meetings and joint participation in online resource development. Supported by NSF‐DUE #1323414.
A Research‐based Laboratory Course Examining the Structure and Function of Alcohol Dehydrogenase Improves Student Confidence in Common Biochemical Techniques
Authors: Huff Mary , Krzysiak Amanda
Published: June 2021

Inquiry‐based learning has been shown to increase student's interest and motivation while improving critical thinking and cooperative learning. At Bellarmine University, we have introduced a semester‐long research project into our second semester Biochemistry course that allows students to study the fundamental relationship between protein structure and function using the yeast alcohol dehydrogenase I (ADH1) enzyme. Students explored the published crystal structure of ADH1 and were asked to identify a mutation that they could create that might affect the catalytic function of the enzyme. Using site‐directed mutagenesis, the mutation was introduced into the ADH1 gene, and the mutant protein was expressed and purified using His‐tag affinity chromatography. Finally, enzyme kinetic studies were performed and the results were compared to wild‐type ADH1. At the completion of the semester, a formal lab report resembling a journal publication was submitted. Student performance was evaluated based on the quality of the formal lab report, individual lab notebooks, and weekly participation. Through this exercise, students used a number of biochemistry techniques and developed research skills that better prepared them for independent laboratory research in their senior year. Student assessment of the familiarity and confidence in both commonly used databases such as pubMED and PDB as well as the biochemical techniques used in this course increased significantly.
Teaching Professional Skills to Chemistry and Biochemistry Majors
Authors: Sarisky Catherine , Hollis W.
Published: June 2021

Our department houses both an ACS‐certified Chemistry major and an ASBMB‐certified Biochemistry major. For a number of years, the department has required completion of a sophomore‐level professional skills course for all majors, meeting 1.5 hours per week. This course includes searching the scientific literature (including the use of PubMed and SciFinder), scientific writing, oral and poster presentation skills, career planning and resume writing, and scientific ethics. Enrollment is capped at 12, to allow each student to give two in‐class presentations. A challenge has been allowing students to pick topics that interest them, that have scholarly sources available, and that can be accomplished by a student presently enrolled in second‐semester organic. Students have particularly struggled to include enough chemistry in their presentations. We will share several strategies that we have used to run a course that serves students from both majors.
Probing the Inflammatory Response Behind Diabetes and Obesity via the Biochemical Characterization of NOD1, an Innate Immune Receptor
Authors: Rivas Thomas , Lauro Mackenzie , Grimes Catherine , Bahnson Brian
Published: June 2021

Innate immunity is an intricate system employed by humans to regulate the trillions of bacteria that live in our body. One protein integral to the innate immune response is called nucleotide‐binding oligomerization domain‐containing protein 1 (NOD1). NOD1 is a cytosolic, membrane‐associated protein that is linked to irritable bowel syndrome, asthma, diabetes, and obesity. These diseases are thought to arise from improper mRNA splicing of NOD1 transcripts leading to a poorly functioning NOD1 protein receptor that can no longer regulate an overstimulated inflammatory response. Activation occurs when NOD1 binds to a bacterial cell wall component called iE‐DAP, which then signals the the NF‐κB cascade to produce pro‐inflammatory cytokines and chemokines.

Current research is trying to describe the binding of iE‐DAP to NOD1, however, these studies almost exclusively use expensive and low‐yielding insect cells to obtain their active protein. This project aims to develop a bacterial expression system to express wildtype NOD1 and the LRR domain, which is the region thought to be involved in ligand binding. Currently I have produced both constructs in high yield from an E. coli system to allow their biochemical characterization through peptidoglycan pull‐down experiments and circular dichroism. Future work aims to obtain crystal structures of NOD1 bound to its ligand and perform NMR studies to observe ligand binding events.
An RNA Research‐based Advanced Biochemistry Laboratory Course: Design, Implementation, and Outcomes
Authors: Mouzakis Kathryn
Published: June 2021

