bluuebunny/crossref_metadata_2025 huggingface dataset:
Shrinking the Mystery of Tsantsa Identification: Hair Analysis of Shrunken Heads from the Denver Museum of Nature and Science
Authors: Oya Mayu , Vinovskis Carissa , Royer Danielle
Published: June 2021

INTRODUCTION

Tribes in South America, such as the Jivaro, were historically known to perform shrinking of human heads as part of their war rituals. Although authentic shrunken heads (tsantsas) are of human origin, imitation tsantsas were later fabricated using animal or synthetic materials. Numerous tsantsas are held in museum collections worldwide, including five in the Anthropology Collection at the Denver Museum of Nature and Science (DMNS). A previous analysis of the tsantsas curated at the DMNS suggests that two specimens may be imitations.

GOALS

The purpose of this study was to microscopically evaluate hairs from each of the five tsantsas in the Anthropology Collection at DMNS, and test the hypothesis that hairs from each specimen are human in origin.

METHODS

Five hairs from five different areas of the head (frontal, right and left temporal, vertex, occipital) were obtained for a total of 25 hairs per specimen. The hairs were mounted for microscopic analysis by researchers blinded to the origin of the samples. Using a brightfield microscope at 40x, images were collected using the Amscope image capture software and analyzed using ImageJ. The following qualitative characteristics were scored and compared to human norms: hair color, medulla features, cuticle thickness, pigment granule distribution, and the presence of scales. Measurements of hair shaft width and medulla width were also recorded for each hair and compared to human reference values for global populations.

RESULTS

The qualitative analysis revealed that all of the hairs exhibited typical human characteristics. For each of the specimens, mean hair shaft and medulla width values were mostly within the normal range for human hair. However, a few exceptions were observed: 68% of sampled hairs from one specimen exhibited a medulla wider than expected for human hair, where the medulla is typically less than one‐third of the shaft width, and in another specimen, 72% of sampled hairs had shaft widths below the normal human range of 60–120um.

CONCLUSION

When considering results from both the qualitative and quantitative analyses, the hairs from each of the DMNS tsantsas are likely to be human in origin. Previous analyses of the facial characteristics of the DMNS specimens suggested that two specimens may be imitations rather than authentic tsantsas. However, the hairs sampled from the two potential imitations appear to be human. It is possible that the genuine human hairs were manually imbedded into the scalp of the fabricated tsantsas; closer inspection of the internal scalp may provide insight. DNA analysis is needed to confirm the human origin of the tsantsas.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in the Effects of Prenatal Exposure to Low Doses of Endocrine Disruptors on Stress‐Related Behavior
Authors: Kaimal Amrita , Cherry Ariana D. , Martin Nicholas M. , Buechter Hannah E. , Hoover‐Smith Jessica M. , Holmes Philip V. , MohanKumar Sheba M. , Mohankumar P.S.
Published: June 2021

Prenatal exposure to endocrine disrupting chemicals (EDCs) has been shown to produce a predisposition to behavioral disorders in later life. Bisphenol A (BPA) and di(2‐ethylhexyl) phthalate (DEHP) are two widely prevalent EDCs in the environment and exposure to these chemicals is unavoidable. We examined if prenatal exposure to these chemicals would produce behavioral changes in offspring. Female rats were orally administered BPA (B; 5 μg/kg body weight), DEHP (D; 7.5 mg/kg body weight), a combination of BPA and DEHP (B+D), or saline (control; 10 μL/kg body weight) during days 6 through 21 of pregnancy. Adult offspring underwent a battery of behavioral tests consisting of the open field task (OFT), elevated plus maze (EPM), and shock probe defensive burying (SPDB). In the OFT, control and B+D‐treated females spent a significantly lower percentage of time in the center zone than control males. These sex differences were not observed in B‐ or D‐treated animals, however, both male and female D‐offspring spent significantly less time in the center compared to control males. In the EPM, B animals displayed sex differences that were not present in control animals, with B females spending significantly more time in the open arms compared to both control and B males. D and B+D animals were not significantly different from controls in time spent in the open arms. No differences were seen in total exploration of the OFT or EPM. Control female animals reared and explored the probe significantly more than control males during SPDB. Sex differences were not present in B‐ or B+D‐treated offspring and were altered in D‐treated offspring. B‐treated female offspring buried less than control female offspring, but did not differ in immobility time or frequency of rearing or probe exploration in the SPDB, indicating a less robust response to threat. B treated male offspring were no different than control male offspring in the SPDB. D‐treated female offspring reared and explored the probe fewer times than female control‐treated offspring. Behavior was similar to control male offspring for each behavior, suggesting a masculinization of behavioral response to threat. D‐treated male offspring buried significantly less and spent significantly more time immobile than control males, suggesting a phenotypic shift to a passive‐coping style in response to threat. Finally, male and female B+D offspring showed a similar behavioral phenotype during SPDB. In conclusion, these results demonstrate that prenatal exposure to EDCs differentially alters behavior in male and female rats.

