bluuebunny/crossref_metadata_2025 huggingface dataset:
Sox2 regulates palate development by promoting basal cell differentiation
Authors: Sweat Yan Yan , Yu Wenjie , Sun Zhao , Sweat Mason Eric , Eliason Steven L , Amendt Brad A
Published: June 2021

With a frequency of 1:500–2,500, cleft lip with or without cleft palate (CL/P) is the most common congenital craniofacial defect. In mice and humans, a complex gene regulatory network (GRN) controls the different processes required for palatogenesis. Understanding how transcription factors act as the modulators of gene expression and function in palatogenesis will lead to better genetic counseling and genetic therapies for CL/P. Our group has shown that conditionally inactivating Sox2, one of the earliest transcription factors required during embryogenesis, results in clefting of the palate and fusion of the tongue to the mandible (ankylglossia).

Objective

To examine the role of Sox2, a stem cell factor, in palatogenesis.

Methods

Using Pitx2cre, Sox2 was conditionally inactivated in the oral epithelial tissue of developing murine embryos. Palate development was studied during the stages of embryogenesis.

Results

The conditional ablation of Sox2 was sufficient to generate clefting in mice. Further examination revealed that Sox2 is expressed in the P63+ basal cell population of the oral epithelium as well as the periderm, which acts to prevent oral adhesions during development. Inactivating Sox2 resulted in defective periderm formation. The loss of an intact periderm resulted in intraoral adhesions that prevented complete palate shelf extension and ankylglossia.

Conclusions

Sox2 is required for periderm differentiation. Inactivating Sox2 results in cleft palate and ankylglossia.

Support or Funding Information

University of Iowa College of Dentistry

NIH, NIDCR

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EFTUD2, The Gene Responsible For Mandibulofacial Dysostosis With Microcephaly (MFDM), Is Required For Implantation And Craniofacial Development In Mouse
Authors: Beauchamp Marie‐Claude , Djedid Anissa , Daupin Kevin , Clokie Kayla , Majewski Jacek , Majewska Loydie Jerome
Published: June 2021

Haploinsufficiency of EFTUD2 is associated with MFDM (mandibulofacial dysostosis with microcephaly), but the etiology of this syndrome remains unknown. Our goal is to determine the tissue and temporal specific expression and requirement for Eftud2 during craniofacial development. We used RT‐PCR and in situ hybridization to examine expression of Eftud2 during embryogenesis. Using CRISPR/Cas9 we designed guide RNAs to generate mice with deletion (Eftud2 del) and conditional mutation of exon 2 of Eftud2 (Eftud2 flox). At embryonic day (E) 7.5 and 8.5 of mouse development, Eftud2 was highly expressed in both ectodermal and mesodermal components of the developing head and craniofacial region, by E9.5 Eftud2 was more broadly expressed, in the body wall and developing heart. Eftud2 del heterozygous mice and embryos were viable and fertile and showed a 38% and a 30% reduction of Eftud2 mRNA and protein expression, respectively. Before the onset of organogenesis, Eftud2 del heterozygous embryos had reduced number of somites when compared to their wild type litter mates, indicating a delay in development. Noticeably, RNA sequencing revealed that Eftud2 was the only transcript significantly affected in these heterozygous mice. Eftud2 del homozygous mutant embryos did not implant and failed to grow and hatch ex vivo. Next, we used the Wnt1‐Cre2 transgenic line to delete exon 2 of Eftud2 specifically in the neural crest cells. Eftud2 flox heterozygous embryos carrying the Wnt‐Cre2 transgene were normal. However, Eftud2 flox homozygous mutant embryos that also carry the Wnt1‐Cre2 transgene displayed hypoplasia of the midbrain and pharyngeal arches starting as early as E9.5. By E11.5, most of these embryos showed an open neural tube, all embryos showed exencephaly at E14.5. Cartilage preparations revealed an absence of cartilage in the head, reduced/or absence of Meckel's cartilage, and abnormal inner ear development. Crosses with the Rosa26R mice reporter line, revealed reduced neural crest cell migration into the pharyngeal region at E10.5. Since the mutation designed is predicted to generate a truncated protein with partial function, our data suggest that normal levels of Eftud2 is crucial during embryogenesis. Future studies are focused on determining the molecular and transcriptional basis of MFDM using this mouse model.