Inquiry‐based experiments are traditionally used in biochemistry laboratory courses to teach students a variety of biochemical concepts and techniques. These laboratory experiments are often completed in multi‐week modules, with each module focusing on specific student learning outcomes. In this context, students often struggle to make connections between modules. Integration of an authentic research experience into these courses allows students to master specific learning outcomes in the broader context of a course‐long research project. A three‐credit research‐based advanced biochemistry laboratory course was designed and implemented at Fort Lewis College. The course goals were to introduce students to modern nucleic acid biochemistry techniques and improve their abilities to communicate complex biochemical topics in written and oral form. In this course, students explored the structure and function of the human t‐cell leukemia virus (HTLV) frameshift site RNAs. Teams of three to four students worked on three related research projects throughout the semester. The first part of the course introduced students to the background of the research topic and basic principles of molecular cloning, in vitro transcription, and RNA purification. Students designed RNA constructs to answer specific questions related to their research topic. These RNAs would ultimately be in vitro transcribed, purified, and used in experiments. Students regularly submitted formal research reports and gave oral presentations on their work. Progress towards achievement of course learning goals was monitored with formative and summative assessments. Students made substantial progress towards achievement of these goals and anonymous post‐semester feedback was overwhelming positive.
A web repository of research literature assessment questions to promote student learning and acquisition of scientific reasoning and inquiry skills necessary for both the MCAT2015 and ASBMB certification
Authors: Jakubowski Henry
Published: June 2021

An increasing emphasis is being placed on scientific inquiry and reasoning skills in both the new MCAT2015 and in new student learning goals and objectives developed by the ASBMB for student certification. This comes as both organizations are moving from recommending required courses to recommending acquisition of competencies and skills by students. For the new MCAT2015, students must have a diverse knowledge of concepts/ideas from the biological/ physical sciences and apply this knowledge to problem solving, assessing experimental design/methodology, and using data and statistical reasoning. An optimal approach to prepare students for this new emphasis is to have them analyze data and draw conclusions from research papers. These changes will require faculty to use more research‐literature based question to assess the students' acquisition of these higher level Bloom's learning skills. To facilitate this change, I have developed a model for the creation of a web‐based collection of assessment questions derived from the research literature. Model assessment questions were created using data, figures and tables derived from The Journal of Biological Chemistry. Questions are targeted to assess the broadly overlapping learning goals recently articulated by the ASBMB for programs accredited in Biochemistry and Molecular Biology, and the learning competencies developed by the Association of American Medical Colleges for the new MCAT2015. Faculty would use, contribute to and help peer‐review the proposed collection. Students would use it to gain skills in understanding and evaluating research design and findings.
Broader Impact Careers for Scientists in Science Education and Communication
Authors: Jacque Berri , Tammen Stephanie , Meiri Karina
Published: June 2021

Employment data from the NIH and NSF show a dramatic overproduction of scientists relative to the number of job openings for permanent positions. This has created a bottleneck where many scientists get stuck in a training whirlpool. At the Center for Translational Science Education (CTSE) at Tufts Medical School we work to bring cutting edge biomedical science education to high school students and teachers around the country, while providing scientists opportunities to explore a range of diverse careers in science education. Many graduate students and postdoctoral fellows come to the CTSE hoping to gain teaching experience or to impact the community through outreach in local high schools. Other scientist work as full time postdoctoral scholars, studying curriculum design and evaluation, as they transition to careers with broader scientific impacts, such as teaching, museum curator, K‐12 outreach project manager, or primary investigator performing science education research. In this way scientists apply their skills to solve urgent public health and science education challenges, while gaining skills and experience to better compete in a diverse range of careers in science education and communication.

