bluuebunny/crossref_metadata_2025 huggingface dataset:
Ontogeny of Southern Cassowary, Maleo, and Helmeted Guinea Fowl Casques: Implications for the Study of Bony Patterning in Non‐avian Dinosaur Cranial Ornaments
Authors: Green Todd , Gignac Paul
Published: June 2021

Cranial ornamentations (e.g. horns, domes, ridges, rugosities, crests, casques)—some intricately comprised of numerous bony elements—are common among living and extinct archosaurs. Yet, the developmental processes and selective regimes that bring about these metabolically expensive and seemingly bizarre structures remains a mystery. Such features have independently evolved at least 11 times among birds and are varied in their external morphology. Keratinous sheathing and obliterated sutures in late‐stage development have led to contradictory interpretations of cranial osteology for these species and has left it unclear whether the internal morphology of underlying bones of different casqued birds is arranged in a similar fashion. Here we compare the ontogeny for three of the independent casque acquisitions among Aves to clarify their constituent parts. To evaluate the null hypothesis that avian cranial ornaments possess similar anatomical patterns, we analyzed developmental series of skulls of southern cassowaries (Casuarius casuarius), maleos (Macrocephalon maleo), and helmeted guinea fowl (Numida meleagris) using μCT imaging. Sampling neonates and juveniles with incipient casques allowed us to track telescoping elements and measure growth. Our results point toward at least two modes of casque ontogeny among modern birds: (1) disunited, in which a midline chondrocranial element grows relatively slowly and posteriad to buttresses lateral dermatocranial bones (i.e. cassowaries), and (2) geminal, in which a rapidly growing casque is built from anteriad right‐left dermatocranial constituents only (i.e. Neognathae). These findings suggest that cassowaries are developmental outliers among extant casqued birds and extinct dinosaurs. Counterintuitively, this work identifies modern cassowaries—previously considered hallmark analogs for non‐avian dinosaurs—as poor osteo‐developmental models for studying the deep history of archosaur casque evolution.

Support or Funding Information

2018 American Association of Anatomists ‐ Visiting Scholarship (Todd L. Green); 2017 National Science Foundation ‐ Major Research Instrumentation Grant (Paul M. Gignac); 2016 Western Interior Paleontological Society ‐ Karl Hirsch Memorial Grant (Todd L. Green); 2015 National Science Foundation ‐ Early‐concept Grant for Exploratory Research (Paul M. Gignac); 2015 National Science Foundation ‐ Division of Environmental Biology (Paul M. Gignac)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neonatal Ectotympanic Bone Variation among Lorisiforms and Tarsiers
Authors: Fricano Ellen E. I. , Smith Timothy D , DeLeon Valerie B
Published: June 2021

Ectotympanic bone morphology is a classic characteristic used to differentiate primates and identify fossil affinities, often depicted as either a ring or tube falling along strict phylogenetic lines. Though ectotympanic bone morphology has been frequently cited in academic literature, the way in which this bone lengthens within and across taxa is still not well understood. The lorisiform ectotympanic bone is often characterized as ring‐like and fused to a slightly expanded petrosal bulla, however some authors have suggested that the degree to which the ectotympanic bone contributes to the external auditory meatus may vary within lorisiforms. The ectotympanic of tarsiers is often depicted as slightly elongate, though not as long as catarrhine primates. It is possible, and likely, that the lorisiform ear canal is longer than the lemuriforms though not as long as tarsiers. However, the degree to which the ectotympanic bone contributes to the ear canal has been a subject of some debate. Thus, can ontogeny resolve the questions surrounding the lorisiform and tarsier ectotympanic bones? Across lorisiforms, is the external auditory meatus derived from the petrosal bone, ectotympanic bone, or some combination of the two at birth?

To address these questions, three genera of neonatal lorisiforms (Galago senegalensis, Nycticebus pygmaeus, and Loris tardigradus) and one species of tarsier (Tarsius syrichta) were qualitatively analyzed. It was found that in all three lorisiform species, the ectotympanic ring is formed by birth. The elongation of the ectotympanic bone in lorisiforms occurs almost immediately postnatally. The lorisiform and tarsier ectotympanic bones extend laterally as simple tubes, without forming the tubercles that are observed at intermediate stages in catarrhine species. The precise bony makeup of the lorisiform external auditory meatus varies among the genera studied. At birth among Loris and Nycticebus, the ear canal is already elongated but likely incorporates contributions from structures derived from the squamosal and petrous namely the post‐glenoid tubercle and the auditory bulla. The tarsier ectotympanic bone closely resembles the Galago at birth, and we suggest that the tarsier cranial proportions may be an underlying factor in the ultimate lengthening of the bone in this taxon.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Disappearing Enamel and Molars: The Evolution of a Dietary Niche Focused on Gums
Authors: Burrows Anne M , Hartstone‐Rose Adam , Nash Leanne T , Silcox Mary T , Selig Keegan R , López‐Torres Sergi
Published: June 2021

