bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethical and Inclusive Design Principles in xR
Authors: Krebs Claudia
Published: May 2022

The past years have resulted in an increased use of technology in anatomy and medical education. The use of augmented and virtual reality, collectively often referred to as extended reality (xR) is moving from an experimental idea to a curricular reality. The confluence of a global pandemic and increased accessibility of emerging technologies has resulted in many xR endeavours and education applications. As we explore the use of these technologies, as we integrate them into our classroom, as we develop applications in these technologies, we need to ask the question about how we balance the technological affordances with our values grounded in ethics and inclusivity. Technology can change how we see the world and it can influence our affective response to education. In anatomy education, when using xR, we need to follow universal design principles in order to make the technology accessible to students of all abilities. An emphasis on the learner experience and intuitive interfaces makes the technology fade into the background and puts the academic content into the center of the learning experience. Deliberate choices of how anatomy is placed in the virtual space and whose anatomy we are visualizing grounds these approaches in an ethical framework. We are at the cusp of a new era in technology use, it is an opportunity to make sure that our way forward will reflect our values and build a compassionate, inclusive, and exciting approach to anatomy education.
Without their Consent: Handling Legacy Collections and Anatomy Teaching Specimens Acquired without Informed Consent
Authors: Geller Pamela L.
Published: May 2022

Legacy collections have proven invaluable for teaching students about anatomy and standard methods. It is safe to presume, however, that ancient and historic decedents never consented to their inclusion in collections or use as pedagogical tools. A utilitarian position—that the use of these human remains serves a higher scientific purpose—becomes even harder to justify when educators acknowledge the historic necropolitical projects and suffering that underpinned the formation of legacy collections. How then to proceed scholastically with such affective and politically charged human remains?

As a complex case study, I consider the Samuel G. Morton Crania Collection. To instruct the medical students who populated his anatomy classes, the Philadelphia physician amassed over 900 crania from 1830 to 1851. After Morton’s death, the Academy of Natural Sciences purchased the collection, where it was all but forgotten. Almost a century later, in 1966, it came under the stewardship of the Penn Museum, its pedagogical purpose resurrected. While Stephen Jay Gould drew attention to the scientific racism of Morton’s research, it was not until the passage of the Native American Graves Protection and Repatriation Act of 1990 that ethical deliberations began in earnest. Here I continue these trains of thought with a discussion of the Afro‐Cuban crania in the Morton Collection.

Morton acquired these decedents from his Cuban colleague Dr. José Rodriguez Cisneros in 1840. The latter designated them “negros bozales,” an indicator of their enslaved status and African origins; additional information about tribe and country was not provided. In summer 2020, in the wake of the murder of George Floyd and the racial justice protests his death catalyzed, these skulls erupted into the public consciousness. There were calls to “Return Them All.” I regard this response as well intentioned but also reactionary and unnuanced. To determine if sustained use is viable where consent is inadequate, for this case and more generally, I bring to the fore two concepts: the agentive corpse and ontological insecurity. Both concepts require educators and researchers to culturally contextualize human remains, as well as attend to the dynamic meanings attached to them—by past communities and their living descendants. With this knowledge in hand, I make some tentative recommendations about the fate of these controversial and highly sensitive human remains.
W. Montague Cobb: Near the African Diasporic Origins of Activist and Biocultural Anthropology
Authors: Blakey Michael L.
Published: May 2022

William Montague Cobb, A.B., M.D., Ph.D. was the first African American Ph.D. in anatomy and physical anthropology. He produced 1,100 publications while a professor at Howard University. Cobb had a profound influence on the civil rights struggle from the 1930s‐1970s, making a special contribution to our understanding of health inequalities due to racial and class inequity. He was the principal biographer of Blacks in Medicine. This paper describes his participation in the legacy of activist and interdisciplinary scholarship of African diasporic intellectuals. He posed this epistemic against the reductionist biodeterminism of his time. Cobb promoted a political economy of health and its appropriate amelioration through public policy.
Evolutionary Innovations of the Feeding Apparatus in Herbivorous Dinosaurs
Authors: Nabavizadeh Ali
Published: May 2022

Although most studies in feeding biology of non‐avian dinosaurs have investigated feeding function and ecology in carnivorous dinosaurs, the complex diversity of feeding in herbivorous dinosaurs has recently been explored in much greater detail. Herbivorous non‐avian dinosaurs, composed of nearly all sauropodomorphs and ornithischians as well as a few theropod groups (i.e., ornithomimosaurs, therizinosaurs, and oviraptorosaurs), show a wide range of craniomandibular feeding mechanisms, each with unique adaptations for processing various forms of plant‐life. Here, I will review over a century of feeding studies in herbivorous dinosaurs, with particular focus on more recent advancements in our knowledge of their feeding evolution and ecology. These studies have primarily focused on the many components influencing the functional morphology of the head and neck of these animals, including: dental morphology, with studies in dental microwear and macrowear, overall morphological analysis of the skull and cervical vertebrae, reconstruction of cranial musculature and other soft tissues of the head, and biomechanics of the skull, with comparative analyses of mechanical advantage of the jaw system and 3D computer modeling in the form of finite element analyses and multibody dynamics analyses with the use of laser scans and CTs of dinosaur skulls. All these studies are instrumental in our understanding of the evolution and diversity of bite forces as well as feeding mechanisms that range from orthal to palinal feeding motions as well as bilateral hemi‐mandibular long‐axis rotation (especially in a variety of ornithischians). The similarities and differences between herbivorous dinosaurs and the herbivores of today (especially mammalian megaherbivores) show a great deal about how evolutionary opportunities and constraints have made a significant impact on the diverse nature of herbivory and its importance in ecosystems around the world.
Osteocyte Control of Bone Quality and Fragility in Skeletal Aging
Authors: Alliston Tamara
Published: May 2022