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 Dendritic Spine Density in Hippocampus Following CA/CPR
Authors: Oya Mayu , Yonchek Joan , Quillinan Nidia
Published: June 2021

INTRODUCTION

Cardiac arrest (CA) occurs when the electrical system of the heart malfunctions. Patients do not receive enough blood to the brain and experience global cerebral ischemia, causing neuronal damage and affecting memory. The hippocampus is the main area of the brain dealing with learning and memory, and the dendrites and the spines of the CA1 neuron have been shown to undergo plasticity during learning and memory. Previous work has demonstrated that global cerebral ischemia causes persistent deficits in CA1 synaptic plasticity, but it is not known whether these deficits are associated with morphological changes. Acute dendritic plasticity (occurring within two to three days) after cardiac arrest/cardiopulmonary resuscitation (CA/CPR) have been shown previously. However, little is known regarding dendritic spine morphology at chronic time periods after CA/CPR.

GOAL

This present study investigated the plasticity of the CA1 neurons and their dendrites and spines in mice after CA/CPR.

METHODS

Adult male mice were given CA by intravenous injection of potassium chloride, and resuscitation was initiated eight minutes after arrest with chest compressions and ventilation. Mice were allowed to survive for one, seven, or 30 days after CA/CPR after which brains were collected. Whole brains were stained with Golgi‐cox staining technique, and sagittal sections were prepared for microscopy. High magnification images were taken on a bright field microscope using QCapture software and analyzed using ImageJ. The dendrites and their spines were observed in the CA1 region, and spine density, soma length, soma width, and soma area were calculated. Spines were also assigned a morphological classification (one to four) that ranges from immature to mature spines.

RESULTS

Our data shows that there is a significant reduction in spine densities at each of the time points after CA, compared to the sham. Furthermore, spine classification shows that CA caused a shift towards more immature spine morphology at 24 hours and 7 days after CA/CPR but shifted towards more mature spine morphology at 30 days after CA/CPR.

CONCLUSION

These data demonstrate that there is morphological plasticity and changes in dendritic spine anatomy at chronic time periods after CA/CPR. These changes likely correlate with synaptic plasticity impairments in the hippocampus.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The uracil‐DNA glycosylase homolog UdgX from M. smegmatis as a potential platform for in vitro protein evolution
Authors: Thomas Sydney Alexandra , Noren Chris
Published: June 2021

A uracil‐DNA glycosylase (UDG) homolog from Mycobacterium smegmatis, UdgX, has been reported to lack uracil excision activity but instead binds uracil‐containing DNA tightly even under denaturing conditions, implying a covalent protein‐DNA adduct (Sang et al., “A unique uracil‐DNA binding protein of the uracil DNA glycosylase superfamily” NAR 43, 8452–63, 2015). To determine whether UdgX could be used as a fusion partner to tether a protein library to its encoding DNA as a platform for in vitro protein evolution, we expressed, purified, and characterized UdgX for its uracil‐binding activity. As expected, UdgX exhibits specific complex formation with uracil‐containing single stranded and double stranded DNA substrates by electrophoretic mobility shift assay (EMSA), but only in the absence of SDS. This implies that the UdgX‐DNA interaction is non‐covalent, contradicting the earlier report. Further work will be necessary to determine whether the noncovalent UdgX‐DNA interaction is sufficiently strong to stably tether a protein library to its encoding DNA.

Support or Funding Information

New England Biolabs Inc.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Transcriptional Signature of Three Staphylococcal Superantigenic Toxins in Human Epidermal Melanocytes
Authors: Srinivasan Seshamalini , Chakraborty Nabarun , Miller Stacy‐Ann , Gautam Aarti , Yang Ruoting , Alkhalil Abdulnaser , Moffatt Lauren T. , jett Marti , Shupp Jeffrey W. , Hammamieh Rasha
Published: June 2021

Staphylococcus aureus, a gram‐positive bacterium, causes food poisoning and toxic shock syndrome through the production of pyrogenic toxins known as Staphylococcal enterotoxins serotypes A‐J (SEA, SEB, etc.) and toxic shock syndrome toxin‐1 (TSST‐1). A subset of these toxins have been classified as potential biothreat agents and could effectively incapacitate the soldiers on the front line. The skin model was considered appropriate for this study as the skin is the first defense barrier for the host. The purpose of this research was to identify toxin‐exclusive network‐focused trajectories sequentially perturbed across time that could potentially characterize superantigenic toxicity and early pathogenesis. Normal Human Epidermal Melanocytes were grown and treated with 25 μg/mL of one of the three toxins, SEA, SEB, and TSST‐1. Cells were collected along with viability counts after being incubated for six different time periods ranging from 0.5h to 48h. Total RNA was purified using TRIzol reagent and oligonucleotide microarrays were performed using Agilent slides containing approximately 50,000 probes. Transcriptomic expression data indicated longitudinal trajectories unique to each of the three toxins. Transcriptomic responses to both SEA and TSST‐1 treatments had similar clustering patterns where early (0.5h, 2h), middle (6h, 12h) and late (24h, 48h) time points grouped together. Interestingly, genes responding to SEB treatment followed a different clustering profile where early (0.5h, 2h) and middle (6h, 12h, 24h) time points clustered together, and the latest time point (48h) was separate. Another interesting observation is that there were no shifts in the PCA landscape between the two late time points (24h, 48h) in response to SEA and TSST‐1 treatments, but this trend was not observed for the SEB treatment, which indicated a major transcriptomic perturbation between the 24h and 48h treatments. Therefore, we concluded that at the given doses, the progression of cellular responses to SEA and TSST‐1 treatment had completed by 24h while the SEB exposure continued to affect cellular responses until 48h.We have identified distinct patterns of transcriptomic regulation for each of the SEA, SEB and TSST‐1 toxins. Many of the differentially regulated genes have important functions related to cell transformation, differentiation, apoptosis and inflammation. Work is ongoing to identify differentially regulated networks with diagnostic and therapeutic implications.