Gummivory, a diet focused primarily on gums, is a relatively rare dietary niche among mammals, but is found in several species of strepsirrhine primates. Getting gums requires either gouging a wound in tree bark to stimulate gum flow or scraping at a semi‐dried gum plug. Both of these acquisition activities focus on the anterior dentition, and the processing of the gums requires minimal mastication with molars. Primate diets focused on leaves and fruits are associated with specific dental signatures, morphological features that can also be used when examining the fossil record. Does gummivory, a relatively low‐quality diet based on foods that are readily available, have a consistent dental signature among primates? How did the evolutionary process of gummivory in primates shape the dentition, if at all? The present study was designed to evaluate these questions. Linear measurements were collected via calipers from select anterior teeth and the last lower molar from lorisid primates that feed primarily on gums and their closely‐related, non‐gummivorous relatives: Loris spp. (n=19), Nycticebus coucang (n=61), Arctocebus spp. (n=8), and Perodicticus spp. (n=40). Mean scaled differences were compared among the four groups using Bonferroni corrected one‐way ANOVA's (alpha <0.05). For some of these taxa, differential enamel thickness in the lower canine, used to represent the toothcomb, was examined via Micro‐CT scans. We found consistent reduction (p<0.05) of the lower last molar in gummivorous taxa relative to the non‐gummivorous taxa. We also found differential distribution of enamel thickness in the anterior teeth (specifically, the toothcomb) with reduced enamel on the lingual surface relative to the labial surface of the toothcomb teeth. This feature is consistent with the findings from previous studies of other gouging mammals, and gummivorous primates. Together, these dental findings suggest a potential dental signature of gummivory in primates: reduction of the lower last molar and differential distribution of enamel thickness in the anterior teeth.

Support or Funding Information

NSERC Discovery Grant and Department of Anthropology, University of Toronto.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mesenchymal Cells Utilize Integrins to Attach to Titanium Implant Surfaces
Authors: LALLIER THOMAS Edward
Published: June 2021

Objectives

Titanium implants have been used for decades to effectively replace missing teeth. Recently, surface modifications have been used to enhance implant osteointegration. Despite these innovations, little is known about how cells interact with modified titanium surfaces. This study aims to examine how mesenchymal cells attached to modified titanium surfaces.

Methods

Human periodontal ligament (PDL) and gingival fibroblasts were evaluated for their ability to survive and attach to three different commercially available implants; Dual thermo‐etched titanium (DTET), hydroxyapatite coated dual transition surface (HADTS) and moderately rough titanium oxide layer with high crystallinity and a high phosphorus content (MRTOP). Subsequently, the cell attachments to modified titanium surfaces were examined for enhancement using serum proteins and inhibition using anti‐integrin as well as anti‐extracellular matrix molecule (ECM) antibodies.

Results

Cells attached to modified titanium implant surfaces in a time dependent manner over 24 hours, with no significant difference between the surface modifications examined. Cell attachment was dependent upon the presence of serum in the growth media. Serum is known to contain several ECM proteins, including; fibronection, fibrinogen, and vitronectin. Integrin β1 antibodies significantly inhibited cell attachment, while αVβ3 antibodies did not. Fibronectin/RGD dependent integrin receptor antibodies (αV and α5) inhibited early attachment (before 8 hours). In contrast, collagen dependent integrin receptor antibodies (α1 and α2) inhibited only later attachment (after 8 hours). Finally, laminin dependent integrin receptor antibodies (α3 and α6) failed to significantly inhibit cell attachment. In addition, fibronectin (and to a lesser extent fibrinogen) antibodies inhibited early cell attachment, while collagen Type I antibodies inhibited later attachment.

Conclusions

Cell attachments to titanium surfaces are at least partially modulated by integrin receptors via ECM proteins coating the titanium surfaces. Cell attachment can be broken down into 2 phases: (1) mediated by serum fibronectin/fibrinogen for the first 8 hours, and (2) mediated by collagens produced by the mesenchymal cells between 8 and 24 hours. Further studies are needed to determine if any of these integrin mediated attachment events effect osteoinduction or osteointegration.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Creating a 3D Model to Study Equine Lameness
Authors: Lee Alexander K. K. , Uhl Elizabeth W. , Osborn Michelle L.
Published: June 2021

