bluuebunny/crossref_metadata_2025 huggingface dataset:
A mixed model for the relationship between latitude and human post‐cranial form
Authors: Savell Kristen R. R. , Katz David C. , Auerbach Benjamin M. , Weaver Timothy D.
Published: June 2021

The general adherence of modern human body proportions to ecogeographic rules is frequently argued to be the result of thermoregulatory adaptation to climate. However, much of the history of human migrations follows the same clines that are associated with trends in body form. It is therefore important to test hypotheses about human adaptation to climate with approaches that account for population history and structure.

In this project, we investigate the relationship between latitude and post‐cranial form in modern humans, with the goal of accounting for population history/structure and providing estimates of effects sizes and error. Using a multivariate quantitative genetics mixed model, we estimate morphological effects associated with latitude for long bone lengths and body size using osteometric data from 121 globally‐distributed populations and geographically matched genetic data representing 28 populations. The model includes a random effect for population structure (genetic relatedness) and a fixed effect for latitude. We found that among‐group variation was tightly correlated between limb lengths and body size measures respectively, but that these trait groups were fairly independent of each other. In addition, only bi‐iliac breadth demonstrates a clear directional effect once population history is taken into consideration, though directional trends skew positive for humeral length and negative for distal limb lengths, supporting previous research.

By disentangling latitudinal effects from population structure using a mixed model approach, we add to the growing body of research exploring these strong underlying associations, and allow for a better understanding of the relationship between environmental and post‐cranial morphological diversity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Colonization of the Lower Urinary Tract by Neural Crest and Development of Pelvic Autonomic Innervation
Authors: Southard‐Smith E Michelle
Published: June 2021

The development of organs occurs concurrently with the formation of nerves that are essential for normal function. In the lower urinary tract, comprised of the bladder and urethra, appropriate innervation is essential for storage and coordinated release of urine at socially acceptable times. Understanding how the innervation of the lower urinary tract develops and the signaling pathways that regulate this process is likely to provide insight into congenital problems and offers a means to regenerate neural inputs in cases of damage or disease. The bladder and urethra receive both sensory and autonomic neural inputs that are derived from the neural crest during development. Sensory innervation of the bladder originates from neural crest‐derived dorsal root ganglia at thoracic and sacral spinal levels. Pelvic autonomic sympathetic and parasympathetic neurons that provide motor effects and play a principle role in coordinating bladder contractility are located within the discrete ganglia that lie near the neck of the bladder and proximal urethra. Formation of these mixed pelvic ganglia from neural crest, the routes of neural crest migration into the fetal urogenital sinus, timing of pelvic ganglia neural differentiation, and signaling pathways that regulate these processes are active areas of investigation that are being rapidly advanced through the use of mouse models. An overview of studies undertaken in transgenic mice to derive a spatiotemporal map of neural crest derivatives in the developing lower urinary tract as well as a timeline of differentiation of pelvic ganglia neurons and initial analysis of signaling pathways active during pelvic ganglia neuron differentiation will be presented.

Support or Funding Information

This work was supported by National Institutes of Health grants R01‐DK078158, RC1‐DK086594, and U01‐DK101038.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Black Spot, Black Death, Black Pearl: The Tales of Bacterial Effectors
Authors: Orth Kim
Published: June 2021

External queues can lead to dramatic changes in host cell signaling. Research in my lab centers around elucidating the biochemical activity of virulence factors, termed effectors, used by bacteria to manipulate eukaryotic signaling machinery. My lab approaches the analysis of host signaling pathways by tapping into the enormous reservoir of bacterial virulence factors that are used by bacterial pathogens to manipulate a eukaryotic cell during infection. Most effectors are predicted to have usurped a eukaryotic activity that is then modified by the pathogen for its own advantage. Gram‐negative bacteria use a type III secretion system (T3SS) as a transport vehicle to deliver the virulence factors from the bacterial cytosol to the target host cytosol. The bacterial pathogen Vibrio parahaemolyticus contains two of these systems, T3SS1 and T3SS2, which are believed to function independently of one another in different hosts. The former is found in all Vibrio parahaemolyticus strains and causes death of host cells within hours of infection by induction of autophagy, followed by cell blebbing and rounding with the subsequent release of cellular contents. The other system, T3SS2, is associated with clinical isolates and mediates invasion, followed by vacuolar escape into the cytosol, proliferation and, finally, lysis of the host cells. To uncover the biological mechanisms used by effectors, we use a broad range of tools, including biochemistry, molecular microbiology, biophysics, structural biology, yeast genetics, and cell biology. We have discovered that pathogens utilize novel posttranslational modifications, including Ser/Thr acetylation and AMPylation. We have also uncovered unique mechanisms that manipulate the dynamics of the actin cytoskeleton, the induction of autophagy, and the maintenance of cell membrane integrity. As some of these mechanisms, such as AMPylation, are used by the host, we went on to discover that eukaryotic Fic‐mediated AMPylation/deAMPylation on the endoplasmic reticulum chaperone, BiP, serves as a rheostat for the unfolded protein response. Future studies will endeavor to understand other crafty ways that host cells can be manipulated by pathogens.

