bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct Effect of Invasive and Non‐invasive Breast Cancer Cells on Vasculogenic Capacity of Endothelial Progenitors
Authors: Lee Hyunsook , Shah Sajita , Kim Eun‐Sook , Moon Aree , Kang Kyu‐Tae
Published: June 2021

Blood vessel formation plays an essential role in tumor growth and metastasis. Cancer cells secrete various pro‐angiogenic factors that activate preexisting vascular endothelial cells as well as induce recruitment of bone‐marrow‐derived endothelial progenitors; endothelial colony forming cells (ECFC). The interactions between cancer cells and endothelial cells have been largely investigated, but it has been rarely studied whether the vasculogenic capacity of ECFC are differently affected by invasive and non‐invasive phenotype of the cancer cells. Thus, we investigated to determine the vasculogenic potentials of invasive and non‐invasive breast cancer cells. MDA‐MB‐231 and MCF7 were applied as invasive and non‐invasive breast cancer cells. Bone marrow‐derived ECFC were isolated from human peripheral blood. Tubular structure number and length as well as spheroid sprout number and length formed by ECFC was increased when treated with MDA‐MB‐231‐derived conditioned medium (CM) compared with MCF7‐derived CM. Migration of ECFC was also increased by treatment of MDA‐MB‐231‐derived CM compared to MCF7. To mimic tumor microenvironment, ECFC were directly co‐cultured with cancer cells. Tube formation of ECFC was enhanced under the co‐culture condition with MDA‐MB‐231 compared to ECFC alone control. Interestingly, ECFC co‐cultured with MCF7 significantly decreased tube formation. Our results suggest that MDA‐MB‐231 and MCF7 promote ECFC vasculogenesis in different manners, which may be the important determinants to characterize invasive and non‐invasive phenotype of the cancer cells.

Support or Funding Information

This research was supported by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2016r1a6a1a03007648), and supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017R1A2B4005463).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Blockade of 20‐HETE synthesis confers resistance to stress‐induced apoptosis in renal tubular cells
Authors: Alexander Larry D.
Published: June 2021

Changes of the levels of 20‐Hydroxyeicosatetraenoic acid (20‐HETE), an ω‐hydroxylation product of arachidonic acid catalyzed by cytochrome P450 4A, have been recently reported to mediate program‐cell death associated with renal ischemia‐reperfusion (IR) injury. In this study, we evaluated the effects of 20‐HETE and a 20‐HETE synthesis inhibitor N‐hydroxy‐N′‐(4‐butyl‐2 methylphenyl) formamidine (HET0016), on stretch‐mediated cell death in renal epithelial (HK‐2) cells. Stretch‐induced apoptosis was detected by measuring histone‐associated DNA fragments using ELISA (Enzyme‐Linked Immunosorbent Assay) and caspase activity assays. A 20‐HETE competitive in vitro ELISA kit was used for the determination of 20‐HETE levels in biological samples. 20‐HETE protein levels were analyzed using Western blot. Here we report that HK‐2 cells express cytochrome P450 (CYP) 4A protein and produce 20‐HETE when stimulated with cyclic stretch. Exogenous application of 20‐HETE increased, whereas inhibition of 20‐HETE production with HET0016 attenuated 20‐HETE‐ and cyclic stretch‐induced apoptosis. Our results also show that 20‐HETE and cyclic stretch causes time‐dependent activation of c‐Jun N‐terminal kinase (JNK) activity, which was significantly attenuated by HET0016. In addition, the JNK inhibitor SP600125 significantly attenuated cyclic stretch‐and 20‐HETE‐induced apoptosis, whereas the extracellular signal‐regulated kinase1/2 (ERK1/2) and p38MAPK inhibitors PD98590 and SB23580, respectively, were without affect. These data provide the first evidence that 20‐HETE is a potent and direct inducer of apoptosis in renal proximal tubule cells. More importantly, these observations demonstrate a critical upstream role of JNK in cyclic stretch‐induced apoptosis dependent of ω‐hydroxylation of arachidonic acid.

Support or Funding Information

This work was supported by a Scholarly Activity and Research Program (SARP) grant to Dr. Larry D. Alexander

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insulin receptor‐related receptor as an extracellular pH sensor regulating bicarbonate excretion
Authors: Petrenko Alexander George , Mozhaev Andrey , Serova Oxana , Deyev Igor
Published: June 2021