Chronic lamenesses in horses are often managed but not effectively treated. We hypothesize that (1) a causal relationship exists between the forces created by aberrant biomechanical positions and movements, and degenerative joint diseases, and (2) adjusting the aberrant biomechanics will treat the lameness. As a first step in testing our hypotheses, we have constructed a computer model using equine CT data to model full body positions and gaits that may be related to various equine lamenesses. 3D equine bone models were reconstructed from equine CT data obtained from the University of Georgia and Louisiana State University using the 3D visualization and analysis software, Avizo®. The bone models were imported into Autodesk® Maya® for repositioning of the full skeleton. In Autodesk® Maya®, a full 3D rig was attached to the model to allow for accurate positioning and animation. The ribbon spine technique was used on the spine and neck of the model to allow for accurate positioning with the ability to rotate and bend the spine. The rig was adopted to be manipulated using controls for easier and quicker positioning. To create a trot animation using the model, still frames from Muybridge were used as reference; and the model was matched to the positioning in the photos. Once key positions were set, an animation could be rendered. To demonstrate the utility of the model, we used it to compare the habitual standing posture of a horse with a left front anatomical club foot to that of a horse with normal front limb anatomy using photographs taken from different angles. The model demonstrated there were whole body postural effects of the anatomical club foot in that the horse's vertebral column was bent around the limb with the club foot and the pelvis tilted so the opposite hip was slightly raised. The limb with the club foot was also rotated laterally, which further exacerbated the club shape of the hoof by increasing the stress impacting its lateral wall. Identification of these secondary postural affects allowed them to be targeted and effectively resolved through rehabilitative exercises, which greatly diminished the negative impact of the anatomic abnormality. Our future goal is to further expand the usefulness of the model for 3D biomechanical analysis of equine lameness and to develop a protocol for 3D model creation that can be adopted and edited for use in various animal or human studies.

Support or Funding Information

Louisiana State University School of Veterinary Medicine Charles V. Cusimano Equine Health Studies Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Visible Humanity
Authors: Spitzer Victor Michael
Published: June 2021

The objective of this work is to utilize Visible Human Project type imaging on a donated human cadaver specimen and integrate that image data and resultant anatomical modeling with the medical history and life experience of the donor for the education of medical and healthcare students. The Visible Human Project (1991) was focused on producing three‐dimensional, photographic image data of optimal human anatomy. Criteria for Visible Human specimens required a young donated cadaver with no visible pathology and no surgical interventions. In 2000, a woman presented with a passionate desire to be a “visible human”. It was eventually agreed (after she examined the freezer and cryomacrotome that would be used to section her) that her wish would be granted if the conditions of her death made it possible and if she would also include her medical and life history including video documentation of her remaining life as part of her donation. Motivation for her body and life history donation was to increase compassion among healthcare workers. In contrast to Visible Human Project criteria, she was 72 years old in 2000, had 26 surgeries including four cervical screws with connecting wires to stabilize her cervical vertebrae and a prosthetic hip. She survived melanoma, breast cancer and severe depression. She was sure and determined she would die within a year. She passed away on February 16, 2015. She was imaged with CT and MR and frozen. A plastic copy of her hip prosthesis was 3D printed from the CT data. The hip region was thawed and the prosthetic hip replaced with the plastic copy to permit sectioning the pelvis. The cervical screws and wires were not removed and were successfully sectioned. The cadaver was blocked into four subunits, each less than 20” in height in a manner similar to the Visible Human. Each block was embedded in polyvinyl alcohol dyed blue. Photographic images with 7,360 × 4,912 pixels (pixel size = 63 microns) were acquired in Nikon raw image format. Data includes over 20,000 transverse cross‐sectional photographs, CT and MR images, medical records and hours of recorded video of her life from 2002 till her death. She was introduced to participants at the Fourth Visible Human Conference in Keystone Colorado, October 17–19, 2002. At that conference, some participants participated in videotaped interviews with the donor.

Segmentation of the photographic images has begun and 3D models of her aged vasculature reveal their tortuosity and pathology. 4K and HD videos of the cross‐sectional photographs have been rendered and will be available for viewing. CT renderings of the whole body and the cervical vertebral fusion will also be presented. This is NOT part of the Visible Human Project and that was made clear to the donor prior to her

Support or Funding Information

Funding provided by Touch of Life Technologies, Inc. and the University of Colorado Department of Cell and Developmental Biology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The “Avocado Lab:” an inquiry‐based laboratory experiment to interrogate the function of polyphenol oxidase in avocado browning
Authors: Peres Shelby , Riley Kasandra
Published: June 2021

In the presence of oxygen, polyphenol oxidases (PPO) catalyze oxidation of phenolic compounds into quinones, the polymerization of which is responsible for the visible browning of cut fruits. PPOs are expressed in diverse organisms including animals, fungi, and plants, and the avocado fruit provides students a unique vehicle for qualitatively exploring this enzymatic reaction. Here, we present a scalable, inquiry‐based laboratory experience that is tunable for non‐science undergraduates to 300‐level biochemistry students. Students are provided with a single Haas avocado and access to an assortment of common laboratory and household reagents including salts, oils, soaps, EDTA, reducing reagents, onions, sugar, acids/bases, etc., as well as the ability to control temperature, light, or exposure to air. Each student designs a well‐controlled experiment to test a hypothesis regarding the effect of one or two variables on avocado browning in a two to three‐hour span. During the incubation time, biochemistry students use the Protein Data Bank and primary literature to better understand the structure of PPO and other molecular components of the avocado, while non‐major students discuss the roles of pH, temperature, and substrate availability on an enzyme. Due to the inherent complexity of fruit flesh, avocado browning is not completely understood and is likely multi‐faceted, leading to opportunities for discussion in both classrooms. The insoluble brown phytomelanins produced in the browning reaction appear on a controllable time scale and are easily depicted in a color photo, so students can create a figure to accompany a qualitative analysis of their experimental results in a single lab period. Furthermore, since avocados are familiar foods and select test reagents are generally recognized as safe, the optimal protocol investigated in the lab can be further applied to best practices in the kitchen in everyday life, promoting the transfer of knowledge learned in the classroom to practical environments.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mentorship for developing course‐based undergraduate research experiences (CUREs): The Council on Undergraduate Research Mentorship for Integrating Research into the Classroom (MIRIC) program
Authors: Wolyniak Michael J , Resendes Karen K
Published: June 2021