Funded through: NIH Grant R25OD010953‐04, a gift from Boston Scientific and a grant from Cubist Pharmaceuticals
Specificity and Activity of Y‐family DNA Polymerases DinB and Pol Kappa
Authors: Antczak Nicole , Walsh Jason , Ippoliti Paul , Beuning Penny
Published: June 2021

More than 100,000 lesions are generated on DNA per cell per day due to such agents as reactive oxygen species and ultraviolet radiation. Y‐family DNA polymerases are specialized to carry out translesion synthesis by replicating past the lesion and are conserved in all domains of life. There are two Y‐family polymerases in E. coli, DinB (Pol IV) and UmuD'2C (Pol V), as well as several in humans including Pol κ (hPol κ, a DinB ortholog). Both DinB and hPol κ are known to bypass minor groove adducts of guanine. Using primer extension assays on DNA substrates containing the minor groove adduct N2‐furfuryl‐dG as well as undamaged templates we found that loop 1 residues of DinB near the catalytic site are important for activity as well as the selection of the correct nucleotide. There is a similar trend in the loop 1 region of hPol κ. In single nucleotide incorporation assays, wild‐type and variants of both polymerases show a tendency to insert incorrect dT as well as the correct dC opposite the N2‐furfuryl‐dG adduct. This is also seen for wild‐type and variants of hPol κ on an undamaged DNA template. These observations suggest that the loop 1 region of both polymerases is important in correct nucleotide incorporation during translesion synthesis. This work is supported by the American Cancer Society.
Mechanisms of Translesion DNA Bypass Across (5′S)‐8,5′‐Cyclo‐2′‐Deoxyguanosine by Sulfolobus Solfataricus DNA Polymerases
Authors: Zhao Linlin , Xu Wenyan , Ouellette Adam , Wawrzak Zdzislaw , Shriver Storm , Anderson Spencer
Published: June 2021

The (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine (S‐cdG) DNA adduct is produced from reactions of DNA with hydroxyl radicals generated from ionizing radiation or endogenous oxidative metabolisms. An elevated level of S‐cdG has been detected in Xeroderma pigmentosum, Cockayne syndrome and breast cancer patients, and in aged mice. S‐dG blocks DNA replications in vitro and in human cells, and is repaired by synergetic actions of nucleotide excision repair and translesion DNA synthesis. S‐cdG induces G to A transition in E coli and in human cells, and causes S‐cdG:T mispair in vitro with several translesion DNA polymerases. However, the molecular mechanisms of translesion bypass of S‐cdG remain elusive. We examined kinetic and structural mechanisms of S‐cdG bypass using model DNA polymerases, Sulfolobus Solfataricus DNA Polymerase I (Dpo1) and IV (Dpo4). Both Dpo1 and Dpo4 can moderately bypass S‐cdG with much lower catalytic efficiencies compared to efficiencies with an undamaged template. Dpo1 and Dpo4 bypassed the lesion in a largely error‐free fashion. Pre‐steady‐state kinetics analyses revealed that the lowered catalytic efficiency of Dpo4 in the presence of template S‐cdG derived from a lower DNA binding affinity and an attenuated formation of the productive enzyme‐DNA‐nucleotide complex. To provide structural insights, we solved two X‐ray crystal structures containing Dpo4, S‐cdG‐containing DNA duplex, and the correct dCTP or incorrect dTTP. Together, our study provided mechanistic insights for the translesion bypass across a highly mutagenic cyclopurine lesion.
A Molecular Dynamics Investigation of the Thermostability of DNA Polymerase
Authors: Modeste Erica , Mawby Lily , Miller Bill , Wu Eugene , Parish Carol
Published: June 2021

DNA polymerase replicates DNA within organisms. Since the development of polymerase chain reactions (PCR) in the early 1980s, DNA polymerase has also become a critical tool in biotechnology. DNA polymerase I from Thermusaquaticus(TaqDNA polymerase) is an ideal prototype for PCR due to its thermostability and activity at high temperatures. However, becauseTaq DNA polymerase lacks of a functional 3′ to 5′ exonucleaseit replicates with a relatively high error rate. In addition, Taq DNA polymerase often begins DNA replication at room temperature, which results in mispriming and nonspecific products. In an effort to improve its functionality as a PCR agent,Kermechiev and colleagues (2003) performed a series of random mutationsand found than an I707L mutation reduced the activity at low temperatures. It is unclear how this structural change affects Taq DNA polymerase activity at low temperatures. Molecular Dynamic studies at high and low temperatures on wild‐type and I707L mutated Taq DNA polymerase were employed in order to better understand the structure‐function relationship in this important enzyme. Following simulation using AMBER MD Algorithm, the I707L mutated Taq DNA polymerase was more dynamic at higher temperatures.
Non‐cognate DNA damage prevents formation of active conformation of Y‐family DNA polymerases DinB and pol kappa
Authors: Nevin Philip , Lu Xueguang , Zhang Ke , Engen John , Beuning Penny
Published: June 2021