Support or Funding Information

HHMI, NIH, Welch Foundation, Once Upon a Time… 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.
Genome editing and plant agriculture
Authors: Voytas Daniel
Published: June 2021

The ability to precisely modify plant genomes through homologous recombination (HR) promises to advance both basic and applied plant biology. However, even with the use of sequence‐specific nucleases, which stimulate HR by creating targeted DNA double‐strand breaks, there are only a handful of studies that report precise editing of endogenous plant genes. Our group has been focusing on two efforts to more effectively modify plant genomes through HR. In one, we are developing new vectors to deliver sequence‐specific nucleases and DNA repair templates to plant cells. Specifically, we have been using geminivirus replicons, which function in both monocots and dicots, to amplify nuclease‐encoding cassettes and DNA repair templates. In a second effort, we are attempting to achieve HR by either genetically manipulating DNA repair pathways or delivering nucleases and repair templates to cells proficient in HR. Progress on our efforts to optimize gene targeting strategies will be reported.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deconstructing lagging‐strand synthesis in vivo
Authors: Smith Duncan
Published: June 2021

Half of all nuclear DNA in eukaryotic organisms must be replicated discontinuously on the lagging strand. Because eukaryotic Okazaki fragments are short – on average ~300 nt – complete replication of the human genome must require the synthesis, processing and ligation of tens of millions of Okazaki fragments.

Each Okazaki Fragment is initiated by the error‐prone DNA polymerase alpha and subsequently extended by DNA polymerase delta. Upon encountering a downstream fragment, Pol delta carries out strand‐displacement synthesis coupled to nucleolytic cleavage of the 5′ end of the displaced fragment to generate a ligation‐competent nicked DNA structure. Several partially redundant nucleases have been proposed to participate in Okazaki fragment processing: the roles of these nucleases have been extensively studied in vitro, but in vivo studies have largely relied on genetic interactions without directly examining nuclease cleavage during OF processing. Here, we describe a systematic, in vivo approach to study Okazaki fragment flap processing in Saccharomyces cerevisiae, using the direct sequencing and analysis of lagging‐strand intermediates to examine both lagging‐strand biogenesis and replication‐fork movement. Additional results detailing the cell‐cycle dependence of lagging‐strand processing will also be discussed.

Support or Funding Information

Supported by NIGMS R01 GM114340 and the Searle Scholars Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New prion strain generation through splenic replication
Authors: Sigurdson Christina J. , Aguilar‐Calvo Patricia , Bett Cyrus , Sevillano Alejandro , Kurt Timothy D. , Lawrence Jessica , Soldau Katrin , Hammarstrom Per , Nilsson K. Peter R.
Published: June 2021

Prion aggregates typically spread from their entry site into the central nervous system. However, distinct prion conformers show varying capacity to penetrate the CNS, as certain fibrillar prions replicate persistently in the spleen without CNS access, leading to silent carriers. Subclinical carriers of variant Creutzfeldt‐Jakob (vCJD) prions in the United Kingdom have been estimated at 1:2000, and vCJD prions have been transmitted by blood transfusion from prion‐infected donors. Yet it remains unclear which types of circulating prion conformers will neuroinvade following a transfusion. To understand how prion conformation impacts brain entry of transfused prions, we challenged mice intravenously to five subfibrillar and fibrillar strains. We found that all subfibrillar prions infiltrated the brain and caused terminal disease, however the fibrillar prions showed reduced CNS entry in a strain‐dependent manner. Whereas one fibrillar prion efficiently spread to the CNS with no apparent strain change, a second fibrillar prion replicated in the spleen and emerged in the brain as a novel strain. The new strain showed an altered plaque morphology, targets in brain, and biochemical properties as compared to the original strain, and these properties were maintained upon further passage. Direct intracerebral passage of prion‐infected spleen also generated the new strain, suggesting splenic replication as a potential source. Taken together, these results indicate that exposure to prion‐contaminated blood or blood products may produce novel prion conformers and disease phenotypes, potentially arising from prion replication by non‐neural cell types or from selection of a neuroinvasive conformer.