Support or Funding Information

Canadian Institutes of Health Research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Craniofacial Growth Studies and Craniofacial Growth
Authors: Sherwood Richard J , McNulty Kieran P , Valiathan Manish , Duren Dana L , Knigge Ryan P , Hardin Anna M , Oh Hee Soo , Leary Emily V
Published: June 2021

Current understandings of human craniofacial growth are based on multiple historic longitudinal growth studies. The extent to which regional variation in growth patterns is present among the various growth studies even within the United States, however, is not well understood. The aim of this study is to compare growth patterns across six of the nation's largest growth studies.

Lateral cephalographs were examined from the historical archives of the Denver Growth Study, Iowa Growth Study, and Oregon Growth Study (available via the AAOF Legacy Collection) as well as the Fels Longitudinal Study, the Michigan Growth Study, and the Bolton‐Brush Growth Study. A set of 44 two‐dimensional landmarks were collected on longitudinal series from participants 6–22 years old. A total of 5,622 cephalographs from 726 males and 5,514 cephalographs from 723 females were assessed. Fifth order polynomials were used to evaluate growth of 11 linear craniofacial measures for each growth study and by sex. Rates of growth and associated growth milestones were calculated from polynomial models and compared across study and by sex. In addition, geometric morphometric analyses were used to assess ontogenetic shape differences among studies using a regression of the shape variables on log centroid size, pooled by study, with subsequent evaluation of residual variation.

Growth curves for the separate studies roughly parallel each other yet show differences in the timing of growth milestones and adult morphology. In general, for each linear trait participants in the Denver study are smaller and participants in the Iowa and Michigan studies are larger than participants in other studies at the same chronological age. Timing of onset and peak growth is similar in all 11 craniofacial measures, although trait values at those growth milestones differ by study. Preliminary results from the geometric morphometric analysis identify a common ontogenetic pattern shared across studies but demonstrate that, for a given size, individuals from the Denver study are more advanced along the common shape trajectory relative to other studies. Although ontogenetic trajectories are roughly similar, overall shape differences are present between study populations.

Variations in growth and overall morphology among these six longitudinal growth study populations demonstrate regional differences in the rate and timing of growth that may be attributable to the ancestry and environmental conditions of study participants. Further study of the mechanisms for these differences in growth will improve the accuracy of craniofacial growth predictions, thereby improving the efficacy of growth modification interventions in pediatric craniofacial treatment.

Support or Funding Information

Funding: NIH/NIDCR R03‐DE021435; NIH/NIDCR R01 DE024732

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SPECC1L‐Deficient Cells Show Impaired Collective Cell Migration Attributes that are Rescued by Upregulation of PI3K‐AKT Pathway
Authors: Saadi Irfan , Goering Jeremy , Isai Dona Greta , Hall Everett , Wilson Nathan , Kosa Edina , Wenger Luke , Umar Zaid , Yousaf Abdul Rehman , Czirok Andras
Published: June 2021