Bone fragility increases with age as the result of the concurrent decline of bone mass, quality, and mechanosensitivity. While the coordinated decline of these behaviors remains unexplained, the role of osteocytes in each of these processes, and the age‐related degeneration of the lacunocanalicular network (LCN) in which they reside, implicate osteocytes in bone aging. Using a combination of imaging and computational modeling, we identify canalicular pruning as a driver behind declining bone functionality with age. Furthermore, these analyses predict novel opportunities to restore function to aged osteocyte networks through expansion of the pericellular space.
Dermatologic Health Disparities
Authors: Glass Donald
Published: May 2022

The term “skin of color” identifies individuals of racial groups with skin darker than Caucasians, such as Asians, Africans, Native Americans, and Pacific Islanders. Patients with skin of color often have distinctive cutaneous and hair characteristics, disorders and reaction patterns. There are skin disorders that disproportionately affect skin of color patients, such as keloids, hidradenitis suppurativa, and central centrifugal cicatricial alopecia – these conditions are underresearched and the underlying causes remain poorly understood. Recent progress in our understanding of the mechanisms underlying these diseases has begun to emerge. In this talk, I will discuss these diseases with an emphasis on keloids; the racial differences in their epidemiology and the recent progress in the genetic and molecular understanding of these conditions.
Fascia Mobility and Musculoskeletal Pain
Authors: Langevin Helene M.
Published: May 2022

The network of fasciae is an important part of the musculoskeletal system that is often overlooked. Fasciae are organized in layers, and fascia mobility, especially along shear planes separating muscles, is critical for musculoskeletal function. Patients with connective tissue hyper‐ and hypo‐mobility disorders suffer in large numbers from musculoskeletal pain. The relationship between fascia mobility and myofascial pain is largely unstudied, but a better understanding of this area could result in improved care for many patients with musculoskeletal pain.
The Vitamin A Paradox: Too Much or Too Little Impacts Development and Disease
Authors: Trainor Paul
Published: May 2022

Vitamin A is essential for proper human embryonic development particularly with respect to brain, craniofacial, limb, and organ, patterning and morphogenesis. Later, in adulthood, vitamin A plays important roles in fertility, immune function, maintenance of vision, and skeletal homeostasis. Surprisingly, deficiency or excess of vitamin A and its metabolic derivative, retinoic acid, can elicit similar effects. Therefore, strict feedback and other mechanisms must exist to regulate levels within a narrow physiological range. We discovered retinol dehydrogenase 10 (Rdh10) as a novel critical regulator of vitamin A metabolism and retinoic acid (RA) synthesis during embryogenesis. Rdh10 oxidizes all trans retinol (vitamin A) to retinal, which is then oxidized by retinaldehyde dehydogenases to retinoic acid. Rdh10 loss‐of‐function mouse embryos exhibit craniofacial anomalies such as choanal atresia, facial clefts, and abnormal branchiomeric muscle development, cardiac malformations including choanal atresia, and gastrointestinal defects such as Hirschsprung disease. Underpinning each phenotype were defects in the development of neural crest cells, a migratory progenitor cell population essential for normal craniofacial and organ morphogenesis. We determined that Rdh10 and retinoic acid signaling are precisely required between embryonic day (E) 7.5‐9.5 in both an intrinsic/autocrine and extrinsic/paracrine manner for proper neural crest cell development. Thus, Rdh10 forms a key nodal point in the regulation of vitamin A metabolism and retinoic acid synthesis and is essential for regulating basic developmental and homeostasis signaling pathways. Furthermore, Rdh10 and vitamin A signaling are both genetic and non‐genetic risk factors in the etiology and pathogenesis of congenital birth defects.
Using Student Movement to Improve Understanding of the Pyruvate Dehydrogenase Complex
Authors: Mullen Davis Melissa A.
Published: May 2022