Support or Funding Information

This study was supported by the National Institutes of Health grants NS069566 (CJS), NS076896 (CJS), and the Ramón Areces Foundation (PAC). The Swedish Research Council grants 2015‐04521 (PH) and 2015‐05868 (PH) and The Göran Gustafsson Foundation (PH).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Treatment of a Mouse Model of Cholestasis with a Thyromimetic Improves Biliary Injury But Exacerbates Hepatocyte Injury
Authors: Kosar Karis , Nejak‐Bowen Kari
Published: June 2021

Chronic cholestasis results from bile secretory defects or impairment of bile flow, and there are few effective medical therapies available. Thyroid hormone T3 and synthetic thyroid hormone receptor agonists are known to cause induction of hepatocyte proliferation during liver regeneration through activation of b‐catenin. However, whether these drugs have therapeutic benefits in cholestatic liver disease is unknown. In this study, we administered GC‐1, a thyromimetic that preferentially acts through the TRb receptor found predominantly in liver, to Mdr2 knockout (KO) mice, which is a commonly used model of sclerosing cholangitis characterized by bile acid (BA) regurgitation, periductular inflammation, and fibrosis. We determined Mdr2 KO mice fed 5mg/kg GC‐1 diet had decreased bilirubin, liver to body weight ratios, serum alkaline phosphatase, but increased serum alanine aminotransferase and aspartate aminotransferase compared to KO mice fed normal diet as early as 1 week on diet. Histologically, KO mice on GC‐1 diet had decreased ductular response, less bridging fibrosis, and fewer SOX9 positive hepatocytes compared to KO on normal diet. Although total liver BA were higher in KO mice fed GC‐1 diet for 2 weeks compared to normal diet, they normalized to KO levels at 4 weeks of diet. To elucidate the mechanism of increased BA accumulation and liver injury, we examined expression of BA transporters and detoxification enzymes. KO mice on GC‐1 diet had decreased bilirubin transport and detoxification genes, such as Mrp2, Mrp3, Cyp2b10, and Oatp4, compared to KO mice on normal diet, with the net result being retention of BA in the hepatocyte. Interestingly, KO mice on GC‐1 diet had decreased total and phosphorylated, active b‐catenin compared to KO mice on normal diet, suggesting an alternate mechanism for GC‐1 activation in this model. Thus, GC‐1 reduces cholangiocyte injury during cholestasis by inducing retention of BA in hepatocytes, causing injury to the hepatocytes; this occurs through as‐yet unknown mechanisms that are upstream of BA transporters and biosynthesis enzymes and appears to be b‐catenin independent.

Support or Funding Information

National Institute of Biomedical Imaging and Bioengineering of the National Institute of Health under Award Number T32EB0010216

UPP Academic Foundation Award 1R01DK103775

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anonymity of Willed Body Donors: Survey Results from First Year Medical Students
Authors: Champney Thomas , Eaton Sarah , Ishteiwy Remma , Ring Sarah , Sheu Jonathan
Published: June 2021

There is current debate about the value of anonymity in willed body donors for first year medical students. Some claim that allowing the students to know more about the donors can enhance their empathy and can provide early experience in the doctor – patient relationship. Others claim that knowing too much about the donors may make the dissection experience more traumatic and can take away from the student's anatomical learning experience. To gain a student's perspective on this issue, first year medical students in two different curricular tracks at the University of Miami Miller School of Medicine were surveyed both before and after they completed their first year gross anatomy course (IRB 20160480). One curricular track provides 150 students with a short, eight week, high intensity anatomy course while the other track provides 50 students with a longer, 6 month, low intensity course. The online survey asked the students to rate how much personal information about the donors they would like to receive. The survey response rate was 76% and 63% for the pre‐ and post‐ short anatomy course with response rates of 58% and 56% for the pre‐ and post‐ long anatomy course. There was no difference in the results between the two curricular tracks and no difference in the results before or after taking the anatomy course. The results found that the students would prefer to have information about the donors that includes their medical history, social history and socioeconomic status (3.7 mean Likert score), but are less interested in having complete information about the donors (name, religion, political affiliation) (2.4 mean Likert score). Comments by the students suggested that knowing too much information could have a negative impact on their ability to perform invasive dissections and could have negative emotional effects. The students felt, however, that knowing the donor's medical and social history would allow them to appreciate and synthesize the anatomical information to a greater degree. These results indicate that students would like more pertinent medical information about their donors, but that they have concerns about knowing the complete information about their willed body donors.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Active Learning Lesson with Three Dimensional Models to Improve Understanding of Pelvic and Perineal Anatomy
Authors: Solis Laura J. , Rahimi Omid B.
Published: June 2021