The life science education community has responded to the recommendations of the American Association for the Advancement of Science (AAAS) Vision and Change document with several initiatives designed to improve the way in which undergraduates learn science. These initiatives have often taken the form of one‐time workshops that generate awareness of and interest in developing authentic research experiences for undergraduate STEM classrooms. However, they have been less successful with respect to generating the sustainable change necessary to bring real reform to undergraduate science education. To create sustainable change, long‐term faculty development initiatives focused on mentorship are needed so that instructors seasoned in developing and implementing course‐based undergraduate research experiences (CUREs) can convey their experiences to mentees interested in using these pedagogical techniques as the centerpiece of their own teaching. The Council on Undergraduate Research (CUR) Biology Division has created the Mentorship for Integrating Research Into the Classroom (MIRIC) program to provide a means for members with an interest in developing improved and sustainable active learning techniques to gain experience in this style of teaching through close, long‐term interaction with a veteran teaching mentor.

Developed from the former ASCB Mentorship in Active Learning and Teaching (MALT) program, MIRIC focuses on the development of instructors who wish to develop a dynamic CURE. Current and future life science instructors pair themselves up with seasoned veterans of CURE development and work with them and their students over the course of a semester or longer to develop a CURE that will allow the mentee to bring authentic research into his or her classes.

In our pilot studies, we collected qualitative and quantitative data based on participant interviews and coding videos of student and instructor actions during classroom activity (Smith et al., 2013), respectively, that suggest that MIRIC mentorships have made positive gains in promoting sustainable active learning techniques among participants. Going forward, we wish to use instruments like the Laboratory Course Assessment Survey (Corwin et al., 2015) and Experimental Design Ability Test (Sirum and Humburg 2011) to assess the effectiveness of the MIRIC laboratory intervention.

Support or Funding Information

Council on Undergraduate Research (CUR)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
UR in Curriculum: DNA Barcoding Research Tool can be Scaled from Traditional to Course‐based Research Project with Ease
Authors: Gadura Nidhi
Published: June 2021

Most of our students at Queensborough Community College, CUNY are first generation, non‐traditional students with a vast majority working full‐time or part‐time to meet family obligations and sustain themselves outside the classroom. As an educator, one has to constantly be mindful of this reality. While a few of our students can follow a traditional one‐on‐one research model, majority cannot. Therefore, the option to bring research to them in their classrooms was considered as part of the CUR (Council on Undergraduate Research) initiative. This pedagogy was reluctantly implemented in honors courses in the Biotechnology degree program.

UR (undergraduate research) has since been institutionalized and used cross curriculum in multiple disciplines. A formal approach has been implemented for faculty development. Authentic research experiences are embedded in the curriculum. I partnered with Cold Spring Harbor DNA Barcoding Lab that runs an Urban Barcoding project. This was originally done to help local high school students with authentic research experience. Later, I requested the Urban Barcoding Project team to allow my Queens borough students to participate in the project. I incorporated a DNA Barcoding experiment in my Genetics course as an honors component. Biotechnology students served as mentors to Genetics students while being closely supervised. This served as a capstone experience for sophomores at a community college. A detailed curriculum and assessment strategy was developed for the DNA Barcoding to be incorporated in the curriculum. The Queens borough honors committee then approved this project and its timeline. Students developed a hypothesis driven project while working in teams of three. They met during free club hours all semester long to complete their lab work and analyze their results. This experience also became part of their resume and helped them tremendously as they applied for senior colleges or competitive summer research programs after graduation. After seeing the significant success of this project, my belief that students learn more when they get to do hands on science was reaffirmed. Students not only retained skills that are transferrable but also gainedvconfidence in their skills and work as a team toward completion of their project.

This experience also made me realize that the DNA Barcoding project can be scaled from two to twenty students with great ease! I have offered DNA Barcoding project in three settings: Traditional mentor mentee experience; Course‐based authentic research experience and Peer‐Led authentic research experience. Pros and cons for all three will be discussed.