The acid‐base balance in the organism at the system level is controlled primarily by the kidneys and lungs. In the kidneys, this function is performed by the intercalated cells of two types; alpha cells excrete excess acids while beta cells excrete excess bases as bicarbonate. In a result of the biochemical analysis of the insulin receptor‐related receptor (IRR), a receptor‐like tyrosine kinase enriched in beta‐intercalated cells, we discovered its ability to activate upon the extracellular treatment with mildly alkaline media. IRR is a close homolog of the receptors of insulin and insiln‐like growth factor but cannot be activated by to their agonists whereas IRR homologs do not respond to extracellular pH changes. This pH‐sensing property of IRR is evutionary conserved from amphibians to mammals. The IRR activation by alkali resembles a typical ligand‐receptor interaction since it is dose‐dependent and saturable, specific in relation to other homologous receptors, defined by the structure of the IRR ectodomain and accompanied by its conformational changes. Mutagenesis revealed two sites in the IRR ectodomain that act synergystically to induce the receptor activation. IRR activation triggers intracellular signaling resembling the insulin receptor pathway. We obtained a panel of monoclonal antibodies against the IRR ectodomain, one of them being able to activate the receptor, and another – to inhibit its response to alkaline media. Altogether, the obtained data suggest that IRR is a cellular pH sensor. We used kcnockout mice to address IRR systemic functions. The knockout mice are viable in normal conditions but fail to excrete excees bicarbonate upon experimentally induced alkalosis. Nevertheless, the increased blood bicarbonate is compensated by CO2 retention that keeps the blood pH within the physiological range. We performed comparative analyses of knockout and wild type mice kidney and brain transcriptomes and identified statistically valid changes in the expression of several proteins, in particular, ion transporters. Also, a behavioral phenotype of the knockout mice was revealed. They appeared to be less socially active and less agressive as compared to their wild‐type littermates.

Support or Funding Information

This work was financially supported by the Russian Science Foundation (grant № 14‐50‐00131)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
P2Y receptor regulation of apical K2P channels involved in K secretion by human mammary epithelial cells
Authors: Srisomboon Yotesawee , Zaidman Nathan A. , Maniak Peter J. , Deachapunya Chatsri , O'Grady Scott M.
Published: June 2021

In this study, the molecular identity and regulation of ion channels involved in K+ secretion by human mammary epithelial cells was investigated. Voltage clamp experiments were performed on human mammary epithelial monolayers basolaterally treated with the pore‐forming antibiotic amphotericin B dissolved in a solution with intracellular ionic composition. Addition of benzamil to the apical solution inhibited the basal current, consistent with inhibition of sodium absorption. Moreover, apical addition of the K2P channel blockers quinidine, bupivacaine and a selective TASK1/TASK3 inhibitor (PK‐THPP) all produced concentration‐dependent increases in apical membrane current resulting from inhibition of apical K+ efflux. Expression of mRNA for nine K2P channel subtypes was demonstrated by qRT‐PCR. Western blots using biotinylated apical membranes and confocal immunocytochemistry showed that TWIK1, TREK1, TREK2, TASK1 and TASK3 were all located in the apical membrane. Purinergic receptor stimulation with apical UTP also increased apical membrane current, however pretreatment with GF109203X, a pan‐selective inhibitor of PKC, inhibited the effect of UTP. Furthermore, PKC activation with phorbol 12‐myristate 13‐acetate, like UTP, also increased the current. We conclude that basal K+ secretion depends on the activity of multiple K2P channel subtypes, some of which are inhibited by purinergic receptor stimulation and activation of PKC.

Support or Funding Information

This study supported by a Royal Golden Jubilee PhD program Fellowship from The Thailand Research Fund (PHD/0204/2551) to YS and CD and by the NIH (R01 AI128729‐01) to SMO.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NADPH Oxidase 2 and Reactive Oxygen Species Mediate Pulmonary Arterial Smooth Muscle Cell Phenotypic Modulation Following Chronic Hypoxia
Authors: Cross Laura Weise , Vigil Maria A. , Jernigan Nikki L. , Resta Thomas C.
Published: June 2021

Chronic hypoxia (CH), which is associated with chronic obstructive pulmonary disease and high‐altitude exposure, increases pulmonary vascular resistance and promotes pulmonary hypertension through mechanisms that stimulate vasoconstriction and arterial remodeling. Vascular remodeling is characterized in part by abnormal migration of pulmonary arterial smooth muscle cells (PASMCs). Based upon previous studies implicating NADPH oxidase 2 (NOX‐2) in the development of CH‐induced pulmonary hypertension, we hypothesized that NOX‐2‐generated reactive oxygen species (ROS) enhance PASMC migration following CH. To test this hypothesis, we harvested primary PASMCs from control and chronically hypoxic (4 wk at 0.5 atm) rats, cultured them in 5% serum DMEM F12 growth media for 3 days and in 0.3% serum media for 24 hr, and then plated the cells onto 8 μm polycarbonate membrane Transwell inserts (Costar). After allowing the PASMCs to adhere for 1 hr, they were treated with the general NOX inhibitor apocynin (30 μM), the specific NOX‐2 inhibitor GSK2795039 (500 nM), the superoxide dismutase/catalase mimetic EUK‐134 (10 μM), or a vehicle control (0.1% dimethyl sulfoxide) for 24 hr. PASMCs were fixed for 20 min in 4% paraformaldehyde, washed with phosphate‐buffered saline (PBS), stained with 1% Coomassie, and washed again with PBS. Transwell inserts were imaged with an upright brightfield microscope (Nikon) before and after the top layer of PASMCs was removed with a cotton swab. Cell migration was expressed as the percent of total cells that migrated through the Transwell membrane. Our results indicate that CH significantly (p < 0.05) enhances PASMC migration, while either inhibition of NOX‐2 or scavenging of ROS significantly blunts this response. We conclude that NOX‐2‐derived ROS contribute to enhanced PASMC migration following CH.