Y‐family DNA polymerases are specialized to copy damaged DNA and are associated with an increased risk of mutagenesis due to their low fidelity. It is believed that the mechanism of nucleotide selection by Y‐family DNA polymerases involves conformational changes preceding nucleotidyl transfer but there is limited experimental evidence for such structural changes. Using hydrogen/deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y‐family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide‐induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N2‐furfuryl‐deoxyguanosine that is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O6‐methyl‐deoxyguanosine that is a poor substrate. Structural changes were localized to regions both near and far from the active site and involved several domains of the enzymes. Our observations suggest that DinB and pol κ utilize a common mechanism for nucleotide selection involving a conserved prechemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.
High Resolution Architecture of Human Transcriptional Activity Revealed by Native Elongating Transcript Sequencing
Authors: Mayer Andreas , di Iulio Julia , Maleri Seth , Eser Umut , Reynolds Alex , Vierstra Jeff , Sandstrom Richard , Stamatoyannopoulos John , Churchman L Stirling
Published: June 2021

Major features of transcription by human RNA polymerase II (Pol II) including pausing, antisense transcription, and alternative splicing remain poorly defined due to the lack of quantitative approaches for visualizing RNA synthesis at nucleotide resolution. We developed a powerful approach for performing native elongating transcript sequencing (NET‐seq) in human cells that exploits a quantitative biochemical purification of engaged Pol II along with the associated nascent RNA. This approach allows the genome‐wide mapping of Pol II positions in a strand‐specific manner and at nucleotide resolution. Application of NET‐seq exposes and classifies the high‐resolution transcriptional architecture of human promoter‐proximal regions. We uncover a novel mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive promoter chromatin configuration, is characteristic of lower‐expressed genes and is coupled to promoter‐proximal pausing. Integration of NET‐seq with high‐resolution genomic footprinting data reveals stereotypic Pol II pausing patterns mediated by transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II recognition of exon structures with different processing fates. Together, human NET‐seq exposes novel features of the topography and regulatory complexity of human gene expression.
Regiospecific function of the C‐terminal domain of RNA polymerase II in yeast
Authors: Fuchs Stephen , Babokhov Michael , Mosaheb Mohammad , Morrill Summer
Published: June 2021

The C‐terminal domain (CTD) of RPB1, the largest subunit of RNA polymerase II is comprised of repeating units of a seven amino acid sequence and is indispensible for function in eukaryotes. The aim of this study was to determine if repeats had specific function, dependent on their location within the sequence, or whether repeats were largely redundant. Using a library of precisely designed CTD mutants, a novel TET‐off system to control RPB1 expression, and phenotypic analysis in yeast we demonstrate that, in fact, different regions of the CTD exhibit different function. Specifically, we show differential sensitivity to various transcriptional inhibitors and can link phenotypes to the presence or absence of phosphorylatable serine residues. This study leads to a novel model for the assembly of CTD‐associating factors on the CTD during transcription. We also begin to use this library to identify changes in CTD‐associating protein recruitment to the CTD and to identify regions of the CTD important for processes such as mRNA processing and histone methylation.
Functional Analysis of FOXC1 in TGF‐β Mediated Epithelial to Mesenchymal Transition
Authors: Berry Fred , Mirzayans Freda , Hopkins Alexander
Published: June 2021