Support or Funding Information

NIH‐BioPREP QCC ‐ Stony Brook Collaboration

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the role of Mre11 gene variants in cancer
Authors: Mateos Cristina , Headrick Ashley , Romero Itzel , Kuehn Sydney , Ray Sreerupa
Published: June 2021

Among the different damages that a cell can undergo, DNA double‐strand break (DSB) is the most detrimental one. DNA contains all the necessary information for a cell's proper functioning and replication, therefore a break that damages both of its strands completely destabilizes the DNA molecules. If not properly repaired this could lead to genomic instability and tumorigenesis. Cells repair DSBs by two repair pathways‐ homology‐directed repair (HDR) and non‐homologous end‐joining (NHEJ). Recently, a back‐up NHEJ pathway has been reported and is referred to as alternate‐NHEJ (Alt‐NHEJ). Although Alt‐NHEJ is advantageous for damaged cells, it can mutate the DNA sequences at the junctions, which may lead to an altered genome that can have severe biological consequences like cancer. Mre11 plays a role in DSB repair pathways as HDR, NHEJ and Alt‐NHEJ. Mre11 possesses single‐strand endonuclease activity and 3′–5′ exonuclease activity which are essential for DNA end‐processing. Recently Mre11 is shown to be overexpressed in breast cancer and high Mre11 expression was associated with a more malignant behavior in breast cancer. Furthermore, germline mutations of the Mre11 gene was identified in a recent screening of hereditary susceptibility to breast and/or ovarian cancer. Mutations in DNA repair genes can have a significant impact on cancer prognosis and treatment. Here we show that cells expressing variants of Mre11 are sensitive to DNA damaging agent bleomycin. Work is ongoing to characterize how the Mre11 variants influence response to various DNA damaging agents and induce genomic instability. This will provide mechanistic insights into the role of the Mre11 variants inducing genomic instability and cancer and has potential to impact the treatment of cancer.

Support or Funding Information

This research was supported by Linfield College Start‐Up Funds and Linfield College Faculty Student Research Collaborative Grant (FSRCG) to SR.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kub5‐Hera deficiency compromises homologous recombination and enhances vulnerability to PARP inhibition in BRCA‐proficient breast cancers
Authors: Motea Edward
Published: June 2021

Purpose

Identification of novel factors that compromise homologous recombination (HR) is key toward innovative strategies to expand the use of poly(ADP‐ribose) polymerase (PARP) inhibitors beyond BRCA deficiency. Our lab discovered that partial loss of Kub5‐Hera (K‐H), a transcription termination factor, stimulates the formation of various BRCA‐related cancers in female mice that was accelerated by single exposure to ionizing radiation (IR, 1 Gy). Here, we investigated how K‐H functionally impacts homologous recombination (HR) and cellular response to PARP inhibition in a subset of BRCA‐proficient breast cancer cell lines

Experimental Methods

The functional impact of K‐H alterations in a subset of breast cancer cell lines were assessed by targeted gene silencing, bioinformatics, mutagenesis techniques, plasmid reporter assays, immunofluorescence, microarray/qPCR analyses, chromatin immunoprecipitation, colony formation assays, rescue experiments and Western blots.

Results

Kub5‐Hera promotes the expression of a critical DNA damage response and repair effector, cyclin dependent kinase 1 (CDK1), which phosphorylates BRCA1 for efficient repair of double‐strand breaks (DSBs). Mechanistically, we demonstrate that K‐H binds RNA Polymerase II (RNAPII) at the CDK1 promoter to efficiently enhance CDK1 transcription. K‐H depletion compromises HR, but upregulates poly(ADP‐ribose) polymerase 1 (PARP1) activity for cancer cell survival. Genetic and pharmacological ablation of PARP1 activity in a subset of BRCA‐proficient cell lines with aberrant K‐H reduction leads to synthetic lethality, which is reversed by re‐expression of wild‐type K‐H but not the mutant with reduced RNAPII binding.

Conclusions

K‐H deficiency promotes genomic instability and a state of “BRCAness” in a subset of breast cancers. These cells may have an exploitable addiction to PARP‐mediated DNA repair for survival that could greatly expand the use of PARP inhibitors beyond BRCA mutations. Collectively, these results suggest that aberrant K‐H alterations may impact overall cellular response and survival to DNA damage and have clinical implications in cancer.

Support or Funding Information

NIH R01 CA210489 (PI: David A. Boothman); Minority Supplement CURE Award CA139217‐05S1 and T32CA124334 to E.A. Motea

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epichlorohydrin‐Induced DNA‐Protein Cross‐Links in Human Leukemia Cells
Authors: Passang Tenzin , Millard Julie T
Published: June 2021

Over 1.5 million tons of epichlorohydrin (ECH) are used annually in the manufacturing of epoxy resins and polymers. A probable human carcinogen, ECH shares structural homology to nitrogen mustards, the oldest class of anti‐tumor drugs. Such bifunctional alkylating agents can react with DNA and proteins, leading to genotoxic effects. Previous work in our lab has shown that treating human leukemia (HL‐60) cells with toxic doses of ECH leads to the formation of cross‐links and triggers apoptosis. In these studies, cross‐linking was quantified via single cell gel electrophoresis (the comet assay), which does not distinguish between DNA‐DNA cross‐links and DNA‐protein cross‐links (DPCs). The goal of this work is to further characterize ECH‐induced DPCs. Nuclear proteins that bind to biotinylated DNA after treating HL‐60 cells with ECH under different conditions are captured via streptavidin beads. Cross‐linked proteins are then characterized through gel electrophoresis. Preliminary data suggest that ECH does induce DPC formation, with greater cross‐linking efficiency at higher concentrations of ECH. Our findings could have significance for understanding the occupational risks of ECH exposure among industrial workers.