Disclaimer: Research was conducted in compliance with all Federal requirements. Views expressed are those of the authors and do not constitute endorsement by the U.S. Army.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Functional Effects of Altering the Nonpolar Core of Hemolysin A
Authors: Stuttgen Gage Michael , Weaver Todd Michael , Grilley Daniel Patrick
Published: June 2021

The protein structure and function paradigm, a foundational tenet of biomolecular science, underlies many infectious diseases. Hemolysin A (HpmA), a hemolytic protein produced by Proteus mirabilis, was used as a model to investigate the protein structure‐function paradigm. HpmA is a member of the two‐partner secretion (TPS) pathway, which is used by gram‐negative bacteria to export predominantly virulent proteins outside of the cell. Through this mechanism, the A‐component (HpmA) is translocated, folded, and activated by its cognate B‐component in the absence of high‐energy bond and electrochemical gradient dependency. All TpsA components are relatively large and can be further divided into two domains, the two‐partner secretion domain and the functional domain. Universally, all known TPS domains harbor a right‐handed, parallel β‐helix architecture and are essential for cognate TpsB‐dependent recognition and secretion. Conversely, functional domains provide TpsA diversification including, cytolysis, host cell adhesion, contact‐growth inhibition, and iron sequestration. A truncated version of hemolysin A (HpmA265) was implemented to define the contributions of the TPS domain toward TpsA structure and function. Recently, our group has further dissected HpmA265 into three sequentially folded structural units termed the polar core, non‐polar core, and carboxy‐terminal subdomains. This research project aims to expand upon our recent results and structurally map the role of the nonpolar core during TPS domain dependent secretion, folding and function. Specifically, residues within the non‐polar core subdomain have been selectively targeted and modified. The structural and functional effects of these site‐selective modifications have been evaluated via chemical denaturation, protease sensitivity and hemolytic assays. Each site‐selective alteration selectively shifts the unfolding transitions attributed to the non‐polar core and carboxy‐terminal subdomain, while leaving the polar core transition unaffected. Furthermore, non‐polar core subdomain destabilization differentially alters secretion levels, while leaving hemolytic activity unaffected. More broadly, these results further define β‐helix TPS domain contribution during gram‐negative bacterial infections like whooping cough, meningitis, and urinary tract infections.

Support or Funding Information

Funding for this research was provided by: University Wisconsin – La Crosse Faculty Research Grant Program (TMW) and University Wisconsin – La Crosse Undergraduate Research and Creativity Grant Program (GMS).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Importance of Salt‐Bridge Formation of Lysine 52 and 54 from Apolipophorin III for Protein Structure and Function
Authors: Tran Angela , Shah Kriti , Weers Paul M.M.
Published: June 2021

Apolipophorin III (apoLp‐III) is an 18 kDa exchangeable apolipoprotein found in insects. The protein is used as a model to better understand lipid transport processes and innate immunity due to the availability of three‐dimensional structures of the protein, allowing a structure‐guided approach. ApoLp‐III from Locusta migratoria contains eight lysine residues, which interact with negatively charged phospholipids found in bacterial membranes. They also help to maintain the structure of the protein by ionic interactions. Denaturation of the protein with guanidine‐HCl showed that when lysine residues at position 52 and 54 were changed into glutamine, protein stability was significantly reduced. To identify which of the two lysine residues are involved in salt bridge formation with neighboring glutamate or aspartate residues, site‐directed mutagenesis was employed to design single lysine to glutamine (KQ) variants. The K52Q and K54Q single mutants were generated using the Quick‐change site‐directed mutagenesis kit. E. coli cells were transformed with the mutant plasmids, and protein expression was induced by IPTG. SDS‐PAGE analysis of the bacterial cultures showed the presence of apoLp‐III in the media, indicating that the proteins escaped the bacteria. Upon purification by reversed‐phase HPLC the structure of the mutant proteins will be assessed using circular dichroism to determine helical content and protein stability, while phosphatidylglycerol bilayer vesicles will be used for functional studies. Identification of the salt‐bridge in apoLp‐III will help to better understand the role of ionic interactions in the structure and function of apolipoproteins.