Elevated expression of FOXC1 has been detected in many metastatic cancers characterised by mesenchymal characteristics. Furthermore in development FOXC1 functions in the formation mesenchymal derived tissues including the axial skeleton and eye that are derived through transforming growth factor beta (TGFβ) signalling pathways. However it is not known whether FOXC1 functions in initiation of EMT by TGFβ signalling. We utilized the mouse mammary epithelial cell line NMUMG, to test what role FOXC1 plays in EMT. Treatment of NMUMG cells with 5 ng/ml of TGFβ induced EMT and was accompanied by a down regulation of the epithelial marker E‐cadherin and an upregulation of mesenchymal markers N‐cadherin and Vimentin and Snail1. Elevated levels of Foxc1 mRNA were detected at 24 and 48 hours of treatment. To test whether this upregulation of Foxc1 was necessary for initiation of EMT we used RNA interference to reduce levels of by 60‐85%. When treated with TGFβ, knock down of Foxc1 had no effect on EMT progression based on morphological assessment, and through changes in gene expression monitored by quantitative PCR and immunofluorescence assays. Elevated N‐cadherin mRNA expression was detected in Foxc1 knock down cells prior to the initiation of EMT. In wound closure assays Foxc1 knock down cells demonstrated an increase in cell migration compared to control cells. We next created an NMUMG cell line expressing Foxc1 under the control of a tetracycline inducible system. When Foxc1 levels were elevated in the absence of TGFβ, NMUMG cells continued to express epithelial markers and an induction in the expression mesenchymal markers (N‐cadherin, Vimentin, Snail1) was not observed. Our data suggest that Foxc1 is not necessary or sufficient for the initiation of EMT by TGFβ in murine cells lines.
Insulin and Angiotensin II Regulate Titin Alternative Splicing via Rbm20
Authors: Zhu Chaoqun , Yin zhiyong , guo wei
Published: June 2021

Objective

This study is to determine whether titin alternative splicing is regulated by insulin and angiotensin II via a muscle‐specific splicing factor, RNA binding motif 20 (Rbm20).

Methods

In primary cultures, neonatal cardiomyocytes (CMs) were isolated from day 1 rats. CMs from wild type (WT), heterozygous (HT), and homozygous (HM) Rbm20 deficient rats were cultured in serum‐free medium and treated with insulin, insulin plus LY294002 (LY) and angiotensin II for 5 days. In in vivo assay, HT rats were treated with streptozotocin (STZ) for 8 weeks. Titin isoforms were detected by 1% SDS‐agarose gel. Band intensity was quantified with imageJ. Western blot and QRCR were performed for protein and gene expression.

Results

Insulin and angiotensin II increased the expression of smaller titin isoform (N2B) in both WT and HT CMs, but no N2B was rescued by insulin and angiotensin II in HM CMs. The phosphorylation of Akt was increased in WT, HT and HM CMs. LY can inhibit Akt phosphorylation and titin isoform transition in both WT and HT insulin treated CMs. STZ treatment decreased the expression of smaller titin N2B in HT rats. The mRNA levels of titin isoforms were consistent with protein level. Rbm20 expression was increased in insulin treated CMs.

Conclusion

Titin alternative splicing has been shown to be largely responsible for the diastolic properties of the heart. Rbm20 is the major regulator of the titin alternative splicing. These results indicated that insulin and angiotensin II regulated titin alternative splicing is Rbm20 dependent, and Akt phosphorylation can enhance Rbm20‐regulated titin alternative splicing.

Supported by NIH P20 GM103432, University of Wyoming Agricultural Experimental Station and USDA‐NIFA
Dissecting Musashi1 role in chromatin remodeling and its consequences to DNA repair and radio‐resistance
Authors: Rosa de Araujo Patricia , Silva Acarizia , Penalva Luiz
Published: June 2021

Musashi1 (Msi1) is an evolutionarily conserved RBP with well establish roles in neuronal stem cell self‐renewal and differentiation. In the context of tumorigenesis, Msi1 has been described to be highly expressed in gliomas, medulloblastoma, lung, colon and breast cancers and linked to a poor prognosis. Its expression is required for tumor maintenance and possibly initiation. We recently established that a decrease in Msi1 expression makes glioblastoma cells more radio‐sensitive: Msi1 KD cells subjected to ionizing radiation showed reduced proliferation as evident from colony formation assays; moreover, an increase in DNA damage was observed via comet assay, γ‐H2AX expression levels and 53BP1 ionizing radiation induced foci.