Support or Funding Information

Supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Transcriptional Dysregulation on the DNA Damage Response in Ewing's Sarcoma
Authors: Rollins Matthew , Schwartz Jacob
Published: June 2021

Ewing's sarcoma is a soft‐tissue bone malignancy characterized by a translocation event wherein the N‐terminal low complexity domain of EWSR1 is fused with the DNA binding domain of the ETS transcription factor Fli1. The resulting EWS‐Fli1 fusion protein drives expression of oncogenes and knockdown of tumor suppressor proteins. More specifically, EWS‐Fli1 acts as transcriptional regulator by helping recruit RNA Pol II to promoter regions, which is subsequently phosphorylated by CDK9 to escape initiation into elongation. Interestingly, Ewing's sarcoma is particularly susceptible to chemotherapy compared to other cancers; however, the underlying reason for this sensitivity to DNA damage has not been elucidated. Given EWS‐Fli1's role in disrupting transcription, it was hypothesized EWS‐Fli1 is concurrently disrupting the DNA damage response. Here, a combination of CDK9 inhibition with DNA damage was found to drastically diminish cell viability in Ewing's cell lines but not in non‐Ewing's osteosarcoma or HEK293 cells. Ordinarily, after DNA damage γH2A.X phosphorylation occurs on histones near sites of damaged DNA early during the DNA damage response to signal DNA damage repair initiation. Further work has shown Ewing's cells undergoing the combination treatment above have a marked decrease in this DNA damage marker γH2A.X as compared to non‐Ewing's cells. Together, these results suggest Ewing's sarcoma cells are sensitized to DNA damage due in part to transcriptional dysregulation.

Support or Funding Information

NIH Grant #214660

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MRE11 Regulation by SIRT2 in DNA Double‐Strand Break Repair
Authors: Zhang Hui , Head Pamela Sara , Bastien Amanda , Li Chunyang , Duong Duc , Seyfried Nicholas , Yu David
Published: June 2021

At the core of MRN (MRE11‐RAD50‐NBS1) complex, MRE11 is an essential player in DNA end resection and homologous recombination (HR) repair in DNA double‐strand breaks (DSBs). However, the mechanisms by which MRE11 is regulated in DSB repair are not completely understood. We have previously shown that SIRT2, an NAD+ dependent protein deacetylase, implicated in aging, metabolism, genome maintenance, and tumor suppression, plays a critical role in directing the DNA damage response (DDR). In this regard, SIRT2 depletion in cells results in hypersensitivity to ionizing radiation (IR) and impaired HR. To identify the global landscape of DDR proteins regulated by SIRT2, we performed proteomic analysis to identify acetylated peptides regulated by SIRT2 in response to DNA damage in cells. Our analysis revealed a network of proteins regulated by SIRT2, including MRE11. We validated that SIRT2 interacts with MRE11. In addition, SIRT2 deacetylates MRE11 in vitro and in cells. SIRT2 depletion impairs the accumulation of MRE11 to DNA damage sites in response to micro‐irradiation. Our results identify MRE11 as a novel SIRT2 deacetylation target, critical for the repair of DNA DSBs by DNA end resection and provide insight into how dysregulation of SIRT2 and MRE11 results in genomic instability and cancer development. Moreover, MRE11 deacetylation by SIRT2 may be targeted as an adjunct to improve the efficacy of ionizing radiation for cancer therapy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA recognition by bZIP transcription factors
Authors: Rodriguez‐Martinez Jose Arcadio
Published: June 2021

Eukaryotic transcription factors rarely function by binding DNA as monomers. However, how transcription factor dimerization impacts DNA recognition is poorly understood. bZIP transcription factors are obligate dimers that regulate gene expression in multiple cellular process. Guided by bZIP dimerization properties, we examined DNA binding specificities of 270 human bZIP pairs. DNA interactomes of for 102 bZIP dimers revealed that 72% of heterodimer motifs correspond to conjoined half‐sites preferred by partnering monomers. Remarkably, the remaining motifs are composed of variably‐spaced half‐sites or “emergent” sites that cannot be readily inferred from half‐site preferences of partnering monomers. Identified cognate sites were validated by in vitro binding assays. Furthermore, bZIP heterodimer‐preferred sites were overrepresented in genomic regions co‐occupied by two bZIP proteins as determined by ChIP‐seq peaks. Focusing on ATF3, we observed distinct cognate site preferences conferred by different bZIP partners, and demonstrated that genome‐wide binding of ATF3 is best explained by considering many dimers in which it participates. Notably, our compendium of bZIP‐DNA interactomes were applied to predict the impact of non‐coding mutations on bZIP‐DNA binding.