Clefts of the lip and/or palate (CL/P) are common anomalies that occur in 1/700 live births. Pathogenic SPECC1L variants identified in patients with rare atypical clefts and syndromic CL/P suggest the gene plays a primary role in face and palate development. Null mutants are lethal at embryonic day 9.5 with defective neural tube closure and cranial neural crest cell delamination. Hypomorphic Specc1lΔ200 mutants die perinatally, but do not show cleft palate. Specc1lnull/Δ200 compound mutants resulted in transient oral adhesions, subepidermal blebbing, and a delay in palate elevation. Palate development requires extensive mesenchymal remodeling especially during palatal shelf elevation. We posit that this remodeling involves collective cell migration (CCM) of neural crest derived palate mesenchymal cells. Live time‐lapse microscopy was performed to visualize in vitro wound‐repair assays with SPECC1L‐deficient U2OS osteosarcoma and primary mouse embryo palatal mesenchyme (MEPM) cells. SPECC1L‐deficient cells consistently showed delayed wound closure. Directional movement of neighboring cells is a hallmark of CCM. In wound‐repair assays, an increase in movement parallel to the direction of wound front propagation results can be measured as an attribute of CCM. Tracking of individual trajectories of SPECC1L deficient cells during wound‐repair indeed shows an increase in cell movement perpendicular to the direction of wound closure. This diminished directionality causes delayed wound closure even though cells are adequately motile. We previously demonstrated SPECC1L to be a novel regulator of PI3K‐AKT signaling, with in vitro and in vivo SPECC1L deficiency exhibiting reduced PI3K‐AKT signaling. Indeed, wound closure delay, and the underlying CCM defect, in SPECC1L‐deficient U2OS and MEPM cells can be rescued by activation of the PI3K‐AKT signaling pathway using 740 Y‐P small molecule activator. Our data are the first to show CCM attributes in MEPM cells, and propose that MEPM cells can be used to model mesenchymal remodeling during palatal shelf elevation. We also show a novel role for SPECC1L in CCM through modulation of P13K‐AKT signaling.

Support or Funding Information

This project was supported in part by the National Institutes of Health grants DE026172 (I.S.). Core support and pilot funding was also provided by National Institutes of Health Center of Biomedical Research Excellence (COBRE) grant (P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence (INBRE) grant (P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (IDDRC) grant (P30 HD 002528).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Validating and Optimizing A Combination of INTACT and FACS Techniques for the Isolation of Mouse Astrocyte Nuclei Upon Ablation of the Atrx Intellectual Disability Gene
Authors: Jiang Yuxuan , Pena Ortiz Miguel A. , Jiang Yan , Bérubéé Nathalie G
Published: June 2021

The ATRX gene codes for the ATRX chromatin remodelling protein, which interacts with DAXX protein to deposit histone variant H3.3 at telomeres and pericentromeric heterochromatin. Mutations of the ATRX gene cause intellectual disability. One noticeable example is the ATRX syndrome, which is characterized by distinctive craniofacial features, severe developmental delays, intellectual disability, and mild‐to‐moderate anemia. It is therefore apparent that the ATRX gene likely plays an important role in the central nervous system (CNS). An astrocyte is a cell type that, by the most conservative survey, has the same abundance as neurons in the human brain. However, unlike neurons, astrocyte function is far less understood, and its role other than a supporting function was not known until a decade ago, when astrocytes were discovered to actively participate in higher neuronal processing through the tripartite synapse. The specific role of ATRX in astrocytes has never been reported before. A challenge of elucidating ATRX function in astrocyte is a lack of an optimized technique to isolate a reasonable number of highly enriched Atrx‐ablated astrocyte nuclei for various sequencing purposes, including ChiP‐seq and RNA‐seq. This project aims to fulfill such research demand by validating and optimizing the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) technique with mouse brain tissue at postnatal day 30. By adjusting various experimental conditions such as tissue homogenization time, the concentration of octylphenoxy poly(ethyleneoxy)ethanol (IGEPAL) in buffer solutions, the dilution ratio of the tissue homogenate and the centrifugation time, a total nuclei yield of 90% was obtained. The structural integrity of nuclei was verified by laser microscopy with nuclei sample stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). However, subsequent enrichment of GFP‐tagged AtrxF/y; Cre+/−;Sun1‐GFP+/− nuclei using anti‐G‐protein coated magnetic bead, as per the original INTACT protocol, is plagued with non‐specific binding of less than 32% specificity. To improve the enrichment factor, a preliminary investigation was performed to validate the use of Florescent‐Assisted Cell Sorting (FACS) to enrich Sun1GFP‐tagged nuclei from the previously‐produced total nuclei preparation. A 92% enrichment of GFP‐tagged nuclei was obtained, demonstrating the promising potential of a combined INTACT‐FACS technique to effectively isolate structurally‐sound nuclei from transcriptionally‐sensitive cell types, within a complex and intermingled tissue environment.