Support or Funding Information

This work was supported by NIH grants R01 HL132883 and T32 HL‐007736 (to T.C. Resta) and K12‐GM‐ 088021 (to A. Wandinger‐Ness).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PhUn in the Snow: 3rd Graders Learn About Exercise Physiology
Authors: Halpin Patricia A.
Published: June 2021

Physiology Understanding (PhUn) week is an ongoing effective outreach program of the American Physiological Society. This annual event for K‐12 students is held every November. During PhUn week, students get to meet a physiologist and perform science activities. This past year a small private parochial school contacted me and asked me to come into the third grade class in January. The event started with an introduction to the job of a physiologist and an explanation of heart function. The students created their hypothesis Exercise increases heart rate and planned their experiment. The correct way to measure heart rate via the carotid artery was demonstrated by the physiologist and students took their baseline heart rate. Then together with their partner the students went outside to perform their experiment. There was fresh snowfall on the playground yet the students were undaunted to perform the exercise portion of the activity. One member of the student pair ran for three minutes around the perimeter of the playground. Upon stopping they measured their heart rate. The second member of the pair repeated the exercise and took their post exercise heart rate. The whole class went back into the classroom to discuss their results. A discussion was held and students stated their data from before (baseline) and after exercise. The students agreed that their data supported their hypothesis. The physiologist explained why the heart rate needs to increase with exercise. It is always a thrill to be able to work with young students and be the first scientist they have ever met. This PhUn week event is certain to create a lasting memory for both physiologist and students alike.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Student Perception and Preference in Learning of Sensorimotor tracts in Neuroanatomy: Evidence for gender differences
Authors: Akle Veronica , Gutierrez‐Herrera Jaime Andres , Vela‐Salazar Flor Milena , Blanco‐Alvarado Miguel Angel , Pena‐Silva Ricardo A.
Published: June 2021

Neuroanatomy learning and teaching continue to be a challenge in any curricula. One reason is that relations among different structures is difficult to visualize. Spatial abilities are useful in learning neuroanatomy, but students' skills differ widely in any given medical class, specially between genders. Thus, building and manipulating physical models has shown to be useful for learning anatomy, as it incorporates basic sensorimotor skills in the learning process. This study examines the spatial abilities and perceptions of undergraduate medical students toward learning sensorimotor tracts. It also proposes that following a self‐guided virtual module, in addition to “threading” the tracts over a physical model of spinal cord and brainstem, which allows visualization of the trajectories, would improve the consolidation of the information, enhance the learning experience of the topic, and contribute to student autonomy.

In a cohort of 70 undergraduate medical students, the perception of difficulties and learning strategies and actual spatial proficiency were measured using a questionnaire and a standardized Mental Rotation Test (MRT) in 2017‐II. A virtual module consisting of interactive videos, equipped with formative quizzes and a training module to practice spinal cord segment identification were developed in Spanish. Construction of the scaffold model for tract threading, instruction video for assembly of the model and student outcomes will be tested in 2018‐I following a randomized experimental crossover design.

Results of the pilot study showed that the strategies used by students range widely from plain repetition to coloring. Analysis of the answers by gender show a difference in the strategies prioritized. While female students prefer drawing the tracts in colors significantly more than men, male students prefer mental visualization of the tracts and dislike drawing with colors pencils. Also, there is a wide range of spatial abilities in the class, according to the MRT. Just as reported in the literature for the rest of the world, the results of women are significant lower than in men (P<0.05).

Preliminary evidence shows that the perceptions toward the difficulty of content, self‐efficacy and motivation differ between male and female students. Also, they utilize different strategies to study the topic. Further study would allow to develop and test tools, such as a 3D model of the spinal cord and brainstem to “thread” the tracts, that would benefit students with different learning styles.

Support or Funding Information

Vicerrectoría Académica, Universidad de los Andes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Age‐ and Occupation‐Based Public Health Considerations Related to Osteoarthritis of the Knee Joint: A Cadaveric Study.
Authors: Immonen Jessica , Siefring Chris
Published: June 2021

Objective

Osteoarthritis (OA) literature makes minimal suggestion regarding age of disease onset or preventative strategies to reduce risk for onset in various populations. In 2005, the Center for Disease Control and Prevention estimated that 33.6% of Americans 65+ years old were affected by OA; this cadaveric analysis suggests this is largely underestimated. The objective of this assessment is to identify at‐risk populations for OA in the knee joint and make recommendations to prevent or delay disease onset.

Design

Morphometric analyses of the articular cartilage of the tibial plateau were performed on cadaver specimens using Image ProÒ software on three age populations and surface area measurements for articular cartilage degradation were compared to donors' reported ages, clinical histories and occupations.