Msi1 has been always defined as a translation regulator. However, our recent data shows that Msi1 is often present in the nucleus of tumor samples and cell lines. Moreover, CLIP assays identified a large number of Msi1 interacting sites in intronic regions. We suggest then that Msi1 displays additional regulatory functions in the nucleus that could equally contribute to its role in tumorigenesis. Supporting this idea, among Msi1′s protein partners identified by yeast two‐hybrid and mass spectrometry are several chromatin remodeling proteins including Ino80C. The Ino80 complex has been associated with transcription factors involved in cell proliferation, differentiation and embryonic development. Moreover, Ino80 interacts directly with γH2AX during DNA double‐strand break repair. We are currently conducting experiments to explore the hypothesis that Msi1 influences radio‐resistance and DSB repair via its association with the chromatin remodeling machinery.
Synthesis and Characterization of Ribonucleic Acid Aptamers Targeted at Aspergillus Fungus Cell Surface Carbohydrates
Authors: Sheridan Megan , Engelke David , Rohlman Christopher
Published: June 2021

Aspergillosis is a disease characterized by the degradation of the respiratory system due to Aspergillus fumigatus fungal hyphae. The slow establishment of the infection yields low rates of detection and high mortality rates in immune‐suppressed patients in Intensive Care Units (ICU) and Internal Medical Wards (IMW). RNA aptamers may can aid in the detection of aspergillosis using the cell surface carbohydrate, beta‐D‐glucan. The aptamers would be selected to bind selectively to cell surface carbohydrates of the fungus, where positive signals would be useful in the identification of infection. This work will describe isolation of RNA aptamers from a randomized sequence population of 1020 to 1024 variants. RNA aptamer pools were synthesized using PCR amplification reactions to combine the DNA template with primers. In vitro transcription was conducted to form the RNA molecules, where selection was conducted in rounds against beta‐D‐glucan. Sequencing and binding assays will be used to characterize the RNA aptamer pool.
Let the cells do the work: Production of novel recombinant pre‐miRNAs in E. coli as therapeutic agents
Authors: Yu Aiming , Wang Weipeng , Chen Qiuxia , Ho Pui Yan , Li Meimei
Published: June 2021

Noncoding microRNAs (miRNAs or miRs) have been revealed as critical epigenetic factors in the control of various cellular processes including cell proliferation, survival and disease progression. However, research on miRNA functions and development of miRNA‐based therapy is limited to the use of synthetic RNA and recombinant DNA agents. Recently we have demonstrated that novel human pre‐miRNA agents can be expressed in Escherichia coli on large scale using tRNA‐based recombinant RNA technology. Milligrams of recombinant tRNA/pre‐miRNA chimeras were readily purified to high degrees of homogeneity (> 95%) using chromatography. We further showed that tRNA‐carried pre‐miRNAs were selectively processed to mature miRNAs in human carcinoma cells, which consequently suppressed the expression of miRNA target genes and modulate cell proliferation, apoptosis and chemosensistivity. Furthermore, recombinant miRNAs were active in controlling tumor progression in xenograft tumor mouse models and well tolerated in animal models. Herein we use hsa‐mir‐34a as an example to present our findings on recombinant pre‐miRNA agents, which shall serve as novel tools for the study on miRNA functions and development of RNA‐based therapeutics.
RAP2.6L mRNA Level in Oil Palm (Elaeis guineensis) Leaf, Inflorescence Explants and Calli Produced From Them
Authors: Mgbeze Godfrey , Shittu Hakeem , Iserhienrhien Anthony
Published: June 2021