Students in introductory biochemistry courses often struggle to visualize the structure of large enzyme complexes and understand the mechanisms of the reactions they catalyze. One such complex, the Pyruvate Dehydrogenase Complex (PDC), catalyzes an essential step in glucose catabolism and is comprised of many copies of three enzymes that catalyze five reactions requiring five unique cofactors. To assist students in learning of the PDC reaction and mechanism, students enrolled in in‐person biochemistry participate in a group activity to act out the mechanism of the complex using instructor‐directed student movement. The effect of acting or observing the activity on learning of PDC structure, mechanism, and function is reported using data from IRB‐approved assessment and survey instruments and compared to data from students in virtual biochemistry courses without an activity. Unique course activities involving student movement can improve student learning of complex topics in biochemistry.
Structure‐function relationships for the lncRNA SChLAP1 in aggressive prostate cancer
Authors: Falese James P. , McFadden Emily J. , Hargrove Amanda E.
Published: May 2022

Advances in deep sequencing technologies have facilitated the identification and annotation of thousands of long noncoding RNAs (lncRNAs) across the transcriptome. LncRNAs are documented to play critical housekeeping roles within the cell and are implicated in a wide variety of diseases, including cancer. While studies into lncRNA function in cancer abound, there are limited examples to date of detailed lncRNA structural analyses to enable understanding of structure‐function relationships. Understanding structure‐function relationships would increase our insight into the noncoding transcriptome and yield potential avenues for targeting lncRNAs implicated in disease. The lncRNA Second Chromosome Locus Associated with Prostate 1 (SChLAP1) has been identified in multiple clinical studies as a predictive biomarker and molecular driver of aggressive prostate cancer. While several protein interactors have been identified for SChLAP1 to date, structural insight into SChLAP1:protein recognition has not yet been explored. We believe that structural analysis of SChLAP1 will assist in designing specific therapeutic strategies to inhibit SChLAP1:protein interactions implicated in prostate cancer. To this end, we performed Selective 2′‐Hydroxyl Acylation analyzed by Primer Extension with Mutational Profiling (SHAPE‐MaP) and dimethyl sulfate (DMS)‐MaP in vitroand in cellulo. This approach yielded the first secondary structure model of SChLAP1, which revealed a complex architecture with a wide variety of secondary structures throughout the length of the transcript. Analyzing our in‐cell probing data with the ΔSHAPE algorithm, we identified protein binding regions within SChLAP1 and mapped them to non‐human primate‐conserved exons within the transcript. Finally, we identified a smaller, highly structured fragment of SChLAP1 that houses multiple putative protein binding sites implicated in prostate cancer. We believe that this region of SChLAP1 is amenable to therapeutic targeting and may be used as a smaller construct for in vitrodevelopment of SChLAP1‐targeting therapeutics. We also believe this fragment may be amenable to 3D biophysical analyses, such as X‐ray crystallography or cryo‐EM, to further enhance structural understanding of SChLAP1:protein complexation. Ongoing work is focused on determining the sufficiency of this fragment for protein recognition and characterizing potential magnesium‐dependent tertiary structures. We believe this work will facilitate the development of specific therapeutic strategies for SChLAP1 and contribute to the growing need of characterizing structure‐function relationships within lncRNAs.
MSL2 Localization to Histone Loci in Drosophila Species
Authors: Xie Mellisa , Hodkinson Lauren J. , Rieder Leila E.
Published: May 2022

Nuclear bodies are membraneless structures containing concentrated regulatory factors that coordinate nuclear processes such as gene expression. The histone locus body (HLB) is a discrete nuclear body that is the main site of histone mRNA production. While many factors of the HLB are known, there are many unknown factors. In addition, while HLB function is highly conserved, it is also unknown how the HLB has changed and evolved in different species. The histone locus (HL) of the model organism Drosophila melanogaster contains ~100 tandem arrays of the five histone genes. While D. melanogaster has one HL, the related species Drosophila virilis, has two HL. We observe localization of the male‐specific dosage compensation protein MSL2 to one of the two D. virilishistone loci using polytene chromosome immunofluorescence, which we do not observe in other Drosophila species. In order to confirm our immunofluorescence observations, we mapped existing MSL2 ChIP‐seq data and discovered that when MSL2 from either species is overexpressed, it targets the H2a‐H2b promoter. We immunostained transgenic flies carrying an H2a‐H2b promoter transgene to determine if MSL2 targets this region when outside the context of the histone locus. We do not see the same HL localization when MSL2 is expressed at wild‐type levels. Finally, we performed RNA isolation and qPCR analysis on four Drosophila species to compare expression levels of msl2. Our results indicate that MSL2 may be playing a different role at the HL in different Drosophila species. Overall, this gives us better insight on histone gene regulation.
The impact of natural aging and ovariectomy on neuroplasticity in female rats: Is all estrogen loss the same?
Authors: Grittner Jessica , Miller Shawn , Dougherty Brendan
Published: May 2022