Results

Data showed that by the 7th decade of life, when patients are in their 60s, articular cartilage degeneration on the tibial plateau had commenced in 100% of specimen. All “homemakers” displayed above average medial tibial plateau degeneration (32.33 ± 24.85%) for their age group while simultaneously reporting pathologies in their clinical history that encourage a sedentary lifestyle. Atrophy or degeneration of crucial ligaments supporting the knee can encouraging sheering and cartilage degeneration. Cadaver knee specimens in the 70–79 years old age group displayed a 10.5% decrease in average ACL circumference (mm) compared to the group of <70 years old specimens (P < 0.001).

Conclusions

This assessment identifies an occupational class with a propensity to develop disease and also identifies a more realistic time frame than previous advisory committees have produced regarding age of disease onset and initiation of preventative measures. Especially given the ACL thins with aging, it is recommended that strengthening of the hip abductors and the musculature supporting the knee commence early in adult life to avoid valgus collapse and shearing at the knee joint.

Support or Funding Information

Internal Grant from Rocky Mountain University of Health Professions

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Facial Canal Dehiscence: Radiological incidence and surgical/clinical relevance
Authors: Guzman‐Lopez Santos , Hernandez‐Trejo Andrea Fabiola , Quiroga‐Garza Alejandro , Elizondo‐Omaña Rodrigo Enrique
Published: June 2021

Introduction

The facial nerve is the most vulnerable structure to injuries in otological surgeries. The dehiscence of the facial canal (Facial Canal Dehiscence FCD) is the main factor that exposes it to iatrogenic injuries. The reported incidence varies between 6–57%.

Materials and methods

High resolution head and neck computed tomography (CT) from a 2 year period were evaluated. Those with previous surgery in the temporal region, trauma, neoplasm, colesteatoma, or pathologies that alter the anatomy, were excluded.

Results

A total of 184 healthy temporal bones were analyzed. FCD was observed in 94 (51.1%) of the bones analyzed. The tympanic portion was the most frequently affected site (96.8%). There were no significant differences in gender, age, and symmetry

Discussion

An imaging study will have superior sensibility for diagnosing FCD, tan dissection in cadaver, reason why incidence may vary from previous reports.

Conclusion

Establishing a presurgical evaluation by means of high resolution CT in order to assess the risk of CDF, would reduce the incidence of surgical injuries.

Support or Funding Information

There was no external funding. All funding provided by the department and medical school

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Dynamics studies of DNA polymerase kappa, a human protein involved in translesion synthesis
Authors: Leong Eleanor Ruth , Benitez Benedetta Sampoli
Published: June 2021

Y family DNA polymerases are a class of enzymes characterized by their ability to copy damaged DNA in a process known as translesion synthesis. They do not eliminate the damage per se, but at least they are able to bypass a stalled replication, avoiding cell death. These polymerases bypass a wide variety of DNA damage, including thymine‐thymine dimers, inter‐strand DNA crosslinks, and other damage caused by reactive oxygen species. Unfortunately, they do not replicate the DNA with high fidelity and some polymerases are more error prone than others. In addition, the kinds of lesions that this group of enzymes can bypass are dependent on the enzyme itself, despite the general architectural similarities of all these enzymes. Human DNA polymerase kappa (pol k) is a member of the Y‐family of DNA polymerases involved in translesion synthesis in humans. Kinetics students have shown that this enzyme can bypass minor groove lesions such as O6‐methylated guanine (O‐metG) but not major groove lesions such as N2‐furfuryl‐guanine (N‐fG). Hydrogen exchange experiments have suggested solvent accessibility changes in various systems, hinting at a mechanism of conformational change upon substrate binding. Through molecular dynamics simulations, we tested the validity of the conformational change hypothesis by comparing the structural changes undergone by pol k in the presence of different templating lesions and with correct and incorrect incoming nucleotides. In addition, binary systems, comprised of just the DNA and the protein without the incoming nucleotide, were examined. Our results show that pol k does not attain a close, catalytically competent state in both the binary and the O‐metG systems, as evidenced by the few contacts between the protein and the incoming nucleotides. We have also identified key residues important for the stabilization of the incoming nucleotide as well as formulated a hypothesis to why the O‐metG lesion does not get bypassed as efficiently as other damages.

Support or Funding Information

This work was supported in part by the Rose M. Badgeley Residuary Charitable Trust Award and the Colette Mahoney Award to BSB.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of split Cas6 for inducible dimerization modules
Authors: Ling Justin A. , Vontalge Emma J. , Sashital Dipali G.
Published: June 2021