The RAP2.6L is a member of the ERF (ethylene response factor) subfamily β‐4 of the ERF/APETALA2 transcription factor gene family. These preliminary studies seek to investigate the mRNA level of RAP2.6L gene in oil palm (Elaeis guineensis) leaf, inflorescence explants and the calli produced from them, with a view to understanding the molecular basis of direct and indirect organogenesis. Callus was induced and proliferated from non‐chlorophyllous leaf and immature inflorescence tissues using Murashige and Skoog medium. This was supplemented with different concentrations of auxins: 2,4‐D and NAA, ranging from 100‐120 mg/l and 170‐200 mg/l respectively. The times taken for callus induction, percentage callusing explants and weight of fresh callus after 20 weeks of culture initiation were recorded. Callus production was seen to vary in time of initial response and quantity produced for both 2,4‐D and NAA treatments. The leaf tissues yielded more callus in less time than the inflorescence tissues. Treatment 100mg/l 2,4‐D was effective in producing callus in 11 % of leaf explants, while 185 mg/l NAA was more effective in producing callus in 8 % of same. The level of RAP2.6L mRNA was measured with reverse transcription polymerase chain reaction (RT‐PCR). The results obtained indicated that there was no significant difference in the RAP2.6L mRNA level among the explants and associated calli, suggesting that both explants were equally amenable to indirect organogenesis.
The Complex Role of Sequence on the Stability and Function of a Thermophilic TIM Barrel Protein
Authors: Chan Yvonne , Zeldovich Konstantin , Matthews C
Published: June 2021

TIM barrel proteins are encoded by highly divergent sequences, but fold to highly conserved (βα)8 structures with varying stabilities and functions. The indole‐3‐glycerol phosphate synthase (IGPS) TIM barrel protein, required for tryptophan biosynthesis in yeast, was selected as a target for investigating how sequence influences structure, stability, and function. IGPS from the S. solfataricus (SsIGPS) thermophile was subjected to the EMPIRIC assay to assess the change in fitness accompanying saturating mutagenesis on the 8 β‐stands and the preceding loops, known to be important for protein stability. The strategy was designed to test the presumption that mutations in the β‐barrel would provide an in vivo estimate of protein stability by decreasing fitness. The majority of the mutations in the β‐strands and preceding loops decreased the fitness, as expected. Surprisingly, mutations that remove any of the four‐fold symmetric TIM barrel βα hairpin clamps provided a fitness advantage. In vitro studies on one such mutant protein, I107K, was found to be destabilized to urea denaturation. A functional assay revealed that the I107K mutation increases the Vmax as well as the substrate binding affinity, indicated by lower Km. We speculate that the decrease in stability from these mutations is accompanied by an increase in the dynamics of the platform and, therefore, an increase in the fitness of a thermophilic protein at a suboptimal temperature in a mesophilic host.

Funding source: NIH GM23303
Association between intrinsic disorder and serine/threonine phosphorylation in Mycobacterium tuberculosis
Authors: Singh Gajinder
Published: June 2021

Serine/threonine phosphorylation is an important mechanism to regulate protein function. In eukaryotes phosphorylation occurs predominantly in intrinsically disordered regions of proteins. While serine/threonine phosphorylation and protein disorder are much less prevalent in prokaryotes, M. tuberculosis has both high serine/threonine phosphorylation and disorder. Here I show that, similar to eukaryotes, serine/threonine phosphorylation sites in M. tuberculosis are highly enriched in intrinsically disordered regions, indicating similarity in substrate recognition mechanism of eukaryotic and M. tuberculosis kinases. Serine/threonine phosphorylation has been linked to the pathogenicity and survival of M. tuberculosis, thus better understanding of how its kinases recognize their substrates could have important implications in understanding and controlling the biology of this deadly pathogen.
Conformational regulation of the C‐terminal random coil in S100A4 by Ca2+ ions
Authors: Katona Gergely , Duelli Annette , Kiss Bence , Lundholm Ida , Bodor Andrea , Petoukhov Maxim , Svergun Dmitri , Nyitray Laszlo
Published: June 2021