In adult female rats, circulating sex hormone levels fluctuate significantly across the normal estrus cycle. The most abundant and neuroactive of these sex hormones is 17β‐estradiol (a form of estrogen). We previously demonstrated that 17β‐estradiol is necessary to permit development of respiratory neuroplasticity induced by acute intermittent hypoxia (AIH), called phrenic long‐term facilitation (pLTF). Female rats only develop pLTF during proestrus when circulating estrogen levels are high, and do not express plasticity during periods of low circulating 17β‐estradiol. Additionally, ovariectomy (OVX) abolishes neuroplasticity, but it can be restored with estrogen supplementation. As part of the natural aging process in female rats, estrous‐cycle related fluctuations in circulating 17β‐estradiol levels gradually cease. Rats settle into a state of persistent diestrus, with low levels of circulating estrogen, similar to human menopause. Accordingly, we hypothesized that a reduction in circulating estrogen, either through ovariectomy (OVX) or with natural aging, would eliminate the expression of pLTF. Four experimental groups of female rats were used to test this hypothesis: (1) aged (>22 months old), ovary‐intact, in persistent diestrus (min of 2, 5‐day cycles), (2) young (3 months old), ovary‐intact, in proestrus, (3) young, OVX (2wk post‐surgery), and (4) time controls (both young and aged). Phrenic neurophysiology was used to assess pLTF in anesthetized, ventilated, and vagotomized rats. Circulating estrogen levels were quantified using an ELISA assay for all groups. Contrary to our hypothesis, preliminary results indicate that aged female rats in persistent diestrus produce robust AIH‐induced pLTF (>250% increase in integrated phrenic amplitude 60min post‐AIH). Full results and mechanistic discussion will be presented.
Reading the message: Connections between the epigenetic landscape bromodomain activity
Authors: Glass Karen C. , Montgomery Cameron , Lloyd Jonathan T. , McLaughlin Kyle , Marunde Matthew R. , Evans Chiara M. , Boyson Samuel P. , Keogh Michael‐Christopher , Frietze Seth
Published: May 2022

Bromodomains are conserved epigenetic reader domains known to recognize acetylated lysine residues on the N‐terminal histone tails protruding from the nucleosome. The 61 human bromodomains are found in 41 different proteins, and have been classified into eight different subfamilies based on sequence conservation. These subfamilies often share unique sequence variations that drive differences in histone ligand preference for specific patterns of post‐translational modifications on core and variant histone proteins. The BET bromodomains, which are an attractive drug target in multiple disease states, have been the focus of many studies to elucidate the molecular mechanism of histone recognition. However, much less is known about non‐BET bromodomain‐containing proteins. In this study, we examined the ligand specificity the family IV ATAD2B bromodomain and compared it to its closely related paralog in ATAD2. We show that the ATAD2B bromodomain recognizes mono‐ and di‐acetyllysine modifications on histones H4 and H2A. A structure‐function approach using X‐ray crystallography, isothermal titration calorimetry experiments, and site‐directed mutagenesis coupled to ligand binding assays identified key residues in the acetyllysine binding pocket that dictate the molecular recognition process. Furthermore, our results demonstrate how cross‐talk between multiple modifications alters the binding activity of the ATAD2B bromodomain. The structure of the ATAD2B bromodomain in complex with a small molecule ATAD2 bromodomain inhibitor revealed that critical contacts required for coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized an alternative splice variant of ATAD2B that results in a loss of bromodomain function. Our results outline the structural and functional features of the ATAD2B bromodomain, and identify novel mechanisms regulating the interaction of the ATAD2B protein with chromatin.
Targeting GCN2 Regulation of Amino Acid Homeostasis in Prostate Cancer
Authors: Cordova Ricardo , Misra Jagannath , Amin Parth H. , Damayanti Nur P. , Carlson Kenneth R. , Klunk Angela J. , Mirek Emily T. , Miller Marcus J. , Anthony Tracy G. , Pili Roberto , Wek Ronald C. , Staschke Kirk A.
Published: May 2022

The Integrated Stress Response (ISR) plays a critical role in the adaptation and survival of tumor cells to exogenous and endogenous stresses. The ISR features four protein kinases (PERK, GCN2, PKR, and HRI), each activated by different stresses, that phosphorylate the eukaryotic translation initiation factor eIF2, resulting in repression of global protein synthesis. Paradoxically, eIF2 phosphorylation also enhances translation of select gene transcripts, including the transcription factor ATF4, which is central for ISR‐directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer stress adaptation. Moreover, eIF2 phosphorylation and ATF4 have recently been suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinases, their mode of activation, and the mechanisms by which the ISR facilitate PCa progression are unknown.

We discovered that GCN2 is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduces growth in androgen‐sensitive and castration‐resistant PCa cell lines in culture and cell line‐derived and patient‐derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2.

A transcriptome analysis of PCa cells treated with a specific GCN2 small molecular inhibitor indicates that GCN2 is critical for expression of SLCgenes involved in metabolite transport. We found that GCN2 inhibition decreases intracellular amino acid levels accounting for reduced growth in PCa cells. Using CRISPR‐based phenotypic screens and genome‐wide gene expression analyses of wild‐type and GCN2‐depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is induced by GCN2 and is essential for PCa proliferation. SLC3A2 engages with many nutrient transporters, allowing for their localization to the plasma membrane. Importantly, expression of SLC3A2 reduced GCN2 activation and rescued decreased amino acid levels and growth inhibition due to loss of GCN2.

Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including 4F2 (SLC3A2), which provide for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising therapeutic targets for both androgen‐sensitive and castration‐resistant prostate cancer.
Adaptation of Native GELFrEE for HDL Particle Size Subtype Separation and Differential Apolipoprotein Proteoform Quantification
Authors: DiStefano Nicholas , Lloyd‐Jones Cameron , Seckler Henrique , Compton Philip , Sniderman Allan , Kelleher Neil , Wilkins John
Published: May 2022

High‐density lipoproteins (HDL) are central to cholesterol transport, and higher concentration of HDL‐bound cholesterol (HDL‐C) is associated with lower coronary artery disease (CAD) risk. Recent reports refute a causal link between these variables, suggesting better understanding of HDL variation is necessary. HDL particles differ in size and size subtypes differ in proteome, lipidome, and functional characteristics. Apolipoprotein A‐I (ApoA‐I), a major determinant of HDL structure and biochemistry, is made up of 15 distinct proteoforms. ApoA‐I proteoforms correlate with HDL efflux, a measure of cholesterol transport efficiency, but it is unknown if ApoA‐I proteoforms vary with HDL particle size. Herein, we adapt a native separation methodology to inquire on the relationship between HDL size subtypes, their function, and apolipoprotein proteoform profiles.

Pooled serum (20μl) from 30 individuals with high, medium and low HDL‐C levels, were loaded to CN‐GELFrEE, a native electrophoretic technique, to separate HDL particles by size. Mid‐resolution and high‐resolution modes were employed. Immunoassays were done to characterize ApoA‐I content and average particle size of the electrophoretic fractions. Proteoform quantification was performed on ApoA‐I‐containing fractions by top‐down LC‐MS. SIM scans were devised for higher sensitivity in ApoA‐I proteoform detection. Custom software matched, scored and quantified proteoform‐specific spectra, and calculated association to particle size.

In both resolution modes, average fraction sizes were roughly linearly correlated to fraction collection time, and size‐range overlap between fractions was small. In mid‐resolution mode 3‐4 fractions contained ApoA‐I, while high‐resolution mode yielded 36 distinct HDL size fractions. Fractions roughly spanned the range between 5 to 11nm, similar to previously reported ranges for HDL particles.

Proteoform quantification revealed significant variation of proteoform profiles in different size‐ranges of HDL. Fatty‐acylated ApoA‐I, a species previously correlated to higher cholesterol efflux, had higher relative abundance (~1.5x to 2x) in the pre‐beta‐1 (5‐7.1nm) and alpha‐3 (9‐11nm) size ranges of HDL, while glycated and oxidized proteoforms were significantly more abundant (~3x to 4x) in the medium size ranges (alpha‐4, alpha‐3 and alpha‐2). Interestingly, the truncated proteoform of ApoA‐I had no significant differences in abundance between different particle sizes.

Our experimental data suggest that the profile of ApoA‐I proteoforms in HDL particles is size‐dependent and thus ApoA‐I proteoforms may be important markers or mediators of HDL size regulation. Notably, the differences in PTM prevalence from medium sizes to large and pre‐beta subtypes may be markers of the pathway of HDL maturation and/or different functions of each subtype.
The Effects of Disabled ARR1 Gene on C. Elegans Expression of Substance Preferences
Authors: Jazari Donnya A. , Sareddy Anika M. , Percer Aidan , Kamon Amalia R. , Wang Jocelyn , Armentrout Quinn , De Lucas
Published: May 2022

A vital component in the mechanisms of processing addictive substances are G‐protein coupled receptors (GPCRs), which function as a mediator of behavioral effects of addictive drugs. Arrestins (ARR‐1) are adaptor proteins that regulate the G protein‐coupled receptor desensitization and internalization, and are a key to the effect of dopamine, opioids, and cannabinoid receptors. The ARR‐1 gene in C. Elegans is shown to be responsible for the internalization of GPCRs. GPCRs send signals downstream to the C. Elegans chemosensory nerves, which regulates their olfactory and gustatory senses. In C. Elegans, inhibition of the ARR‐1 gene is shown to affect worms' abilities to desensitize from substances such as alcohol. Using an ethanol tolerance assay, this project aims to analyze the relationship between neural plasticity and its relationship to Arrestin. We hypothesize that ARR‐1 is integral to alcohol sensing, and to investigate this, we use the Arrestin deficient strain of C. Elegans, RB660, wtN2, and an ethanol preference assay. We examine worm behavior using camera‐tracking and represent the data using a heat map.
The Multifaceted Subunit Interface of Malate Dehydrogenase
Authors: Keene Megan , Hanson Isabella , Armendariz Daniel , Botros Natalie , Blythe Hannah , Bell Jessica , Bell Ellis
Published: May 2022