Support or Funding Information

This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers GM089564, 8UL1GM118979‐02, 8TL4GM118980‐02, and 8RL5GM118978‐02.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying Residue‐Residue Contacts via Deep Mutational Scanning
Authors: Hinds Emily C. , Romero Philip A.
Published: June 2021

Structural knowledge is critical for understanding a protein's biological function and interactions, aiding drug design and other advances in medicine and biotechnology. However, there are many proteins for which structural data are either of poor quality or completely absent, and the inefficiency of experimental structure determination necessitates computational approaches to address this gap. The accuracy of de novo folding simulations in full‐length structure prediction can be improved by incorporating information about individual pairs of contacting residues. But if the full structure is not known, often the true structural contacts are also unknown. These contacts can be inferred from an evolutionary alignment of sequences by identifying residues that are co‐mutated, but this approach is greatly limited by the amount of data available. With the rise of ultrahigh‐throughput functional screening and sequencing techniques, large alignments of functionally active sequences can be generated to provide comprehensive mutational data. Here, we demonstrate that contact information can be obtained from a set of active β‐glucosidases identified via deep mutational scanning. To identify residues for which mutations are coupled and therefore may be in contact, we calculated mutual information for all residue pairs in the set of active sequences. Within this set, relatively high mutual information correlates with residues closer in structure. Preliminary de novo modeling simulations in Rosetta show that only a subset of true contacts are necessary for fold‐level predictive accuracy. We expect additional work to confirm that the predicted contacts of β‐glucosidase support accurate de novo structure prediction. Thus, we have formed a foundation to investigate the applications of ultrahigh‐throughput functional screening in protein structure prediction. With a wealth of sequences, quality contact information can be more readily acquired, fueling accurate structure prediction for continued medical and biotechnological development.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining Chaperone Requirements for the Propagation of Heterologous Poly‐Glutamine Aggregates in Saccharomyces cerevisiae
Authors: Killian Andrea N. , Cole Sierra J. , Hines Justin K.
Published: June 2021

Amyloid‐based yeast prions are heritable aggregates of misfolded protein that can be passed on to daughter cells following fragmentation by chaperone proteins including Hsp70, Hsp104, and the Hsp40 Sis1. Yeast prions usually exhibit an amyloid structure, forming cross‐beta sheets of protein sections known as the prion‐forming domain, or PrD. In vivo, long tracts of glutamine have been shown to form amyloid structures. Previously Alexandrov et al. created a set of polyQ sequences with a single heterologous amino acid interspersed every fifth residue (QQQXQ), fused to the MC regions of yeast Sup35 (responsible for the prion [PSI+]) and expressed in a sup35‐ΔN strain of Saccharomyces cerevisiae. These polyQX sequences replace the PrD of Sup35, and form amyloid aggregates in vivo that propagate in an Hsp104‐dependent manner. Here we report our efforts to test the J‐protein chaperone requirements of these aggregates, first by repressing levels of Sis1 to determine if any polyQX aggregates are maintained without Sis1. Preliminary results suggest several QX constructs are dependent upon Sis1 for propagation, demonstrating that these aggregates propagate in an Hsp40/Hsp70‐dependent manner. Subsequent replacement of wild‐type Sis1 expression with several well‐studied Sis1 variants will allow the determination of the importance of specific regions of Sis1 for the propagation of these aggregates. We anticipate that this line of investigation will provide significant insight into the ways in which amino acid content of a PrD affects the interaction of the resulting prion with chaperone proteins.

Support or Funding Information

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Copper and Zinc on PSN gene expression in a Drosophila Alzheimer's Model
Authors: DeGouveia Kelsey , Rogers Ryan Patricia
Published: June 2021

Based on the current trajectory of Alzheimer's disease, the number of Americans affected are said to increase by 13.5 million people, approximately 16% of the elderly population, by the year 2050. [1] Alzheimer's disease is characterized by the presence of amyloid‐β plaques and neurofibrillary tangles resulting in loss of cognition, memory, behavior, and locomotion.[2] Amyloid‐β plaques are proteolytic fragments of the amyloid precursor protein (APP), which is a resultant of processing through the amyloidogenic pathway. [2,3,4] It is believed that elevated consumption of copper or zinc will increase or decrease the toxicity of amyloid‐β in the brain respectively due to its interactions with presenilin, a component of the γ‐secretase complex responsible for the cleavage of APP.[2,3,4] In order to begin mapping out the amyloidogenic pathway, levels of the PSN gene, the Drosophila melanogaster ortholog responsible for coding for the presenilin protein. In order to begin mapping out the amyloidogenic pathway, levels of the PSN gene will be quantified over various time points and under different dietary conditions. It is hypothesized that increased copper consumption will upregulate the expression of PSN, promoting the amyloidogenic cleavage of APP, therefore speeding up the progression of the disease, while zinc will have an opposite effect and potentially break down amyloid‐β aggregates. The proposed experiments will ultimately develop a more efficient model of Alzheimer's disease in Drosophila melanogaster by monitoring physical and genetic effects of dietary changes.