S100A4 or metastasin plays a central role in the progression of metastatic diseases. Like many other examples, the protein S100A4 contains ordered and disordered regions. The aim of this study was to determine how the disordered C‐terminal region influences the binding to Ca2+ and in turn how Ca2+ binding alters the dynamics of the C‐terminal random coil. The combination of small angle X‐ray scattering, pulsed field gradient NMR measurements revealed a conformational change upon binding in the wild‐type protein, but not in the C‐terminal deleted mutant. Comparative isothermal titration calorimetry measurements also indicated that Ca2+ ions bind more tightly to the C‐terminal deleted mutant than the wild‐type protein. Molecular dynamics simulations provide a rational: in the wild type protein the positively charged C‐terminal random coil region interacts strongly with the EF hand residues when they do not coordinate Ca2+ , whereas the C‐terminal region is more free to move in the Ca2+‐bound form of the protein. The C‐terminal random coil never appear to be completely static, but occupies a different conformational space depending on the functional state of is Ca2+ binding protein. Thus this study provides a unique insight to the regulation of random coil regions.
The Prion Switch Unravelled
Authors: Salzano Giulia , Tran Thanh Hoa , Giachin Gabriele , Benetti Federico , D'Angelo Paola , Legname Giuseppe
Published: June 2021

The post‐translational conversion of the cellular prion protein (PrPC) into the misfolded, pathogenic scrapie isoform (PrPSc) plays a key role in prion diseases. PrPC interacts with metal ions, in particular copper, through the octarepeat and non‐octarepeat (non‐OR) binding sites. In order to gain insights into the structural determinants involved in PrPSc formation we investigated the effect of human PrPC (HuPrP) point mutations linked to the genetic form of human prion diseases. Previously, we carried out a structural investigation to determine the high resolution NMR structure of the truncated recHuPrP(90‐231) carrying the E219K polymorphism, the fCJD‐linked V210I and the GSS‐causing Q212P mutations. Such structural studies led to the preliminary conclusion that the structural disorder of the β2‐α2 loop region is a key pathological feature. Here, we evaluated the effect of the pathological mutations on the N‐terminal unstructured domain. We used synchrotron‐based X‐ray absorption structure (XAS) technique to study the coordination geometries of Cu(II) and Cu(I) at two pH values (5.5 and 7.0) in different recHuPrP(90‐231) constructs carrying pathological mutations. We clearly showed that mutations and pH exchanges cause a dramatic modification on both Cu(II) and Cu(I) coordinations in the non‐OR region. Our XAS study highlights the importance of the non‐OR region for prion conversion and proposes a cellular model in which PrPC coordinating copper with one His may be more prone to the conversion at acidic condition. Thus, the non‐OR region may act as the long‐sought‐after prion switch.
Safe Inhibition of Amyloid Proteins Toxicity without Protein Specificity
Authors: Bitan Gal
Published: June 2021

The distinctive 3D structures of proteins have been optimized by a hundred million years of evolution allowing them to recognize their substrates with very high specificity. Thus, drugs targeting enzymes/receptors must compete specifically and bind with high affinity, typically, in the nanomolar range. In contrast, abnormal self‐assembly of amyloid proteins into toxic oligomers and fibrils was not optimized and may, in fact, be a process evolution selected against. Therefore, the forces mediating the abnormal assembly process are substantially weaker. This is why the resulting oligomers are metastable and the nucleation of amyloid fibrils is a rare event. Consequently, we hypothesized that safe remodeling of the abnormal assembly process to inhibit formation of toxic species could be achieved using compounds that bind with moderate affinity and low specificity. We reasoned that binding of such compounds to stable proteins would not affect their structure or function. We tested this hypothesis using “molecular tweezers” that bind selectively to exposed Lys residues. Supporting our hypothesis, a lead tweezer named CLR01 was found to remodel the assembly and inhibit the toxicity of multiple amyloid proteins in vitro and in vivo without affecting normal physiology. CLR01 showed beneficial therapeutic effects in animal models of Alzheimer's disease, Parkinson's disease, and familial amyloid polyneuropathy and was found to have a high safety margin. The data show that misfolded‐protein clearance enhancers, such as CLR01, act by a unique mechanism that is specific to a process rather than to a particular protein, representing a novel approach to designing drugs for the currently cureless amyloid‐related diseases. The presentation will discuss the mechanism and present unpublished data demonstrating the unique action and therapeutic potential of our approach.