Support or Funding Information

This work was supported by a Dean's Undergraduate Research Opportunity Program award to Yuxuan Jiang and the Canadian Institutes for Health Research MOP#142369.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Co‐expression Network Analysis of Altered lncRNAs and mRNAs in Diabetic Cardiomyopathy using Human iPSC‐derived Cardiomyocytes
Authors: DHANASEKARAN ANURADHA , PANT TARUN , MISHRA MANOJ , BAI XIAOWEN , GE ZHI‐DONG , BOSNJAK ZELJKO
Published: June 2021

Introduction

Mounting evidence in the past have substantiated the role of long‐non coding RNAs (lncRNAs) in diabetic cardiomyopathy (DCM). However, the pathogenesis of DCM remains unknown.

Aim

This study aimed to investigate the effect of the pro‐diabetic stimulus on the profile of lncRNAs and mRNAs, and the underlying co‐expression signaling network associated with DCM disease pathogenesis.

Results

Out of 40,173 lncRNAs and 20,730 mRNAs being screened, 1582 mRNAs and 2046 lncRNAs were differentially regulated (fold change>2.0, P<0.05) between DCM and control group of which more than half were intergenic and antisense lncRNAs. The subgroup analysis revealed more than 411 lncRNAs being co‐expressed with neighboring genes. To further investigate the potential regulatory role of these lncRNAs in gene regulation and the pathogenesis of DCM we constructed lncRNA‐mRNA co‐expression network which showed overall 48,155 significant co‐expression network connections. Moreover, in the DCM derived network, we had a comparatively greater number of nodes and connections than that in the control group. Majority of the dysregulated mRNAs of DCM co‐expression network were significantly enriched for the GO terms including actomyosin structure organization (GO:0031032), muscle contraction (GO:0006936), membrane raft assembly (GO:0001765), drinking behavior (GO:0042756), Focal adhesion (GO:0005925), skeletal muscle tissue development (GO:0007519), and cell‐matrix adhesion (GO:0007160) which have been associated with DCM in the past.

Conclusion

This is the first study to investigate the genome‐wide lncRNA expression profile and co‐expression patterns with mRNA in DCM using microarray technology. These results provide comprehensive data about the pathways and regulatory mechanisms associated with DCM and indicate that lncRNAs may play a crucial role in DCM.

Support or Funding Information

This work was supported, in part, by a National Institutes of Health research grant P01GM 066730 (to Dr. Bosnjak) from the United States Public Health Services, Bethesda, Maryland, USA & DST‐UKEIRI DST/INT/UK/P‐50/2012 (to Dr. Anuradha Dhanasekaran) from the Department of Science and Technology, New Delhi, India.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of RpoS by RbsD in Escherichia coli
Authors: Peterson Celeste , Dorlean Shaina , Parsons Dylan , Tran Thomas , Dey Arup
Published: June 2021

In stressful environments, RpoS a stationary phase regulator, accumulates in E. coli and induces a state of dormancy. A screen for regulators of RpoS was carried out with an overexpression DNA library and an RpoS’‐’LacZ fusion. Overexpression of the rbsD gene, which codes for a ribose pyranase necessary for efficient metabolism of ribose, was found to reduce RpoS levels. We next used a series of LacZ fusions and RT‐qPCR to determine how rbsD affects RpoS. We found that rpoS mRNA levels were modestly lowered in the rbsD overexpressed strain. However, a much larger effect was seen in the post‐transcriptional level as determined by an RpoS’‐’LacZ protein fusion. Furthermore the effect of overexpression of rbsD was diminished in a fusion that did not have the RpoS untranslated hairpin loop. In summary overexpression of rbsD resulted in decreased RpoS translation in a manner that depended on the RpoS hairpin loop.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long noncoding RNAs in immune response and inflammation
Authors: Mandal Subhrangsu S , Obaid Monira
Published: June 2021