Novice students have difficulty understanding the anatomy of the pelvis and perineum due to the complex arrangement and limited visibility of structures in these areas. Previously, medical students at UT Health San Antonio studied these regions using dissection, prosection, peer teaching, and radiographs. We combined 35‐minute sessions with three dimensional (3D) models made with simulated anatomical contents using arts and crafts materials, and a case study utilizing digital anatomy software, BodyViz, to the traditional teaching methods. The effectiveness of the additional 3D active learning tools was assessed by comparing student exam performance, following their participation in the sessions, with the exam results from the previous three academic years. The results demonstrated that students, with access to the 3D tools, slightly reversed a decreasing trend on exam average scores and showed significant improvement in the identification of specific structures in the pelvis and perineum. The study also suggested a slight advantage in student attendance to the active learning lesson following exposure to cadaveric prosections as compared to those attending the active learning lesson prior to the laboratory session. In the current academic year, the 3D models alone were utilized by medical students in two optional active learning lesson sessions to evaluate the model's effectiveness in improving their understanding of the anatomy. In a class of 217 students, 53.4% of students chose to participate in the active learning lesson using the model. Out of those students struggling in the course, 69% chose to attend the active learning sessions. Overall, students had positive comments about the impact of the models and the session in their learning. Student performance on exam topics related to the pelvis and perineum is tracked and compared to medical student averages in previous medical classes. The active manipulation of the 3D models and the additional time dedicated to the active learning exercise gives students the capability to study the complex spatial relationships of structures and improve their performance on course exams. Our models show great potential as an optional teaching method in enhancing medical student learning of the anatomy of the pelvis and perineum.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Learning styles among medical students: Kinesthetic learners approach to learning anatomy.
Authors: Hernandez Jessa , Vasan Nagaswami
Published: June 2021

According to Vark's modalities, there are 4 different learning styles that an individual can use: visual, auditory, read/write and kinesthetic. Each student uses one or more of these that helps them the most in learning new material. The Visual learners prefer depictions of information as figures and charts; Auditory learners prefer to hear the information as lectures and discussions; Read/Write learners prefer information presented as text‐based and the Kinesthetic learners prefer concrete experiences that connect the material to reality. Most people have a multimodal approach to learning. Traditional teaching of anatomy includes lectures and dissections. Lectures and dissections fall short for kinesthetic learners in the that their concrete experience is limited to one cadaver or a prosection lacking applicability to the widespread model. A recent study showed that 20% of the medical students are kinesthetic learners. Many studies evaluated new ways to teach anatomy including computer‐assisted learning and using live models. None of these, however, aid the kinesthetic learner. Ever since Da Vinci's drawings and Andreas Vesalius' illustrated anatomical textbook, art has been a large part of learning anatomy. Being a kinesthetic learner, I found a way to use art and modeling to create a concrete experience that satisfies my learning style and help me learn anatomy: Crochet. Through crochet I was able to take what I was learning in lecture and dissection and recreate it. To do this I dissect, study a structure and create a 3‐D mental model. This way, I was able to gain a broader visuospatial understanding of the anatomical concepts. The presentation will include number of drawings and crochet models.

Support or Funding Information

Supported by the Cooper Medical School of Rowan 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.
Findings of the Anomalous Fibularis Digiti Quinti Muscle and Implications of its Associated Variable Presentations from Cadaver Dissections
Authors: Brechtel Benjamin Stilwell , Dao Tung V. , Chaney Morgan E. , Belovich Stephanie J. , Siesel Kathy J. , Fredieu John R.
Published: June 2021

From our cohort (KSUCPM 2017) of 26 cadavers dissected by podiatric medical students for lower‐extremity anatomy, we observed bilateral and robust fibularis digiti quinti (FDQ) tendons in a 99‐year‐old female. Here, we describe the case, incidence of the FDQ tendons from dissections, and their variability as a component of anomalous lateral leg musculature. Due to variability, we have considerations for the anthropological and clinical significance of our findings. In both feet of our case, the FDQ tendon arose from the fibularis brevis tendon proximal to the lateral malleolus, but did not separate completely from the fibularis brevis tendon until passing through the inferior fibular retinaculum. On the lateral dorsum of the foot, the FDQ passed through a third fibular retinaculum formed by the fibularis tertius tendon, and inserted onto the extensor sling of the fifth digit. Our case is considered fully present FDQ. Of the 52 limbs dissected in the KSUCPM 2017 cohort, 17 limbs (33%) showed a fully present FDQ, 20 limbs (38%) exhibited an FDQ in a rudimentary form, and 15 limbs (29%) lacked an observable FDQ. Ongoing 2018 cadaver dissections are also represented. Functionally, human bipedality requires less dexterity than that of nonhuman primates in the routine use of hindlimbs. Therefore, we interpret the high variability of the FDQ, including its absence in many feet, as a relaxation of natural selection. The clinical significance of our findings suggests that the presence of a fully developed FDQ anomaly can be involved in pathology. In addition, physician awareness of the FDQ is important for surgical considerations.