Cas6 endoribonucleases are essential for guide RNA biogenesis during CRISPR‐Cas (clustered regularly interspaced short palindromic repeats‐CRISPR associated) immunity, and have exquisite specificity for short structured RNAs that form in the repeating regions of CRISPR transcripts. The Cas6 protein from Thermus thermophilus contains two domains, an N‐terminal domain (NTD) containing the endoribonuclease active site, and a C‐terminal RNA‐binding domain (CTD). The modular nature of this protein makes it a potential platform for molecular scaffolding by splitting the two domains and inducing dimerization through RNA binding. Here we show that the Cas6 NTD and CTD can be split into two separate proteins, and that the domains retain their RNA binding and cleavage activity. Fusion of the domains to the C‐termini of cyan or yellow fluorescent protein (CFP or YFP) enabled measurement of dimerization in the absence or presence of RNA using fluorescence resonance energy transfer (FRET). We find that wild‐type Cas6 NTD and CTD dimerize even in the absence of RNA. However, introduction of mutations in the interface between the two domains disrupts this interaction. The interface mutations do not disrupt RNA binding or cleavage, and addition of RNA restores dimerization of the two domains. These results demonstrate that split Cas6 can be developed into a molecular scaffolding platform that can be controlled through the addition of RNA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Chromatin on Genome Accessibility and Cleavage by CRISPR‐Cas9 in vivo
Authors: Verma Surbhi , Yarrington Robert , Trautman Jon , Schwartz Shaina , Carroll Dana
Published: June 2021

CRISPR‐Cas9 is a prokaryotic immune surveillance complex, which has been adapted for use in eukaryotes as a revolutionizing RNA‐guided genome editing system. Despite its widespread success, differences in organization and structure of the prokaryotic versus eukaryotic genomes may limit its full utility. The eukaryotic genome is highly compact and organized into chromatin with stretches of DNA wrapped around histone protein cores (nucleosome). Additionally, for proper gene regulation, chromatin is further divided into nucleosome bound (NB) and nucleosome depleted regions (NDR). To make genome editing more efficient, we need to understand how chromatin may impact in vivo target accessibility and editing by CRISPR‐Cas9. Nucleosomes have been demonstrated to limit Cas9 accessibility in vitro, and indirect evidence suggests this may also be true in cells. To address this question directly, we are using Saccharomyces cerevisiae as a model system, as it has well‐characterized nucleosome locations and well worked out chromatin regulation. In particular, we are studying the cell cycle‐regulated HO (homothallic switching endonuclease) promoter in its native state as well as in mutants that contain constitutive NDRs in place of native NB regions. We find that several in vivo target sites within strongly positioned nucleosomes (NBs) are cut less efficiently than nearby nucleosome‐depleted regions (NDRs). Furthermore, when nucleosomes are displaced from NB targets by genetic manipulations, Cas9 cleavage efficiency increases. This indicates that reduced Cas9 cutting efficiency at NB targets is due to reduced accessibility to DNA rather than problems with Cas9 cleavage activity or poor guide‐RNA design. Developing an understanding of how genomic factors limit Cas9 accessibility will empower us to perform more precise and regulated genome modifications and to potentially design Cas9 variants with better chromatin access. Findings from the current study will help in making future genome targeting by CRISPR‐Cas9 more effective.

Support or Funding Information

National Institutes of Health

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Characterization of RNA Aptamers Targeted at Aspergillus Cell Surface Carbohydrates
Authors: Woodhead Nicole M. , Silvestri Sabrina S. , Engelke David R. , Rohlman Christopher E.
Published: June 2021

Aspergillosis is a disease characterized by the degradation of the respiratory system due to Aspergillus fungi. The slow growth rate of the infection allows it to establish a strong foot‐hold within the body, making it difficult to treat, and making early detection vital to successful therapy and patient survival. Traditional methods for the detection of Aspergillus in the body can be extremely difficult and invasive. Additionally, these methods provide only minimal rates of detection and high mortality rates in immuno‐suppressed patients. RNA aptamers have the potential to bind selectively to cell surface carbohydrates of the fungus Aspergillus, and aid in the detection of Aspergillosis, where fluorescent detection could be used to increase the sensitivity of ligand binding and the identification of infection. In this study, RNA aptamers targeted at the Aspergillus cell surface carbohydrate Galactomannan were selected from a 40 nucleotide randomized sequence population. Aptamer template pools were synthesized using PCR amplification to generate the initial cDNA pool. RNA aptamers were synthesized via in vitro transcription of the template pool utilizing modified NTPs. Successful aptamers were selected through rounds of binding to Galactomannan, followed by repetition of the amplification and transcription process. We will describe the characterization of the sequence diversity of the aptamer pool as well as its binding characteristics. We continue to refine the current pool of aptamers in order to obtain aptamers with high affinity for the carbohydrate Galactomannan. This research will help the advancement of our project goal of developing medical diagnostics and analytical biosensors which can detect the presence of Aspergillus.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Significance of the Human T‐cell Lymphotropic Virus Type‐1 pro‐pol Frameshift Site Pseudoknot
Authors: Williams Marcus Alonso Cee , Cooper‐Sansone Andrew , Chadeayne Devon , Mylroie Elena , Contreras Uriah , Joe Natalie , Mouzakis Kathryn
Published: June 2021