Support or Funding Information

T32HG002760

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Mitochondrial DNA Transcription by Post‐Translational Modifications of Mitochondrial Transcription Factor B2
Authors: Senti Mackenna L , Bostwick Alicia M , Dittenhafer‐Reed Kristin E
Published: June 2021

Mitochondrial DNA (mtDNA) encodes for 13 protein components required for oxidative phosphorylation (OXPHOS), the primary cellular energy production pathway. While the critical roles of mitochondria in metabolism and cellular function are well established, the mechanisms regulating expression of mtDNA‐encoded genes are poorly understood. Defects in the regulation of mtDNA transcription are implicated in numerous pathologies such as neurodegenerative disorders and cancers. mtDNA is complexed to proteins in structures known as nucleoids, of which some proteins play critical roles in transcriptional regulation. Our objective is to determine whether reversible post‐translational modifications (PTMs) of nucleoid proteins, including lysine acetylation and serine/threonine/tyrosine phosphorylation, regulate mtDNA transcription. Our central hypothesis is that PTMs alter the function of these proteins and provide a means of regulating mitochondrial gene expression. We highlight our work on the characterization of mitochondrial transcription factor B2 (TFB2M), focusing on the mutagenesis, bacterial expression, purification, and analysis. Site‐directed mutagenesis was used to alter the amino acids known to be post‐translationally modified to amino acids mimicking either the modified or unmodified state. Purified mutants and wild type TFB2M were analyzed in a mtDNA binding assay to determine the effects of PTMs on TFB2M function. Initial data suggest that TFB2M relies on modified or unmodified states of key PTM sites to regulate its ability to bind to mtDNA. Characterization of these sites is critical for determining TFB2M's role in mtDNA transcriptional regulation. Future work involves additional screening of TFB2M mutants, optimization of the mtDNA binding assay for analysis of other nucleoid proteins, and in vitro transcription assays to assess mutant protein functionality on mtDNA transcription.

Support or Funding Information

This research is based upon work supported by the National Science Foundation Division of Molecular and Cellular Biosciences under Grant No. 1814845.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Cataract‐associated RNA‐Binding Protein Celf1 Post‐transcriptionally Regulates the Key Eye Transcription Factor Pax6
Authors: Weatherbee Bailey , Sandeep Aryal , Siddam Archana , Gautier‐Courteille Carole , Viet Justine , Paillard Luc , Lachke Salil
Published: June 2021

The lens is a transparent tissue that focuses light on the retina allowing high‐resolution vision. Loss of lens transparency is defined as cataract, the leading cause of blindness worldwide. An estimated 25–50% of congenital cataract cases arise from underlying genetic alterations, making it important to characterize cataract‐linked genes and their pathological mechanisms.

Recently, our lab demonstrated that deficiency of the gene Celf1 causes cataract in fish and mouse, indicating its conserved function in vertebrate lens development. Celf1 encodes an RNA‐binding protein (RBP) that directly binds to specific mRNAs and control them on multiple “post‐transcriptional” levels, impacting their alternative splicing, decay or translation into protein. An important unresolved question is whether a post‐transcriptional regulator like Celf1 controls key transcription factors (TFs) in lens development. To address this fundamental question, I generated and characterized lens‐specific Celf1 conditional knockout mice (Celf1cKO/lacZKI). Using immunofluorescence assays, I demonstrate that Celf1cKO/lacZKI lenses exhibit abnormally high protein levels of the homeodomain‐TF Pax6. Further, by performing Celf1‐antibody coupled RNA‐immunoprecipitation (RIP) followed by RT‐qPCR, I demonstrate that Celf1 protein directly associates with Pax6 mRNA.

Next, I investigated if Celf1 coordinately functions with other RBPs to exert combinatorial control over gene expression. I performed co‐immunoprecipitation to demonstrate that Celf1 is associated with another RBP, specifically HuR/Elavl1 in lens cells. Finally, by performing HuR/Elval1‐antibody coupled RIP‐RT‐qPCR assay I show that HuR/Elavl1 binds to Pax6 mRNA.

These data indicate that the RBPs Celf1 and HuR/Elavl1 directly interact and bind to Pax6 mRNA to mediate combinatorial control of Pax6 protein expression in mouse lens development. Alterations of Pax6 levels are known to cause severe eye defects in human and animal models. Thus, my research uncovers a novel RBP‐based molecular mechanism for regulation of Pax6 expression in the lens, in turn, informing on a new pathway for the pathology of eye defects, including cataract.

Support or Funding Information

NEI/NIH RO1 EY021505, Milton H. Stetson Memorial Undergraduate Research Fellowship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of a Bacterial Three‐Hybrid Assay for RNA‐Protein Interactions
Authors: Hegner Courtney L , Wang Clara , Mansky Rachel , Berry Katherine E
Published: June 2021