Support or Funding Information

Research supported by Kent State University College of Podiatric Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding Shape Factors That Influence The Tibial Tunnel Length in Anterior Cruciate Ligament Repair
Authors: Congdon Kimberly , O'Mary Kevin , Jergensen Jason
Published: June 2021

Anterior cruciate ligament repairs are performed over 100,000 times annually in the US. A common surgical method is a bone‐patellar tendon‐bone graft, which requires the placement of a tibial tunnel through the proximal tibia. Mismatches between the length of the tibial tunnel and the length of the graft have been reported to occur 26% of the time or more, and require procedural modifications during surgery. Most methods for placing the tibial tunnel rely on consideration of patient height and/or angle of placement. Little is known about the variables that affect mismatches. Better understanding how the shape of the proximal tibia impacts the length of the tibial tunnel will allow for improved surgical technique and could lower the number of mismatches, and perhaps even graft failures. We examined 33 right proximal tibiae from donor cadavers (15 females, 18 males) via three‐dimensional scans that allowed us to take 6 linear measurements to describe the dimensions of the proximal tibia. We also used the directions for a popular tibial tunnel guide to simulate a tibial tunnel length on all individuals. Our analysis found the length of the tibial tunnel is influenced by multiple shape components that vary by sex. Specifically, male tibial measurements were always significantly larger than female measurements (p<0.05), there were a greater number of significant correlations among measurements in the male tibia than the female tibia, and the length of the tibial tunnel is more closely correlated to anterior‐posterior measurements in females in contrast to males, where it is more closely correlated to mediolateral measurements. These findings suggest there are sex‐based differences in the shape of the proximal tibia that may confound tibial tunnel placement. The development of surgical techniques should incorporate knowledge of shape‐based variables, and future research should continue to explore sex‐specific foundations of variable outcomes in clinical treatments.

Support or Funding Information

This project was funded by the Basic Sciences Department, College of Osteopathic Medicine, Touro University Nevada

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomical Variations of the Axillary Artery of Human Cadavers
Authors: Mathis Monica , Marshall Jonathan , Hammer Leah , Chambers Paige , Rosario Martin G.
Published: June 2021

Anatomical variation is demarcated as the standard discrepancy in the structure and morphology of body arrangements, for instance, arteries and nerves. Numerous variations are benign and can be attributed to changes in the embryologic development. The axillary artery (AA) is an extension of the subclavian artery. Typically, the AA extends from the first rib (outer border) to the teres major muscle (lower border). The pectoralis major divide the AA into three parts, for identification purposes, above (first part), underneath (second part) and below the muscle (third part). From the second part the lateral thoracic artery branches, supplying the pectoralis and serratus anterior muscles. The thoracodorsal artery branches out of the third part of the AA to supply the latissimus dorsi and subscapularis muscles.

PURPOSE

Report anatomical variations in the axillary arteries explicitly on the second and third division.

METHODS

The AA's were dissected by students of the Physical Therapy program at Texas Woman's University Dallas under the supervision of the Anatomist of the same program. The primary technique used was blunt dissection for the identification of the AAs. Out of Ten human anatomical specimens, Three exhibited variations of the AA.

RESULTS

First variations we identified was lateral thoracic artery branched out of the third division of the AA and not the second. Furthermore, from the third division we identified that out of the subscapular artery we had branches for latissimus dorsi (two branches), subscapularis (Thoracodorsal artery) and the Serratus Anterior (two branches) muscles (Lateral thoracic).

CONCLUSIONS

It appears that the axillary artery had several deviations from its standard structures. Clinically this variation might not cause any impairment. However, the knowledge of this different arrangements can help prevent unwanted difficulties throughout several interventions.
The H3K36 histone methyltransferase SETD2 as a target of leukemogenic chemicals: molecular and cellular studies.
Authors: BERTHELET Jérémy , DUVAL Romain , BUI Linh‐Chi , MATHIEU Cécile , PETIT Emile , DUPRET Jean‐Marie , GUIDEZ Fabien , CHOMIENNE Christine , RODRIGUES‐LIMA Fernando
Published: June 2021

SETD2 is a key histone modifying enzyme that is responsible for the trimethylation of lysine 36 of histone H3 (H3K36me3) in mammals. During transcriptional elongation in normal cells, H3K36me3 marks the zone on exons for accurate gene transcription and plays an important role in the regulation of alternative splicing. In addition, SETD2‐mediated H3K36me3 is required for DNA repair and recruitment of other epigenetic enzymes. Several studies have shown that loss of function mutations in SETD2 are involved in cancer development. In particular, SETD2 inactivating mutations have been shown to be critical drivers in leukemia development.

Benzene (BZ) is a chemical of great industrial interest. BZ is also known to be a major hazardous air pollutant originating from anthropogenic (road transport, tobacco smoke, etc) and natural sources (forest fires). BZ is classified as a class I human carcinogen. Environmental and occupational exposure to BZ lead to bone marrow malignancies such as leukemia. The leukemogenic effects of BZ relies on its metabolization in lungs and bone marrow cells into reactive metabolites, in particular benzoquinone (BQ). However, most of the key molecular and cellular mechanisms underlying its hematotoxicity are not fully understood.