Programmed ribosomal frameshifting (PRF) is a common viral mechanism used to regulate the levels of viral enzymatic and structural proteins. PRF events are stimulated by cis‐acting RNA elements within the viral transcript, occur during translation, and change the ribosomal reading frame. Each PRF occurs at a frameshift site that includes a “slippery” sequence, a spacer, and a downstream structure. The human T‐cell Lymphotropic Virus Type‐1 (HTLV‐1) retrovirus uses two, independent ‐1 PRF sites to express three of its viral enzymes. While the structure was previously predicted to be a pseudoknot, the importance of this structure to frameshifting has not been established. In this work, we are examining the importance of the frameshift site structure to HTLV‐1 pro‐pol frameshift stimulation. Specifically, we examined how mutations that change the RNA structure impact ‐1 PRF efficiency. We hypothesized that mutations that disrupted the pseudoknot structure would decrease the frameshift efficiency. Likewise, mutations that restored the pseudoknot structure should have no impact on frameshift efficiency. To test these hypotheses, we measured the frameshift efficiencies for several variant frameshift sites using an in vitro dual‐luciferase frameshift assay. Preliminary data suggests that when the pseudoknot structure is disrupted, there is a significant decrease in frameshift efficiency. Surprisingly, a mutation that restored pseudoknot formation increased the frameshift efficiency. These results are significant because they suggest that the pseudoknot structure plays a critical role in frameshift stimulation.

Support or Funding Information

NIH NIGMS SCORE SC2 Award (1SC2GM121197‐01) and Research Corporation for Science Advancement Cottrell Scholar Award (23983)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Protein Translation Initiation by Estrogen
Authors: Holz Marina K.
Published: June 2021

Translational control of gene expression is critical for cellular homeostasis. We focus on eukaryotic initiation factor 3 (eIF3), a large translation initiation factor complex composed of 13 subunits (eIF3a‐eIF3m) in human cells. Overexpression of several eIF3 subunits (eIF3a, b, c, h, i, and m) or underexpression of two eIF3 subunits (eIF3e and f) are observed in a variety of malignancies, including breast, prostate, lung, cervical, and gastric cancers.

Estrogens are key hormones regulating the development and function of reproductive organs in all vertebrates, including puberty onset, fertility, and the estrous cycle. Estrogens also play important roles in many non‐reproductive functions in mediating cardiovascular function, bone metabolism, embryonic and fetal brain development, and other critical biological processes. Estrogen signaling is critical for the development and progression of several types of cancer. Estrogen receptor(ER)a is a nuclear transcription factor that upon binding to its ligand estrogen can promote expression of growth and survival genes, and inhibit expression of transcriptional repressors, antiproliferative and proapoptotic genes.

We hypothesized that in addition its transcriptional role, estrogen‐ERa signaling acts to regulate protein synthesis. We observed that ERa regulates both eIF3 levels and activity, and therefore rearranges the translational machinery to promote the synthesis of specific factors involved in malignant transformation. Our results indicate there exists regulation of eIF3 transcription by ERa. Moreover, estrogen controls the ERa/eIF3 interaction, subsequently directing the assembly of translation initiation complexes and mRNA translation.

In conclusion, estrogen may act as a master regulator of gene expression. In addition to its genomic activity in control of transcription, non‐genomic estrogen signaling regulates protein synthesis, and leads to increased levels of proteins that mediate estrogenic functions.

Support or Funding Information

CA151112‐02, RSG‐13‐287‐01TBE

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chemotropic Receptor Deleted In Colorectal Cancer (DCC) Prevents Translation Initiation By Directly Inhibiting Ribosome Function
Authors: Filbin‐Wong Megan E. , Gonen Tamir , Kieft Jeffrey S.
Published: June 2021

Axonal outgrowth and guidance is dictated by a variety of chemotropic signals. Netrin receptors at the growth cones of developing neurons function to attract or repel outgrowth during commissure formation. The dependence receptor Deleted in Colorectal Cancer (DCC) is activated by netrin‐1, and dimerization initiates a variety of responses, including activation of Src tyrosine kinases and focal adhesion kinase. Prior to netrin‐1 activation, the monomeric C‐terminal tail of DCC binds to 80S ribosomes and prevents polysome formation. DCC activation and dimerization releases 80S ribosomes for translation initiation, presumably to promote axonal outgrowth. The C‐terminal tail of DCC is a 35 kDa protein that is composed of three domains (P1‐3) and is predicted 91% unstructured. Dimerization is facilitated by the P3 domain, while the P1 domain binds to the 60S subunit protein, ribosomal protein L5 (rpL5). Yet, the exact mechanism of how the DCC C‐terminal tail inhibits translation is unknown. Titrating the full‐length C‐terminal tail into translation‐competent lysates, we found that the tail specifically inhibits eukaryotic translation, and inhibits protein synthesis of messages that use both cap‐dependent and internal ribosome entry site translation initiation. Hence, we propose that the DCC C‐terminal tail functions primarily through binding the ribosome, not eukaryotic initiation factors. In fact, we have evidence from co‐sedimentation and gel filtration chromatography studies to show that the full‐length tail binds to the small and large subunits, as well as the 80S ribosome in the absence of mRNA. We propose a mechanism by which the C‐terminal tail blocks the decoding groove of the 80S ribosome to stall the apo‐80S complex. Our hypothesis is supported by data showing the P1 domain (and its interaction with rpL5) is not solely responsible for the decrease in translation, and thus the P2‐3 domains are also important for function. Collectively, our results support the idea that the DCC C‐terminal tail inhibits translation by effectively preventing ribosomes from binding to messages, until receptor dimerization and release of 80S ribosomes. Thus chemoattraction via netrin‐induced DCC receptor dimerization might lead to a global (not message‐specific) increase in localized protein synthesis at axonal growth cones.