The mammalian immune system orchestrates innate and adaptive immune responses that are complex biochemical processes regulated by various protein and lipid mediators such as cytokines, chemokines, hormones, growth factors, and others. The emerging evidences suggest that non‐coding RNAs (ncRNAs) play vital roles in regulation of immune responses. In a recent study, we discovered that lncRNA HOTAIR plays critical roles in immune response and inflammation. HOTAIR expression is induced in immune cells (macrophages) upon treatment with lipopolysaccharide (LPS) or peptidoglycan, which are components of bacterial cell membrane. HOTAIR is required for LPS‐induced expression of cytokines and pro‐inflammatory genes. HOTAIR regulates activation of NF‐κB and its target gene (IL‐6 and iNOS) expression via facilitating the degradation of IκBα. Thus, HOTAIR appear to be a critical player in NF‐κB activation in macrophages suggesting its potential functions in inflammatory and immune response. Beyond HOTAIR, we also performed RNA‐Seq analysis on macrophages and identified novel lncRNA associated with immune response and inflammation. Overall, lncRNA appear to be play critical roles in immune response and inflammation and are misregulated in inflammatory disease.

Support or Funding Information

National Institute of Health‐ 1R15HL142032

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural polymorphism in actin filaments modulates gelsolin binding
Authors: Lee Myeongsang , Kang Hyeran
Published: June 2021

The conformational states of actin filaments determine their biological functions and numerous actin binding proteins tightly control filament structure. Gelsolin is an actin regulatory protein that remodels actin cytoskeleton by severing and capping the barbed‐end of an actin filament. A recent study demonstrates that actin filaments exist in multiple conformations with varying tilted states, suggesting structural polymorphism in the filament. Yet, it is unknown how polymorphic nature of actin filament structure affects gelsolin binding. Here we investigated the interactions between the segment 1 domain of gelsolin and filaments with distinct structural states identified in the recent experimental study, utilizing molecular docking and the explicit molecular dynamics (MD) simulations. Our simulation results show that the filaments with different tilted states influence the binding modes of gelsolin segment‐1 domain. Analysis on buried area and the number of hydrogen bonds in the actin‐binding interface indicate that distinct filament structure results in different binding affinities of gelsolin. Distributions of salt‐bridge interactions between actin subunit and gelsolin may explain molecular basis of the filament structure‐dependent gelsolin binding affinities. This study suggests that structural polymorphism in actin filaments may play an important role in modulating interactions between actin filaments and binding proteins.

Support or Funding Information

This study was supported by the UCF start‐up fund and In‐House grant for Hyeran Kang. We acknowledge the computational time from UCF stokes cluster.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Yeast Polygalacturonase Activity is Resistant to Extreme Temperature and pH
Authors: Caspers Mary Lou
Published: June 2021

Polygalacturonase cleaves the α1–4 galactosidic bonds of polygalacturonic acid (PG), a major component of fruit mucilage, to produce galacturonic acid. A unit of polygalacturonase activity is defined as the number of mmol of galacturonic acid produced per min at pH 5.0. Polygalacturonase is stable to submersion in a boiling water bath for 1 hour or 10 minutes at 200 °C. When the polygalacturonase assay is conducted at a pH 3 or 14, no loss of activity is noted. Addition of 0.3 M β‐mercaptoethanol abolishes polygalacturonase activity and so this concentration of β‐mercaptoethanol is used as an assay control. Dithiothreitol is able to reduce polygalacturonase activity in a dose‐dependent manner. A 38 % reduction in enzyme activity is seen at 81 mM dithiothreitol and this increases to 95 % inhibition at 162 mM dithiothreitol. Tris(2‐carboxyethyl)phosphine at 50 mM completely inhibits polygalacturonase activity. β‐Mercaptoethanol, dithiotheitol and tris(2‐carboxyethyl)phosphine all reduce the disulfide bonds formed between the side chains of cysteine residues. This suggests that the resistance of polygalacturonase to denaturation by high temperature and acid or base is due to the stabilization of its tertiary structure by disulfide bonds. (I gratefully acknowledge the work of Melissa L. Zuteck, Edwin A. Gay, Joshua Henning, Ayah Bakri, Bianca Yaldo and Glenn R. Oppenlander who assisted in this work while undergraduates.)