Support or Funding Information

This work was supported by funds from the Department of Sciences at Wentworth Institute of Technology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relating Collapse to Sequence and Behavior in Glutamine‐Rich Domains
Authors: Garelli Colton J. , Scott Adam S. , Ellis Jamie P.
Published: June 2021

Intrinsically disordered proteins, or IDPs, are ubiquitous throughout eukaryotic proteomes with particularly important roles in biosignalling and transcription regulation. Within this category, glutamine‐rich (Q‐rich) domains appear commonly within transcription activators. Q‐rich domains are known to collapse rather than fold upon binding and have a high tendency to aggregate. Their behavior appears to be mediated by solvent interaction with some evidence that water is trapped within the collapsed domains. These traits appear important in gene activation, yet effective pathways to evaluate the behavior of Q‐rich domains remain elusive. In this work, we develop fluorescence methods to experimentally observe the solvent exchange between bulk media and trapped water within collapsed Q‐rich domains. In conjunction with experimental observation, we have evaluated constructs for known parameters including disorder, solubility, and aggregation propensity to directly align experimental results with available prediction systems. We composed a library of disordered constructs from known transcription activators and repressors. Specifically, we selected peptides from within the Auxin response factor (ARF) family of proteins in Arabidopsis thaliana to utilize a biologically characterized genome with natural sequence variation including percent glutamine content. The control used was κ‐casein, an inexpensive protein that has been previously shown to collapse and trap water. We optimized a general expression, purification, and labeling protocol to isolate the library of constructs with a solvent‐sensitive fluorophore. Our methods allow us to directly observe both solvent exposure and exchange on constructs of varying glutamine content. This work provides important new tools to measure and compare Q‐rich domains. These tools can be applied to clarify the relationship between sequence and behavior in Q‐rich domains, specifically the biophysical implications of peptide‐solvent interactions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering the PETase Enzyme to More Efficiently Break Down PET Plastics
Authors: Duplan Amanda , Hall Bonnie L.
Published: June 2021

Polyethylene terephthalate (PET), a thermoplastic polyester resin containing a high ratio of aromatic components, is widely used in plastic products. PET is unreactive and difficult to degrade, and thus overproduction and improper disposal methods have caused the world's oceans and ecosystems to be polluted with plastic waste. In 2016, Yoshida et al. discovered Ideonella sakaiensis, a bacterium able to use PET as a carbon source for growth. The enzyme PETase was found to break down PET plastics, but at a slow rate. Our goal is to engineer a PETase with a higher efficiency of PETase hydrolysis. The first step in the process is overexpression and purification of the PETase protein. Colorimetric and liquid chromatography assays will be optimized for determining the rate and efficiency of PET breakdown. PETase mutants will then be engineered to improve enzyme activity under the desired conditions. An optimized PETase enzyme capable of degrading plastics could be the first step to repairing our polluted ecosystems.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical Characterization of Aptenodytes forsteri Cytochrome P450 19 (Aromatase)
Authors: Zarate‐Perez Francisco , Velázquez‐Fernández Jesús B. , Jennings Gareth K. , Shock Lisa S. , Lyons Charles E. , Hackett John C.
Published: June 2021

Cytochrome P450 19 (aromatase) catalyzes the conversion of androgens to estrogens in a sequence of three reactions that each depend on NADPH and O2. As the terminal enzyme in estrogen biosynthesis, it is critical for the maintenance of myriad tissues and has emerged as a successful target for the treatment of estrogen‐dependent breast cancer. Aromatase is a phylogenetically‐ancient enzyme and its breadth of expression in other species has highlighted distinct physiological functions. In songbirds, estrogen production is required for programming of neural circuits controlling song. Aromatase also plays a role in sex determination of fish, reptiles, and amphibians. Biophysical characterization of the human enzyme has been limited by low yields from recombinant expression systems. Accordingly, we sought to identify an aromatase homolog with enhanced expression properties in the absence of stabilizing ligands. This work describes the expression, purification, and biophysical characterization of Aptenodytes forsteri (Emperor penguin) aromatase (afCYP19A1). Using human cytochrome P450 reductase as a redox partner, the enzyme kinetic parameters for turnover of androstenedione were like the human enzyme and mass spectrometry analysis conformed that afCYP19A1 likewise catalyzes the transformation through 19‐hydroxy‐ and 19‐oxoandrostenedione intermediates. Equilibrium binding studies revealed that androstenedione and anastrozole had the highest affinity for the enzyme and were closely followed by 19‐hydroxy‐androstenedione and testosterone. The affinity of 19‐oxoandrostenedione was an order‐of‐magnitude lower. Ligand‐binding kinetics were determined by stopped‐flow UV‐visible spectroscopy. Both androstenedione and testosterone where determined to bind to the enzyme in a step‐wise fashion with a spectroscopically‐detectable intermediate. In summary, these studies describe the first biophysical characterization of a non‐human aromatase that displays strikingly similar enzyme kinetics and ligand binding properties to the human enzyme despite bearing 75% sequence identity. In addition, they provide access to an alternative model to study the catalytic mechanism of the enzyme using methods requiring substantial amounts of ligand‐free enzyme.