Here we provide evidence that SETD2 is a target of BQ, the prime hematotoxic metabolite of BZ. Using a specific HPLC based SETD2 assay, we found that the methyltransferase activity of SETD2 is readily inhibited by BQ in vitro and in cellulo in an irreversible manner (kinact=300 M−1.s−1). This inhibition is due to the covalent modification (arylation) of cysteine residues present in Zn2+ finger motifs (AWS and post‐SET domains) by BQ with concomitant release of Zn2+. Accordingly, exposure of cells (K562 or HEK293T) to BQ leads to global loss of H3K36me3.

The H3K36 histone methyltransferase SETD2 is increasingly recognized as a tumor suppressor gene, which can facilitate the initiation and maintenance of leukemia. Our data suggest that BQ, the major hematoxic metabolite of benzene, can impair the functions of SETD2 which may contribute to benzene‐dependent leukemogenesis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of CoREST Complex‐Chromatin Interactions with Chemical Tools
Authors: Wu Mingxuan , Hayward Dawn , Kalin Jay H. , Song Yun , Schwabe John , Cole Philip A.
Published: June 2021

Chromatin remodeling and histone modifications play essential roles in the regulation of gene activation or silencing in physiologic and disease processes. Among the histone modifying enzymes, histone deacetylases (HDACs) and lysine specific demethylase 1 (LSD1) have been shown to be critical in transcriptional regulation and are considered drug targets in cancer and other diseases. The CoREST complex contains HDAC1 (or HDAC2) and LSD1 but the interplay between subunits of this complex and their interactions with nucleosomes is poorly understood. We have employed engineered sortase to generated histone H3 containing post‐translational modifications (PTMs) that ligates H3 N‐tail depsipeptides and globular histone H3 with excellent conversions and all native sequence. These semisynthetic histone H3s possessing various combinations of H3K4‐dimethyl and N‐tail Lys acetylation marks were incorporated into reconstituted mononucleosomes and investigated as substrates with the purified core CoREST complex. These experiments revealed an interdependence of particular PTMs in CoREST processing and regioselective CoREST deacetylation. In addition, we prepared nucleosomes containing N‐propargyl and hydroxamic acid Lys analogs as probes of CoREST interaction. These unnatural warheads enhanced the stability of CoREST complex‐nucleosome complexes which should facilitate biophyiscal characterization.

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overlap Extension Polymerase Chain to Create a New Expression Vector and the Protein Binding Affinity Analysis using MST of Eph Receptors
Authors: Bowman Deanna , Muller‐Greven Jeannine , Smith Adam , Buck Matthias
Published: June 2021

Erythropoietin‐producing human hepatocellular (Eph) receptors are a subfamily of protein tyrosine kinases (RTKs) that possess a sterile alpha motif (SAM) domain. These receptors are important for cell‐cell communication, proliferation, cell growth, and migratory responses. They are also over‐expressed in cancer cells and tumor vessels. Eph receptors are thought to be involved in tumor progression and metastatic spread. The behavior is dependent on the density of the receptor and the presence of ephrin ligand in opposing cells. Oligomerization of the EphA2 receptor plays a key role in its function. Dimerization and oligomerization are part of the activation of RTKs and the response to ligand stimulation. Restricting the cluster size of active EphA2 affects the signaling output, and preformed, ligand independent dimers are one possible source of aberrant signaling. One way to study the EphA2 receptors is to produce the intracellular domain (ICD) in E.coli. However the protein expression is very low and thioredoxin (Trx) is needed as a fusion tag to improve the level of soluble protein. For clustering and dimerization study as well as binding study of the ICD domain to the plasma membrane, the large Trx tag of 110 amino‐acids may block the binding hindering the results and should be removed. A highly sequence‐specific cysteine protease from Tobacco Etch Virus (TEV) was used to cleave the fusion protein by inserting the TEV protease recognition site between the Trx tag and EphA2ICD. Genetic engineering using overlap extension polymerase chain was used to create this new expression vector fusing the Trx tag at the N‐terminus of EphA2 with the insertion of a TEV protease recognition site in between. The EphA2 fusion gene was then cloned into the pET28a expression vector. The new construct was then transformed into E. coli strain BL21 and the recombinant EphA2 was expressed as a soluble and active protein. The protein was purified using the Nickel‐NTA affinity resin. After protein purification, cleavage conditions of the Trx tag using TEV protease were optimized and EphA2ICD cleaved and uncleaved was tested for its ability to bind liposomes using Microscale thermophoresis (MST). The MST shows a higher binding affinity of the cleaved protein to the liposomes compared to the uncleaved protein indicating some obstructive effect of the Trx tag. This new expression construct can be used to analyze the role of Eph receptors a more accurate way. Further investigation of the structural determinants of oligomerization through the manipulation of the receptor sequence will lead to a better understanding of the role that EphA2 plays in metastasis and tumorigenesis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the role of the interface on activity and regulation of gMDH
Authors: Garcia Nina Marie , Schwabe Michael , Graham Sasha , Bell Ellis
Published: June 2021