Non‐coding RNAs regulate gene expression in every domain of life. In bacteria, non‐coding small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by protein chaperones. While much is known about the mechanism of a few model RNA chaperone proteins, significant gaps remain in our understanding of how other bacterial RNA‐binding proteins interact with RNA to regulate gene expression. While there are numerous biochemical methods to study bacterial RNA‐protein interactions, new genetic methodologies would be a powerful complement to current tools. We have recently developed a bacterial three‐hybrid (B3H) assay that detects the binding of E. coli sRNAs with multiple RNA chaperone proteins. This assay couples the transcription of a genetic reporter to the interaction of a DNA‐bound “bait” RNA (e.g. an sRNA) and an RNAP‐fused “prey” protein (e.g. Hfq). Successful interaction between RNA bait and protein prey stabilizes RNA polymerase (RNAP) at a test promoter and activates reporter gene expression. This B3H assay offers a straightforward path to identify and interrogate mutations in these RNA‐binding proteins with molecular phenotypes of interest. Currently, only ~40% of known sRNA‐chaperone interactions are detectable in the system. In order to maximize the utility of our bacterial three‐hybrid assay, a larger proportion of known RNA‐protein interactions must be successfully detectable. Current efforts are focused on optimizing the signal of the B3H assay so that weaker RNA‐protein interactions can be reliably detected. Here, we show results from our efforts to improve the assay's signal‐to‐noise ratio by tuning the sequences, spacing, and the concentrations of the B3H assay components. We have observed an improvement in the assay's signal in response to systematic alterations of the assay components. With an optimized B3H assay, we aim to discover new sRNA‐binding proteins and uncover the regulatory roles sRNAs and these proteins play in biology.

Support or Funding Information

Mount Holyoke College and the Luce Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Binding affinity of TWIST2 wild type and mutant proteins and their potential role in the Barber‐Say Syndrome
Authors: Crespo‐Hernández Noe , Torres‐Bracero Alexandra , Rodriguez‐Pou Yesenia , Cadilla Carmen L
Published: June 2021

Barber Say Syndrome (BSS) is a rare autosomal dominant disease, classified as an ectodermal dysplasia, affecting the eyelids and skin as well as exhibiting dysmorphic facial features. BSS exhibits variable phenotypes. Molecular analysis of patients with BSS identified mutations in the TWIST2 gene, involving amino acid substitutions of Glutamate 75 to Glutamine (E75Q) or Alanine (E75A).TWIST2 is a transcription factor of the bHLH superfamily, belonging to the class II group. Glutamate 75 is a highly conserved amino acid in the basic region of bHLH proteins, which is responsible for nucleotide binding in both class I and II groups. Both the I and II bHLH classes are known for their specificity towards canonical E‐box (CANNTG) DNA response elements. The E75Q and E75A mutations have been suggested to alter the DNA‐binding activity of TWIST2, leading to both dominant‐negative and gain‐of‐function effects. In this study, we expressed His‐Tagged TWIST2, and the E75Q and E75A mutant proteins, in E. coli plysBL21 cells. Subsequently, proteins were purified by affinity chromatography and yields between 1.2 to 1.7 mg/L of pure recombinant protein were obtained from 2 liter cultures. Protein affinities towards known E‐boxes in the human TNFa, MCK1 and POSTN genes were measured using Biolayer Interferometry technology. TWIST2 wild type equilibrium constants were in the range of 0.003573 to 0.7929 uM. The equilibrium constants of the E75Q and E75A mutants were three to six order of magnitude higher when compared to the wild type protein. Therefore, the TWIST2 wild type protein showed higher binding affinities towards double‐stranded oligonucleotides containing E‐boxes, while the pathological variants found in BSS patients showed lower affinities to the same oligonucleotides. These results suggest that basic region variants of the TWIST2 glutamate 75 may cause gene dysregulation by their reduced capacity to bind their cognate DNA response elements.

Support or Funding Information

These studies were partially supported by grants U54MD007600 (NIMHD RCMI) and R24GM061838 (NIGMS RISE).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydrogen Sulfide (H2S) Inhibits Insulin Amyloid Fibril Formation
Authors: Torres Alani Aldarondo , Rosario Alomar Manuel F. , Garriga Juan López , Lednev Igor K.
Published: June 2021

Insulin from bovine pancreas (IBP) is one of about thirty human proteins that form amyloid fibrils. These amyloid fibrils obtain a b‐sheet structure and assemble into insoluble fibrils, causing many amyloidosis diseases. Amyloidosis caused by insulin protein is an acquired systemic disease. Developing procedures to eradicate amyloid fibril precursors is key to detaining amyloid fibril formation. To explore amyloid inhibition, native insulin protein was exposed to different concentrations of H2S, and protein unfolding was induced. Atomic Force Microscopy (AFM) imaging evidenced overlaying spherical structures, as the concentration of H2S increased in each sample as opposed to the fibrillary structures in the control sample. 3D AFM contour plots of the overlaying spheres due to protein‐H2S interaction show smoother peaks of greater height contrary to the sharp smaller peaks of the insulin amyloid fibrils. Results from Far UV‐Circular Dichroism spectra suggest that insulin protein in presence of H2S conserves its native insulin structure. These results are independent of pH value and are observable at physiological pH. Deep Ultraviolet Resonance Raman Spectroscopy further explains protein‐H2S interaction suggesting the formation of trisulfide bridges in insulin‐H2S sphere structures. Results suggest H2S inhibits insulin amyloid fibril formation by detaining the development of insulin fibril precursors.

Support or Funding Information

RISE‐E‐BASE Program UPRM

NIH Grant Number: 1R25GM127191‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.