Support or Funding Information

National Institutes of Health National Institute of General Medical Sciences [Grant R01GM114168]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Tumor Necrosis Factor Receptor Binders using Yeast Surface Display
Authors: Vunnam Nagamani , Szymonski Sophia , Sachs Jonathan N. , Hackel Benjamin
Published: June 2021

Tumor Necrosis Factor Receptors (TNFRs) are essential regulators of the immune system. Dysregulation of TNF receptors play a role in the pathology of many autoimmune diseases. Available treatments for TNF‐related diseases cause adverse side effects by unintentionally inhibiting ligand binding to off‐target receptors. In our previous work we showed an alternative therapeutic strategy for inhibition of the pro‐apoptotic activities of TNF receptors by specifically targeting receptor‐receptor interactions, without interrupting ligand binding. In the current study, we employed two small protein scaffold libraries, T7 phage gene 2 protein (Gp2) and affibody, to discover binders that specifically modulate the signaling activities of death receptor 5 (DR5) and TNFR1. Using yeast display and directed evolution, we will identify Gp2 and affibody variants with high binding affinity, stability and specificity to DR5 and TNFR1. These small protein scaffolds can be used as therapeutic agents in TNFR related diseases.

Support or Funding Information

NIH 1R01 EB023339‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of Chimera Prolyl Endopeptidases to Determine Role of Domains in Substrate Size Specificity
Authors: Alakija Femi , Clack Beatrice
Published: June 2021

Eurygaster integriceps Puton (commonly known as the Sunn pest) is a member of the family Scutelleridae. It is a major pest of wheat and is commonly found in Middle East, northern Africa, Russia, and Eastern Europe (Critchley, 1998). The Sunn pest secretes a proteolytic enzyme, a prolyl endopeptidase (spPEP) into the wheat grains with subsequent degradation of gluten (Darkoh et al., 2010), the major protein in wheat. The spPEP is a unique prolyl endopeptidase in the family of S9 serine proteases because it recognizes large proteins (gluten) as substrates ranging from 30kDa to greater than 140kDa. All other prolyl endopeptidases are purported to bind only peptides shorter than 30 amino acids in length. PEPs consist of two conserved domains, a hydrolase domain which includes the catalytic triad (Ser554, Asp641, and His680) and a seven bladed β‐propeller domain (Juhász et al., 2006). To understand the role with which the two domains have on substrate specificity, chimeras were constructed in which domains were swapped between the sunn pest prolyl endopeptidase (spPEP), mammalian prolyl endopeptidase (hPEP), and the bacteria prolyl endopeptidase (bPEP). Forward and reverse primers identical within the hinge region of either spPEP, hPEP, or bPEP connecting the two domains were used in polymerase chain reaction (PCR) to amplify the vector and the respective domain of each PEP. Ligation independent cloning (LIC) was used to clone the insert DNA into the LIC vector (pNYCOMPS‐LIC‐ccdB‐FH10T+(N‐term) expression vector). The product chimeras include spPEP hydrolase/hPEP β‐propeller, spPEP hydrolase/bPEP β‐propeller, hPEP hydrolase/spPEP β‐propeller, hPEP hydrolase/bPEP β‐propeller, bPEP hydrolase/spPEP β‐propeller, and bPEP hydrolase/hPEP β‐propeller. Each of the chimeras were expressed in BL21 (DE3) pTFe. The cell lysates were used to determine the enzyme activity for each of the chimera using Gly‐Pro‐p‐nitroanalide (GPpNA) as substrate.

Support or Funding Information

Office of Research and Sponsored Programs, Stephen F. Austin State University, and Ed & Gowen Cole, Nacogdoches. Texas.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rational Design of pH Sensitive MS2 Virus‐Like Particles for Drug Delivery Applications
Authors: Alvarez‐Benedicto Ester , Hartman Emily , Francis Matthew B.
Published: June 2021

Virus‐like particles (VLPs) have emerged as promising drug delivery and imaging scaffolds. VLPs made from the MS2 Bacteriophage Coat Protein (CP) enter cells via receptor‐mediated endocytosis and are believed to accumulate in the endosomal pathway. This pathway involves a decrease in pH levels that can potentially trigger the disassembly of acid sensitive VLPs, leading to cargo release. However, MS2 CPWT VLPs are highly stable in the endosomal pH range (4.5–5.5), which could slow endosomal release of cargo within the cell. Here, we set out to identify an acid‐sensitive variant of the MS2 CP that could lead to faster cargo release in the acidic environment of the endosome. We identified a previously‐unknown double mutant, CPT71H/E76C, that exhibits significant acid sensitivity in vitro at the endosomal pH range, in contrast to MS2 CPWT. The population of CPT71H/E76C VLPs was measured after a 4 h incubation at 37°C in a pH range from 1.0 to 7.3 with High Performance Liquid Chromatography using a Size Exclusion Chromatography column (HPLC‐SEC). Acid screens showed CPT71H/E76C to have 50% less well‐formed VLP at pH 4.9. These mutations alter a key hydrogen bond interaction, which could be the reason for increased acid sensitivity. To explore this hypothesis, capsids were alkylated with N‐ethylmaleimide, a moiety that could restore the hydrogen bond between the residues, and screen for acid sensitivity. Results showed modified capsids had an acid sensitivity similar to CPT71H, suggesting the hydrogen bond is involved in this property. Acid sensitive CPT71H/E76C could potentially improve the delivery of drugs to cancer cells.