Most Malate Dehydrogenases are dimeric though there are a small number (for example the LDH like Apicomplexan Malate Dehydrogenases) with tetrameric structures. A variety of studies have shown the oligomeric structure to be important for the overall activity of the enzyme with roles from stability to allosteric interactions being suggested. To explore our overall hypothesis that regions of the subunit interface would be tailored to specific functions we initiated an examination of the cross interface interactions of malate dehydrogenases from a wide variety of organisms whose crystal structures are available. Using the Hawkdock MM/GBSA routine we determined the residues involved in the interface in each structure. For the dimer interface we found between 24 and 35 residues involved depending on isoform. Mapping these residues onto structures revealed 5 areas of interaction, I to V with regions II – V being spatially conserved in all structures (figure 1). Region I was found only in chloroplastic versions of MDH. While the general areas of contact are conserved the detailed nature of the interactions revealed significant differences including inclusion of a conserved repulsion in region II in all Isoforms. These observations led to a series of hypotheses concerning the roles of specific regions and residues in isoform families. We focused on the cross interface interactions potentially related to citrate regulation of the organelle isoforms, and identified a unique cross interface interaction between the sidechain of D87 and the helix dipole of helix T268‐286 on the opposite subunit adjacent to a D92‐Y273 interaction across the interface. The role of L269/S266 in this helix and adjacent loop which we hypothesized form a cross interface molecular switch, was explored using site directed mutagenesis which showed that mutation of either residue to alanine disrupted either oxaloacetate or NADH saturation, overall catalytic activity and citrate inhibition. These results suggest a cross interface network (figure 2) in organelle malate dehydrogenases. Comparison of interface residues from structures with and without Citrate bound show that region III and V show the largest changes induced by citrate with some residues showing greater and others lesser contributions to the interface and are consistent with the observed network of interactions and suggest that changes across the interface might be induced by citrate interacting with S266. Interestingly the tetrameric MDHs from Ignacoccus Islandicus and the apicomplexans shares many but not all of these residues, lacking in particular D92, T268 and Y273. Knock in mutants of the Ignacoccus Islandicus and the Plasmodium falciparum MDHs studied both computationally and with enzyme kinetics shed further insight on the potential roles of these residues and the evolution of cooperativity in MDH.
Directed Evolution of Antibacterial Nanobodies Using Novel Antigen Production Strategies
Authors: Crysler Anna , Streu Craig
Published: May 2022

Antibiotic resistance has been a reality ever since the discovery of penicillin, yet with the overuse and misuse of antibiotics since the 1940s, antibiotic resistant infections are on the rise. Compounding the problem, over the past few decades antibiotic discovery and development has slowed. As a result, we are facing a crisis that is expected to create up to 10 million deaths globally by 2050 without urgent action. Selective pressure from antibiotics allows bacteria to generate a variety of resistance mechanisms, creating infections that can become severe and difficult or impossible to treat. Even many antibiotics deemed “last resort” are diminishing in effectiveness due to the emergence of highly resistant bacteria. Among these “last resort” antibiotics are glycopeptides that target bacterial cell wall synthesis in Gram‐positive bacteria. Specifically, a precursor molecule to peptidoglycan synthesis, known as lipid II, is a common target for glycopeptides. To evade these antibiotics, the structure of lipid II is often modified to prevent glycopeptide binding. As bacterial resistance to the glycopeptides spreads, new antibiotics must be made to combat these differences in lipid II molecules. Most approaches to increasing the availability of new antibiotics fall into two categories—reducing time required for discovery and reducing cost of development. This research outlines a novel strategy for antibiotic design using directed evolution techniques. By taking advantage of selective processes, much like those that drive resistance, directed evolution can be used to develop novel antibiotics that meet both these needs. In addition to keeping up with the pace of antibiotic resistance, directed evolution also offers the opportunity to drive more personalized medicine in healthcare. Streamlining the discovery process may ultimately make it possible to take bacterial samples from patients and rapidly create antibiotics specific to their infections. Single domain antibodies, or nanobodies, are a class of molecules with favorable properties for use as antibiotics given their size, stability, and potential antigen specificity. This poster focuses on the directed evolution of nanobodies for use as antibacterial molecules against lipid II in Gram‐positive bacteria. In addition, it outlines a unique antigen selection strategy that is expected to be broadly applicable to a wide range of Gram‐positive pathogens.
Allosteric networks regulate function in multi‐enzyme complexes
Authors: Boehr David D. , D'Amico Rebecca , O'Rourke Kathleen , Bosken Yuliana , Sahu Debashish , Chang Chia‐en
Published: May 2022

Networks of noncovalent amino acid interactions propagate allosteric signals throughout proteins. Tryptophan synthase (TS) is an allosterically controlled bienzyme in which the indole product of the alpha subunit (αTS) is transferred through a 25 Å hydrophobic tunnel to the active site of the beta subunit (βTS). Previous nuclear magnetic resonance and molecular dynamics simulations identified allosteric networks in αTS important for its function. Here, we show that these allosteric networks change across the catalytic cycle of αTS, such that chemical catalysis is associated with specific conformational dynamics and networks not observed in the substrate‐ and products‐bound states. Moreover, substitution of a distant, surface‐exposed network residue in αTS enhances tryptophan production, not by activating αTS function, but through dynamically controlling the opening of the indole channel and stimulating βTS activity. While stimulation is modest, the substitution also enhances cell growth in a tryptophan‐auxotrophic strain of Escherichia coli compared to complementation with wild‐type αTS, emphasizing the biological importance of the network. Network residues thus represent ideal targets to optimize mutagenesis strategies in protein engineering studies directed towards changing enzyme function and regulation, especially in multi‐enzyme complexes.
An Enhanced 1D Electrophoresis Simulation with Pedagogical Tools
Authors: Bernhardt Tyler , Seyfarth Matthew , Hanson Robert M. , Roberts Rebecca , Craig Paul A.
Published: May 2022