Support or Funding Information

I thank the Department of Chemistry and Biochemistry, University of Detroit Mercy for support of this project

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Initial characterization of DszA for the high throughput validation of rationally designed DszC mutants
Authors: Sanchez Misael , Truong Sharon , Vey Jessica
Published: June 2021

Sulfur dioxide production through fossil fuel combustion is a negative contributor to atmospheric pollution and human health. Currently, the preferred method for the removal of sulfur containing compounds from fossil fuels is though hydrodesulfurization. This process however, continues to be costly and inefficient in targeting certain common aromatic organosulfur contaminants like dibenzothiophene (DBT). Fortunately, the biodesulfurization (BDS) pathways in R. erythropolis and other bacterial species have shown promise in the removal of sulfur from such organosulfur compounds. However, improvements are still necessary for this pathway to become competitive and industrially applicable. Our current laboratory objectives are to rationally design the BDS pathway in R. erythropolis to decrease substrate specificity, starting with the first enzyme in the pathway DszC. To facilitate high‐throughput validation of our rationally designed DszC mutants, we will generate a strain of E. coli that harbors the three downstream biodesulfurization genes (DszA, DszB, and DszD). Here, we present our preliminary overexpression and biochemical characterization of the other enzymes in the BDS pathway, focusing on DszA, a flavin‐dependent monooxygenase. DszA crystals have also been produced and are being optimized for co‐crystallization with dibenzothiophene sulfone (DBTO2) substrate to accurately identify the substrate active site.

Support or Funding Information

Research Initiative for Science Enhancement (RISE), National Institute of Health (NIH), California State University Northridge

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insights into gasdermin pore formation from the structure of a pre‐pore
Authors: Xia Shiyu , Ruan Jianbin , Fu Tian‐Min , Wu Hao
Published: June 2021

Downstream of inflammasome activation, gasdermin D (GSDMD) mediates IL‐1β secretion and pyroptosis by forming membrane pores. Structural investigation into GSDMD pore formation will provide new mechanistic knowledge about the GSDM family and facilitate the treatment of GSDMD‐associated diseases. We hypothesize a dynamic model where upon caspase cleavage of autoinhibited GSDMD, GSDMD‐NTs dissociate from GSDMD‐CTs for lipid binding and oligomerization into a pre‐pore, followed by β‐barrel insertion to form a membrane pore. In light of our recently resolved structure of the mouse GSDMA3 membrane pore, we aim to determine the structure of a GSDM pre‐pore. We arrest the pore formation process at the pre‐pore stage by generating a GSDMA3 mutant defective in membrane insertion while functional in lipid binding and oligomerization. Cryo‐electron microscopy (Cryo‐EM) analysis reveals a pre‐pore conformation where GSDMA3‐NT subunits oligomerize into a soluble ring without extended transmembrane β strands.

Support or Funding Information

This work was supported by US NIH grant DP1HD087988 (H.W.), R01Al124491 (H.W.), and Charles A. King Trust Postdoctoral Fellowship Program (J.R.). We thank the University of Massachusetts Cryo‐EM Core Facility and NCI National Cryo‐EM Facility for data collection.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Intermolecular Interactions of a Cataract‐Causing Protein: γB Crystallin
Authors: Faraone Julia , Bushunow Symeon , Fadden Aaron , Mills Jeffrey L , Thurston George , Michel Lea Vacca
Published: June 2021