Support or Funding Information

Howard Hughes Medical Institute, Janelia Farm Visiting Scientists Program & Metropolitan State University of Denver Provost Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Numerous Cellular Pathways Modulate Non‐Imported Mitochondrial Protein Abundance
Authors: Shakya Viplendra P.S. , Barbeau William , Knutson Christina , Hughes Adam L.
Published: June 2021

Mitochondria are a central hub of metabolism in cells, and their functional decline leads to the onset of numerous metabolic and age‐related disorders. A key question regarding the link between mitochondria and disease is how exactly do changes in mitochondrial function lead to cellular toxicity? To date, most studies have focused on the idea that loss of particular mitochondrial functions is what drives toxicity. However, recent studies have identified a previously underappreciated culprit for toxicity associated with mitochondrial impairment: the accumulation of unimported mitochondrial precursor proteins. Mitochondria are protein‐rich organelles, with ~1000 proteins in yeast, and ~1500 in mammals. Out of ~1,000 mitochondrial proteins, 99% are encoded in the nucleus, translated in the cytoplasm, and imported into mitochondria post‐ or co‐translationally. The majority of these proteins require the mitochondrial membrane potential for import, which is generated by the electron transport chain on the mitochondrial inner membrane. Thus, under conditions of mitochondrial impairment, hundreds of mitochondrial precursor proteins fail import, and their accumulation leads to proteotoxic stress for the cell termed mitochondrial precursor overaccumulation stress (mPOS). We sought to develop a comprehensive understanding of the cellular pathways that operate to mitigate toxicity of unimported mitochondrial proteins. Using S. cerevisiae as a model system, we performed fluorescence microscopy and western blot based screens on ~500 GFP‐tagged mitochondrial proteins under conditions of mitochondrial protein import failure. Through these screens, we have identified at least three potential quality control mechanisms cells utilize to dispose of these proteins, and determined that loss of these systems leads to cellular toxicity under conditions of mitochondrial impairment. Our current work is focused on delineating the protein machinery involved in these quality control systems, and defining the sequence features that drive mitochondrial precursor proteins toward a given fate. Overall, these studies are a step toward understanding the role of mPOS in metabolic diseases, and will help to explore new facets of mitochondrial‐associated disorders.

Support or Funding Information

United Mitochondrial Disease Foundation, NIH R35 GM119694. Searle Scholars Program, Glenn Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Function and Evolution of Ubiquitin HECT Ligases
Authors: Kim Elizabeth D. , Ronchi Virginia P. , Klein Jennifer M. , Summa Christopher M. , Haas Arthur L.
Published: June 2021

The blueprint for enzyme structure and function resides in its amino acid sequence. Within evolutionarily‐related protein superfamilies, functional specificity and speciation are argued to arise through co‐evolving subsets (sectors) of sequence positions. Herein we investigate the functional divergence and evolution of the family of Homologous to the E6AP Carboxyl Terminus (HECT) E3 ligases. The HECT ligase superfamily is catalytically promiscuous despite sharing a common, well‐studied catalytic cycle. Ubiquitin chain length and linkage specificity varies among family members. We use an evolutionary approach to pinpoint residues that underlie concerted interactions of HECT E3 ligases. Statistical Coupling Analysis (SCA) is a bioinformatics predictive method that identifies residue sectors that change in tandem during evolution within a multiple sequence alignment. Our SCA results revealed a network of co‐evolving residues within the catalytic domain that are functionally and thermodynamically coupled. Furthermore, our results suggest that the HECT domain has evolved independently of the N‐terminal targeting domains. We also find that SCA uncovers potential allosteric signaling pathways for HECT ligase enzyme function.

Support or Funding Information

[This work was supported by the School of Graduate Studies at LSU Health Sciences Center to A.L.H. and E.D.K.]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anticancer Effect of Cucurbitacin B on Head and Neck Cancer
Authors: Ganapathy Aravinda Krishna , Horita Megan Ann , Selvamani Vijayalingam Ann , Subramanian Thirugnana , Chinnadurai G Ann , Ezekiel Uthayashanker
Published: June 2021

Head and neck cancer, which includes epithelial malignancies of various origins of the pharynx, larynx, sinuses, oral cavity and nasal cavity, is the sixth most common type of cancer in the world. The primary treatment modality for head and neck cancer involves surgery and aggressive concomitant chemo and radiotherapies. These treatments are associated with significant complications, which are potentially life‐threatening. We wanted to test plant‐derived compounds for anticancer activity against head and neck cancer. Some phytochemicals have been shown to exhibit anticancer effects, and inhibit tumor development and growth in animal models. We screened several phytochemicals on two head and neck cancer lines, SCC‐40 and UM‐SCC‐47 to identify potential anticancer compounds. We identified cucurbitacin B to be a highly potent inhibitor of cell proliferation and viability. Cucurbitacin B inhibited proliferation of both SCC‐40 cells and UM‐SCC‐47 at very low concentrations (0.5–2 μM). The mechanism of inhibition appears to be by induction of apoptosis and autophagy in the treated cells.