Interfaces between subunits often play important roles in communicating activation or inhibition critical for an organism's survival. The molecular mechanism of such interactions has not yet been fully understood. Glyoxysomal malate dehydrogenase (gMDH) is a homodimeric enzyme whose interface is thought to participate in the regulation both by citrate and by substrate inhibition as well as a proposed reciprocating subunit mechanism of the overall catalytic cycle. The interface contains a variety of electrostatic (some favorable, some repulsive) and non‐polar interactions. To understand the mechanism of subunit interactions we have probed the role of individual sidechains at the interface by site directed mutagenesis to determine the effects on stability, activity, and regulation. L269 and S266 appear to form a relay system that connects the active site to the interface. We have constructed, expressed, and purified two mutants, L269A and S266A. Biochemical and biophysical techniques were used to evaluate the structure and stability of the mutants. Size exclusion chromatography and chemical cross‐linking experiments demonstrate that L269A and S266A maintain a dimeric quaternary structure. Comparison of CD spectra of the mutants to the native showed similar secondary structure and, via thermal melts at 222nm, TM values. To study, the role of these residues in gMDH activity, initial rate kinetic studies were performed and showed that S266A and L269A have a lower affinity for OAA while, unlike the native enzyme, the L269A mutant is not saturated by NADH over experimentally accessible ranges. This, coupled with the decreased activity of both mutants, suggests that the residues play a key role in the binding of NADH and regulation of gMDH by OAA. This was further supported by fluorescence based thermal shift (FTS) studies that demonstrated how NADH binding is greatly decreased in L269A. Thus, the results suggest that subunit interactions mediated at least in part by S266 and L269 are part of a relay system between active sites in the homo‐dimer that regulates activity and inhibition of gMDH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Microprotein‐Protein Interactions via APEX Tagging
Authors: Chu Qian , Rathore Annie , Diedrich Jolene K. , Donaldson Cynthia J. , Yates John R. , Saghatelian Alan
Published: June 2021

Microproteins are peptides and small proteins encoded by small open reading frames (smORFs). Genomics and proteomics technologies have led to the recent discovery of hundreds to thousands of new microproteins. Emerging evidence demonstrates that these microproteins have fundamental roles in various biological processes including metabolism, apoptosis, and development, highlighting the value of characterizing these molecules. The identification of microprotein‐protein interactions (MPIs) has proven to be a successful approach to the functional characterization of these genes; however, traditional immunoprecipitation methods result in the enrichment of non‐specific interactions for microproteins. To address this issue, we tested and applied an in situ proximity tagging method that relies on an engineered ascorbate peroxidase (APEX) to elucidate MPIs. The results demonstrated that APEX tagging is superior to traditional immunoprecipitation methods for microproteins. Furthermore, the application of APEX‐tagging to two uncharacterized microproteins, C11orf98‐MP and MIEF‐MP revealed that they interact with nucleolar proteins nucleophosmin and nucleolin (C11orf98‐MP) and acyl carrier protein in mitochondria (MIEF‐MP) respectively, demonstrating the ability of this approach to identify novel hypothesis‐generating MPIs.

Support or Funding Information

Q.C. is a postdoctoral fellow funded by the George E. Hewitt Foundation for medical research. This study was supported by the NIH with an NCI Cancer Center Support Grant P30 (CA014195 MASS core, A.S.) and R01 (GM102491, A.S.), the Leona M. and Harry B. Helmsley Charitable Trust grant (#2012‐PG‐MED002, A.S.), and Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.). Imaging work in this study was supported by the Waitt Advanced Biophotonics Core Facility of the Salk Institute with funding from NIH‐NCI CCSG: P30 014195, NINDS Neuroscience Core Grant: NS072031 and the Waitt Foundation. We also want to thank the Cancer Center Support Grant P30 CA014195 for purchasing and providing access to the MGC collection.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New assays reveal opposing effects of 17b‐estradiol in the presence and absence of ovaries on the cardiac network of calmodulin‐binding proteins
Authors: Patton John Harrison , Kaster Kyle Nicholas , Gebert‐Oberle Briana , Clayton Sarah , Giles Jennifer , Tran Quang‐Kim
Published: June 2021