Electrophoresis is a fundamental tool for biochemistry and molecular biology research. Our team has updated a popular 1D electrophoresis simulation to give instructors a choice ‐ they can use the built in protein mixtures or they can add their own protein mixtures and use the simulation to focus on particular learning goals and objectives in their classroom and laboratory courses. The simulation retains many of its original features: variable voltage and % acrylamide, built in molecular weight standards, animation of the pipetting process, and a built‐in plot for estimating molecular weights. Features in the updated simulation include (a) an expansion from two to ten wells, (b) drag and drop options for loading proteins on the gel, (c) a choice of ten built‐in protein sets and learning objectives designed for the BASIL curriculum (https://basilbiochem.github.io/basil/index.html), and (d) the option to introduce custom designed protein mixtures with associated learning objectives. The simulation, formerly a Java applet, was redeveloped using java2script/SwingJS and now runs both as an independent Java application and as a browser‐based JavaScript app.
Biological Evaluation of 3‐Aryl‐4‐indolylfuranones as potential anti‐cancer agents
Authors: Mowery Patricia , Dexter Molly S. , Hermann Andrew , Hurysz Brianna , Manning Maegan , McNulty Matthew , Pierce Kelsey
Published: May 2022

The American Cancer Society estimates that this year there will be approximately 1.9 million new cases of cancer and nearly 610,000 cancer‐related deaths. The need for new and innovative anticancer drugs is increasingly crucial. One attractive anti‐cancer target is tubulin, which is involved in many cellular functions including cell shape, mitosis, migration, and movement of organelles. While anti‐tubulin drugs have been used to treat cancer for ~70 years, they tend to be complex molecules (derived from natural products), plus suffer from multi‐drug resistance, low solubility, toxicity issues, and/or the lack of multi‐cancer efficacy. These limitations highlight the continued need for the discovery and development of new and novel drugs to enter the pipeline, especially with the significant increase in understanding of tubulin and the mode of action of inhibitors.

Using an interdisciplinary approach with undergraduate researchers, we previously developed a furanone‐containing small molecule, PY‐407‐C. The compound demonstrated strong (sub‐micromolar) anti‐proliferative activity in U937 cancer cells. It was submitted for NCI‐60 (National Cancer Institute) screening, which showed nanomolar activity against a variety of cell lines with tubulin as the potential target. We confirmed that it inhibited tubulin polymerization in vitro. We now report on a suite of compounds that are regioisomers of PY‐407‐C and with various moiety modifications. This work has identified new compounds with strong cytotoxicity and important structure‐function information. Overall, these studies provide basic research on small molecule inhibitors of cancer.
Anionic membrane phospholipids: A New Class of Chemokine‐Binding Site Important for both Apoptotic Cell Clearance and Antibiotic Activity by Chemokines
Authors: Pontejo Sergio , Murphy Philip
Published: May 2022

Chemokines are small cytokines that orchestrate immune cell migration by binding to cell surface glycosaminoglycans (GAGs) and activating G protein‐couple receptors (GPCRs) expressed by leukocytes. We have discovered that chemokines selectively bind with high affinity to anionic membrane phospholipids, including phosphatidylserine (PS) and cardiolipin (CL). This adds to GPCRs and GAGs a third class of high affinity membrane binding site for chemokines and expands the scope of chemokine action to new areas of biology dictated by the specific biological niches occupied by their phospholipid binding partners. PS, for example, is usually restricted to the cytosolic face of the phospholipid bilayer of the plasma membrane of healthy eukaryotic cells, but it is flipped to the outer leaflet of the membrane of apoptotic cells and apoptotic extracellular vesicles (ApoEVs), where it acts as an ‘eat‐me’ signal recognized by phagocytes for apoptotic cell clearance. In this regard, we have recently demonstrated that ApoEVs generated in vivo from mouse thymus induce phagocyte migration using endogenous chemokines bound to the vesicle surface via PS. Our results indicate that PS‐bound chemokines on ApoEVs may serve as ‘find‐me’ signals that recruit phagocytes toward apoptotic cells for the removal of dying cells. This is a new PS‐dependent mechanism for chemokine delivery by extracellular vesicles. On the other hand, CL is absent in the plasma membrane of eukaryotic cells but it is a major and naturally exposed anionic phospholipid in the bacterial plasma membrane. As such, we have found that chemokines act as potent direct bacteria‐killing agents by binding to bacterial CL. This finding identifies the molecular mechanism for antimicrobial action by chemokines and launch new avenues of research in antimicrobial therapies and bacteria‐host interactions. In short, we have identified chemokines as the first example of a family of soluble cytokines whose functions are regulated by direct interaction with membrane phospholipids.
Remembering Our Past: Teaching the History of Anatomy at Indiana University
Authors: Brokaw James J. , Azim Homaira M. , Bendinger Jonathan E. , Byers Kelsey T. , Dufeau David L. , Hanson Danielle C. , Organ Jason M. , O'Loughlin Valerie D.
Published: May 2022