Support or Funding Information

AG and MH are supported by DeNardo Education and Research Foundation Fellowship. Financial support for this study was provided by a Seed Grant from Saint Louis University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Decorating with Amino Acids: Biosynthetic Investigation of a Unique Polyphenol Antibiotic
Authors: Whiteley Andrew , Shah Nina , Horta Calista , Petukhova Valentina , Sanchez Laura , Watts Katharine Rose
Published: June 2021

As bacteria become increasingly resistant to current antibiotics, the need for research to create new antibiotics is crucial. The natural antibiotic molecule TLN‐05220 is biosynthesized by Micromonospora echinospora ssp. challisensis NRRL 12255 and exhibits bioactivity against antibiotic‐resistant bacteria in the same range as vancomycin. It is a hexacyclic aromatic polyketide that also contains a unique nitrogen‐containing piperazinone ring, which is found in only a few other natural products. The original report of TLN‐05220 includes a draft genome sequence of the producing strain, and a potential gene cluster for construction of the polyphenol core. It is also hypothesized that a non‐ribosomal peptide synthase (NRPS) is responsible for the production of the piperazinone ring. Here we propose an alternate M. echinospora strain that produces TLN‐05220 as demonstrated through investigation of its genome and mass spectrometry analysis of its secondary metabolites. Through bioinformatic analysis of this strain's genome, we found a strikingly similar biosynthetic gene cluster for production of a hexacyclic polyphenol to that proposed in the original report. However, there are no NRPS genes within or nearby this cluster. Instead, we have identified two asparagine synthetases that lack a highly conserved N‐terminal cysteine residue ‐– suggesting instead that they act as amino acid ligases, incorporating an entire amino acid entity instead of the transfer of a nitrogen atom. Therefore, we hypothesize that alanine, glycine, and diaminoproprionic acid are incorporated into the structure by these amino acid ligases and other tailoring enzymes. We will confirm their incorporation by feeding 13C‐labeled amino acids to cultures of M. echinospora on semi‐solid agar plates and analyzing the culture by Matrix Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increasing Active Learning in the Biochemistry Classroom: Using Group Quizzes to Stimulate Discussion
Authors: Moore Laura J.
Published: June 2021

A variety of pedagogical methods (such as POGIL and teaching through case studies) that encourage discussion are particularly adaptable for biochemistry courses. However, these methods often fall short when students in the classroom are not motivated to engage in discussion of the material. In the Monmouth College Biochemistry classroom, group multiple‐choice quizzes have been used to stimulate discussion and collaborative work. Students are assigned to a group of 3 or 4 students; each student takes an individual multiple‐choice quiz prior to working on the group quiz. The individual quiz allows each student to organize their thoughts before taking the quiz as a group thereby avoiding the issue of group dynamics and students who move through the quiz at different rates. The group quiz is self‐scored using an IF‐AT scorecard. With these low stakes quizzes, almost all students actively engage in the discussion of the material and take an interest in what is being discussed. Student feedback suggests that this process enhances their ability to work collaboratively and increases their comprehension of the material. Finally, due to the multiple‐choice format of these quizzes and the increased comprehension of the material, students have shown an improvement on the ACS standardized biochemistry exam since this method has been implemented.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Zyflamend Induces Apoptosis in Pancreatic Cancer cells via modulation of Endoplasmic Reticulum stress and Autophagy
Authors: Puckett Dexter , Alani Dina , Chahed Samah , Frankel Victoria , Whelan Jay , Bettaieb Ahmed
Published: June 2021

Background

Pancreatic cancer is the fourth leading cause of death in the United States with over 53,670 new cases and 80% mortality in 2017. The 5‐year relative survival rate for patients with pancreatic cancer is 8.2%. Currently available therapies have only moderate efficacy, but notable limitations. Hence, there is an urgent need for alternative therapeutic approaches. Zyflamend, touted as a natural herbal extract to treat inflammation, is credited as being able to benefit many other conditions such as cancer including oral, mammary, bone, skin, colorectal, and prostate cancer. However, its effects on pancreatic cancer in particular, remain largely unexplored.

Objective

The objective of this study is to investigate the effects of Zyflamend on pancreatic cancer survival and decipher the underlying molecular mechanisms

Methods

Pancreatic cancer cells were treated with Zyflamend and key signaling pathways involved in the survival and proliferation of cells were investigated.

Results

Zyflamend treatment caused a dose‐dependent decrease in cell proliferation, consistent with an increase in apoptotic cell death. The effects of Zyflamend were mediated, at least partially, through alterations in autophagy and endoplasmic reticulum stress.

Conclusion

These studies identify Zyflamend as a potential novel approach to treat pancreatic cancer.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.