Reduced estrogen concentrations following menopause are associated with increases in the incidence of cardiovascular disease. Calmodulin (CaM) is required for the activities of numerous cardiac proteins yet is not sufficiently expressed for its binding targets. We have begun to examine the effects of 17b‐estradiol (E2) on the cardiac network of CaM‐binding proteins. Female rats received sham surgery or ovariectomy, followed by treatment with vehicle or E2 for 2 weeks. E2 treatment in sham animals increases the interaction between endogenous CaM and the cardiac α adrenergic receptor type 1A. Surprisingly, ovariectomy substantially increases this interaction, while E2 replacement now reduces it. To examine the effects of these treatments on the population of cardiac CaM‐binding sites that are unsaturated by endogenous CaM, lysate from left ventricle was subjected to saturating Ca2+ concentration and processed through a CaM sepharose column. Flow through contained CaM‐binding sites saturated by endogenous CaM, while sepharose‐bound fraction represented endogenously unsaturated CaM‐binding sites. The fractions eluted from the CaM sepharose were subsequently used in competitive binding assays using purified CaM and a CaM biosensor. E2 treatment in sham animals increases the number of unsaturated CaM‐binding sites. Ovariectomy further increases this number, while E2 treatment now reduces it. The data indicate that E2 treatment exerts opposing effects in the presence and absence of ovaries on specific endogenous CaM‐target associations and the number of endogenously unsaturable Ca2+‐dependent CaM‐binding sites in the heart. These results suggest that estrogen replacement may have unpredictable functional outcomes.

Support or Funding Information

Supported by NIH Grant HL112184 to Quang‐Kim Tran and Grants from the Iowa Osteopathic and Educational Research Funds to Sarah Clayton and Quang‐Kim Tran.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Spatial Compartmentalization of Akt/mTOR Signaling: What Happens in the Nucleus
Authors: Zhou Xin , Zhong Yanghao , Zhang Jiao , Shyy John Y. ‐J. , Zhang Jin
Published: June 2021

The Akt/mTOR pathway is a central player in regulating distinct and diverse cellular processes, such as cell growth, survival, and metabolism. We hypothesize that spatial compartmentalization is a critical mechanism for achieving signaling specificity of Akt/mTOR signaling. To examine this spatial regulation of Akt/mTOR signaling within subcellular compartments, we developed a series of genetically encoded fluorescent protein‐based molecular tools, including Akt activity reporter (AktAR2) and mTORC1 activity reporter (TORCAR), to directly visualize the kinase activities dynamics of Akt and mTORC1 in single living cell. We found that cellular mTORC1 activity is intricately regulated in a signal‐ and location‐specific manner. Surprisingly, both growth factor and amino acid surrogate induce mTORC1 activity in the nucleus, in contrast to the current thinking that mTORC1 is not active in nucleus. To further dissect the regulatory mechanisms of nuclear mTORC1 activity, we developed Akt‐STOPS (Akt Substrate‐based Tandem Occupancy Peptide Sponge), which allows perturbation of Akt signaling at distinct subcellular compartments. Expression of Akt‐STOPS in the nucleus suppressed the growth factor stimulated nuclear Akt activity without significant inhibition of Akt activity in the cytosol. Strikingly, expression of Akt‐STOPS in the nucleus largely diminished the growth factor‐induced nuclear mTORC1 activity, and down‐regulated the transcription by mTORC1‐dependent RNA pol III, suggesting that growth factor induced nuclear mTORC1 activity is dependent on nuclear Akt activity. These results have led to a new model of regulation of mTORC1 in nucleus. Taken together, we developed new molecular tools to interrogate the spatial control of Akt/mTOR signaling and uncovered new insights into the spatial compartmentalization of Akt/mTOR signaling.

Support or Funding Information

This work was supported by NIH grants R35 CA197622 and R01 GM111665.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bacteriological Evaluation of Essential‐Oil Based Products from a Local Small Business
Authors: Santos Anael A. , Butler Russ , Gillen Craig , Santos Amanda B. , Santos Fernanda B.O.
Published: June 2021

All‐natural, essential‐oil based products are becoming a significant segment of local small business. In fact, an increasing number of consumers are choosing organic plant‐based alternatives over the cosmetics currently available. Independently of the source of ingredients, cosmetics should be tested before becoming available for purchase. It has been recognized that microorganisms can grow and reproduce in cosmetic products, and may cause spoilage or chemical changes in these products, which can result in injury to the user. Standards for the analyses of food and cosmetics have been established by the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM). The present study followed these analytical guidelines to ensure the quality and safety of All‐Natural products (defined as such by the business owner) from a local small business. The tested products included a lotion, a diaper balm, and a bug spray. Two bottles of each product from different batches were randomly chosen and used for testing. Each bottle of each product was tested separately using the Aerobic Plate Count (APC) method and the total coliform analysis according to the BAM. After incubation in the appropriate agar plates, no colonies were found. Therefore, all plate readings from all products were below detectable limits of these methods. These results indicate that the products meet the FDA's requirements for distribution to, and safe use of, the consumer.

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