Most students pursuing careers in anatomy or related disciplines have a limited understanding of how, over the centuries, the intricate structure of the human body came to be known. To provide students with the relevant historical perspective, we developed a team‐taught survey course in the history of anatomical sciences—including gross anatomy, histology, neuroanatomy, and embryology—from antiquity to the present. Taught entirely via Zoom during the Spring semester of 2021, History of Anatomy (2 semester hours credit) met once per week for approximately 2 hours. Enrollment consisted of 5 undergraduate students majoring in Biology (2), Human Biology (2), or Anthropology (1), as well as 3 graduate students pursuing either a master’s degree in Clinical Anatomy (1) or a Ph.D. in Anatomy Education (2). Three of the students had no prior coursework in anatomy. Through assigned readings, lectures, and discussions, the class explored the work of the great anatomists and their discoveries. A particular emphasis was placed on the evolution of anatomy as a discipline and the cultural influences, scientific controversies, and ethical dilemmas facing its practitioners. Syllabus topics included critical appraisals of the role of gender, race, and ethnicity in anatomical discovery. A key feature of the course was the opportunity for students to engage in robust discussions about such controversial issues as: Eurocentric biases in our understanding of human anatomy and the untold story of Muslim contributions to anatomical knowledge well before Vesalius; Competing claims of priority for who “discovered” the pulmonary circulation; The underappreciated role of women in the history of anatomy and medicine; The ethical quandary of teaching anatomy from archival fetal specimens obtained before the era of informed consent; Accusations that famed anatomist William Hunter used the bodies of murdered pregnant women to create his anatomical atlas of the gravid uterus; Complicity of Eduard Pernkopf and other Nazi‐era anatomists in the unethical use of executed victims to obtain images for a renowned anatomical atlas. All students were assessed through weekly homework (written responses to study questions), a mid‐term writing assignment, and a term paper about an historical topic of the student’s choosing. Graduate students had the additional requirement of a class presentation about their term paper topic. The end‐of‐course evaluation suggested that the course was well‐received by the students (mean Likert score = 4.63 on a 5‐point scale; n = 6). Based on this positive reception, we plan to offer History of Anatomy again on a recurring basis. We believe that by knowing our history, both the good and the bad, future practitioners of anatomy and related disciplines will be less likely to perpetuate the biases and ethical transgressions of earlier eras.
Itaconate is a negative regulator of hepatic lipid metabolism during sepsis
Authors: Mainali Rabina , Quinn Matthew
Published: May 2022

Sepsis is a life‐threatening clinical implication with increased uncontrolled host immune response to an infection, mortality, morbidity, and financial burden worldwide. Due to its critical role in metabolic reprogramming in inflammation, the Irg1/itaconate has received much attention as an immunomodulator. However, understanding of itaconate’s anti‐inflammatory and immunometabolic activities in response to infections is mostly limited to immune cells. By employing multi‐omic approaches, here we show that in the context of sepsis, a disruption in TCA cycle flow drives hepatic itaconate accumulation, indicating potential non‐immune functions. However, the functional role of itaconate in this central organ critical to maintaining systemic metabolism is yet to be elucidated. To gain more insight into its physiological role in the liver during sepsis, we subjected wild‐type and whole‐body Irg1 knockout mice to sepsis via cecal slurry injection. In conjunction with our previous findings, we find wild‐type septic mice develop hepatic steatosis. Interestingly, global Irg1 knockout mice develop a more severe form of a hepatic steatotic phenotype and a significant increase in hepatic lipid burden compared to wild‐type counterparts in response to sepsis. This data demonstrates itaconate as a negative regulator of hepatic lipid accumulation in the context of sepsis. However, the exact molecular mechanism by which itaconate interacts with regulators of hepatic lipid metabolism during sepsis is yet to be uncovered. As an anti‐inflammatory metabolite, itaconate limits the glycolytic response in activated immune cells. Similarly, invitro, we find that 4‐OI, an itaconate derivative, antagonizes LPS induced glycolysis in hepatocytes. Given our findings of heightened lipid accumulation in septic Irg1 knockout mice, we further hypothesize glycolysis to be elevated and fueling de novolipogenesis via the production of acetyl‐CoA. Indeed, unbiased metabolomics data show heightened hepatic lactate levels indicative of hyperactivated glycolysis in knockout septic mice. Mechanistically, we find elevated gene and protein expression of lactate dehydrogenase, the enzyme that facilitates the conversion of pyruvate to lactate, driving this accumulation of lactate seen in septic Irg1 knockout mice. In summary, our preliminary findings thus far highlights itaconate as a negative modulator of hepatic glycolysis as well as de novo lipogenesis in restraining sepsis‐induced hepatic steatosis.