Support or Funding Information

NIH‐ MARC (5T34GM007821‐38) and the Amgen Scholars 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.
Utilization of multiple lipid sources in the production of biodiesel by using lipase nanoparticles from Candida rugosa in a nonaqueous system
Authors: Rivera Hector A. , O'Neill Crystal A. , Sharma Rohit K. , Thapa Bibek , Bovea Vanessa B. , Griebenow Kai
Published: June 2021

One of the major challenges for the sustainable fatty acid methyl or ethyl ester (FAME/EE, a.k.a biodiesel) production is the smart utilization of low‐cost feedstocks, e.g., waste cooking oil (WCO), brown grease, or algal lipids. Although traditional catalyst systems could be used to transform such feedstocks into biodiesel, some well‐established drawbacks are associated with their use. Alternatively, lipases can convert the FFA‐rich feedstocks into FAME/EE in a manner with less environmental impact. However, low enzyme activity in organic solvents and high cost remain challenging for the industrial application of lipase nanoparticles. In this study, we are addressing these drawbacks of lipase by developing lipase nanoparticles (LNP), cross‐linked lipase nanoparticles (CLNP), and immobilized LNP on iron oxide nanoparticles (IONP). Lipase from Candida rugosa was selected as an economic widely available source and purified to ca. >90%. Different formulations of nanoparticles were formed, and detailed surface and morphological characterizations were performed by using the various microscopy techniques, e.g., scanning and transmission electron microscopy, and atomic force microscopy, dynamic‐light‐scattering, and magnetic properties by the vibrating‐sample magnetometer. For structural characterization, Fourier‐transform infrared spectroscopy and X‐ray diffraction were used. Identification of fatty acid esters was done by gas chromatography coupled to a mass spectrometer. The activity of the LNP, CLNP, and LNP‐IONP formulations increased 10‐, 20‐, and 32‐fold in n‐hexane, 18‐, 42‐, and 54‐fold in 1,4 dioxane, and 13‐, 29‐, and 43‐fold in THF, respectively, compared to free lipase. Detailed analysis of fatty acid ester formation was performed using the different combinations of substrates (WCO and brown grease, methanol and ethanol), lipase formulations (free lipase, LNP, CLNP, and LNP‐IONP), and solvent systems (hexane, 1,4 dioxane, THF, and t‐butanol). The nanoparticulate lipase formulations converted a high percentage of fatty acid esters into biodiesel in less time and the overall operational stability of the system improved considerably.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential modulation of microtubule stability by inhibitor‐bound Kinesin‐5
Authors: Kim Catherine , Kim Elizabeth , Liu Liqiong , Wojcik Edward
Published: June 2021

Kinesin‐5 forms a homotetramer that mediates the assembly of the bipolar mitotic spindle apparatus in many eukaryotes. Thought primarily to crosslink and slide antiparallel microtubules during spindle morphogenesis, this motor protein has been the target for the development of small molecule anti‐mitotic agents as potential cancer therapeutics. Most of the discovered effectors bind to a single site formed by loop‐5 wherein they mediate allosteric inhibition of the motor ATPase activity and motility. All of the loop‐5 inhibitors block ADP release and are therefore argued to all inhibit the motor in a cognate manner. Furthermore, at the single molecule level, the inhibitors have been shown to trap the monomeric motor domain in a weakly microtubule‐bound state that exhibits one‐dimensional diffusive movements along microtubules in vitro. Herein we show that, despite these similarities, different loop‐5 pocket inhibitors can elicit remarkably distinct impacts on the motor‐bound microtubule dynamics. We observe that monastrol‐bound human Kinesin‐5 motor domains can drive the net depolymerization of microtubules in vitro, while the STLC‐bound motors do not. Furthermore, while monastrol and STLC both induce metaphase arrest with mono‐polar spindles in cultured cells, monastrol also causes a significant decrease in microtubule density in these spindles. We conclude that the monastrol‐inhibited motor appears to drive microtubule depolymerization in vitro and in vivo. Therefore, loop‐5 pocket inhibitors of human Kinesin‐5 are not all synonymous, and can modulate a range of microtubule interaction which likely will impact their clinical efficacy.

Support or Funding Information

This work was funded by grants from the National Institutes of Health (R01GM097350) and the School of Graduate Studies at LSU Health Sciences Center – New Orleans.

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 Ramachandran Outlier Prevalence for X‐ray Crystallographic Model Structure Validation
Authors: Vavrinak David A. , Weiss Charles
Published: June 2021

There are numerous errors in published crystal structures, and the prevalence of Ramachandran outliers can be a valuable tool in identifying these errors. However, it is necessary to know which outliers are valid in a PDB file and which ones result from errors in the X‐ray crystallographic model structure. To address this problem, the Python language and NumPy library were used to compile data on approximately eight thousand high‐quality X‐ray crystal structures of proteins published in a public database to extract relevant data to provide insight concerning the probability and validity of a Ramachandran outlier assignments. Ramachandran analyses were performed by Phenix using the Phenix/Python application programming interface. The data collected include each residue's identity, Ramachandran outlier evaluations, phi and psi angles, outlier score and type, and information regarding the location of the residue in the protein. The above information along with metadata about the protein crystal structures are stored and maintained in an HDF5 file. The results of the data analysis using machine learning and other statistical methods will be presented.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nano‐biomaterial and functional genomics research
Authors: Mydin Rabiatul Basria S. M. N.
Published: June 2021

Nanotechnology has become a foremost research interest, which provides an opportunity to explore functional devices at nanometer regime and make it possible to design biomaterials compatible with nanoscale topography. The nanometric scaled topography of biomaterial plays a critical role in cellular stability and cell survival. This nanostructure surface has been shown to modulate diverse cellular responses of cell adhesion, migration, proliferation and differentiation. As for medical implant surface technology, this nano architecture structures may be beneficial for nano‐mechanical‐adaptation response which activates gene regulation involved in cytoskeleton remodelling of extracellular matrix alterations, plasma membrane modulations, locomotory behaviour, intracellular signalling response for cell metabolism in mechanosensitivity activities. On the other hand, for therapeutic research the present cell‐nano‐biomaterial study has provided profound understanding in regulation of cellular survival and cell growth signals, activation of DNA damage and DNA repair mechanisms and activation of redox regulator pathways. More research should be conducted to explore nanomaterial–nanomolecular interactions by performing SNP and DNA methylation studies as this knowledge is important in explaining the gene regulation and self–renewal potential of nano‐biomaterial interaction in the cell systems. Furthermore, comprehensive study using confocal laser scanning microscopy coupled with specific markers are needed for the understanding of cell migration on nano‐biomaterial surface especially on the cytoplasmic spreading behaviour involving filopodia and lamellipodia. Likewise, the metabolomics and bio‐kinetics of nanomaterial studies are also examples of important research areas to be explored. The valuable antioxidant properties of some nano‐biomaterials have created research gaps in product life cycle, circulatory system, half‐life of the antioxidant properties and other possible chemical reactions from the nanomaterials. Knowledge of these aspects will create opportunities for better understanding which may help researchers to develop better nano‐biomaterials products to be used in medicine and health‐line services.

Support or Funding Information

The author are thankful to the Ministry of Education (MOE) Malaysia for funding this work under Transdisciplinary Research Grant Scheme (TRGS) grant no. 6769003 and Universiti Sains Malaysia (USM) for USM‐Short Term Research Grant (304/CIPPT/6315073).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
HE4 Promotes Events Associated with Metastatic Ovarian Cancer Via Regulation of the Extracellular Matrix
Authors: Gaudet Hilary , Ribeiro Jennifer , Khan Mehreen , Schorl Christoph , James Nicole , Oliver Matthew , Singh Rakesh , DiSilvestro Paul , Moore Richard , Yano Naohiro
Published: June 2021

Human epididymis protein 4 (HE4) has diagnostic and prognostic abilities for epithelial ovarian cancer. The Risk of Malignancy Algorithm (ROMA) uses HE4 serum levels in combination with other factors to detect and monitor ovarian cancer. Several studies have demonstrated a role for HE4 in invasion and migration. Here, we further investigated the role of HE4 in events associated with metastatic ovarian cancer. Treatment of OVCAR8 ovarian cancer cells with recombinant HE4 (rHE4) promoted invasion, haptotaxis toward a fibronectin substrate, and adhesion onto fibronectin. In addition, overexpression of HE4 or treatment with rHE4 promoted the upregulation of transcripts coding for extracellular matrix proteins, including LAMC2 and LAMB3. Deposition of laminin‐332, the heterotrimer comprising LAMC2 and LAMB3 proteins, was increased in OVCAR8 cells treated with rHE4 or conditioned media from HE4‐overexpressing cells. Enzymatic activity of matriptase, a serine protease that cleaves laminin‐332 and contributes to its pro‐migratory functional activity, was enhanced by rHE4 treatment. Focal adhesion kinase signaling was activated in OVCAR8 cells treated with conditioned media from HE4‐overexpressing cells and focal adhesions were increased in cells treated with rHE4 in the presence of fibronectin. These results indicate a role for HE4 in mediating events associated with metastatic ovarian cancer.

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