Gamma crystallin proteins can be found in the eye lens, helping to maintain transparency through their intermolecular interactions. Because crystallins remain in the lens after their initial synthesis, mutations caused by prolonged exposure to environmental stresses can have permanent consequences, including aggregation and liquid‐liquid phase separation of the crystallins. These structural deviations can cause clouding of the lens, a condition known as cataracts. With the use of light scattering and nuclear magnetic resonance (NMR) spectroscopy, we aim to study the detailed interactions between crystallins. We have expressed and isolated recombinant bovine Gamma B crystallin protein in Escherichia coli and performed T1/T2 NMR experiments to measure the longitudinal and transverse relaxation times of the protein. These experiments allowed us to analyze the crystallins in terms of the attractive forces between molecules and how those forces change with the manipulation of environmental conditions (temperature, pH). Analysis of our current data suggests higher rates of intermolecular interaction at higher concentrations of crystallins and lower temperatures.

Support or Funding Information

RIT and NIH Grant EY018249

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the Mechanism of Hemolytic Function and Type Vb Secretion by a Gram‐Negative Pathogen
Authors: Wade Gina , Grilley Dan , Weaver Todd
Published: June 2021

Proteus mirabilis is a pathogenic opportunistic Gram‐negative bacterium that produces and secretes a toxic protein called hemolysin A (HpmA). P. mirabilis utilizes a type Vb, or two‐partner (TPS), secretion system to simultaneously transport and activate HpmA across its outer membrane and release it into the blood of a host where it destroys red blood cells (hemolysis). Like all other TPS proteins, HpmA contains an N‐terminal TPS domain (≈300 residues) essential for secretion and a separate C‐terminal functional domain (>1200 residues) responsible for virulent functions. Our goals are to determine how secretion of HpmA is prompted by its folding and identify regions of HpmA critical to its hemolytic function. In the absence of secretion, the unfolded full‐length HpmA requires a folded TPS domain to act as a template protein to achieve its hemolytically active form. We term these Template Assisted Hemolytic Activity (TAHA) assays. Performing TAHA assays with genetically altered HpmA, we have quantitated the importance of different regions on HpmA folding and function. We have determined the borders of the hemolytic functional domain, marking the first identification of a functional region on a TPS hemolysin. Other hemolytic proteins are known to adopt a modular method of function. To determine if the function of HpmA is dictated by similar modules, we have created small deletions and amino acid randomizations within the hemolytic functional region. Separately, by rationally designing deletions and variants within the TPS domain of full‐length HpmA, we have determined that the folded template recognizes specific sequences within the unfolded TPS domain to initiate folding. This supports our N‐terminal first secretion model and suggests a continuity of structure and linkage of folding between the TPS domain and the remainder of HpmA. A continuous β‐helical structure starting from the TPS domain could explain this link between domains of secretion and function. To test this link and characterize formation of secondary structure during templated folding, we have performed structural studies using CD spectroscopy. By identifying regions necessary for hemolysis and characterizing how TPS domain folding is required to induce hemolytic activation, our results have expanded our working model for HpmA secretion and function.

Support or Funding Information

This research was supported by an Undergraduate Research and Creativity grant and a Dean's Distinguished Summer Fellowship to GW.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unraveling the Biosynthesis of the Essential Lipoyl Cofactor in Staphylococcus aureus
Authors: Dill Zerick Robert , McCarthy Erin L , Booker Squire J
Published: June 2021

Staphylococcus aureus (S. aureus) is the leading cause of infections acquired in a hospital setting. In recent years, the rise and spread of methicillin‐resistant S. aureus (MRSA) that has evolved resistance to antibiotics previously successful at eradicating the infection has become a major global health concern. Thus, the search for novel antibiotic targets has been the focus of many research efforts. One potential new target is the biosynthesis of the lipoyl cofactor, which plays an essential role in the oxidative decarboxylation of various α‐keto acids and breakdown of glycine. The second step of the de novo pathway for the biosynthesis of the lipoyl cofactor, the attachment of two sulfur atoms to C6 and C8 of an n‐octanoyl chain connected to a target lysyl residue on a lipoyl carrier protein, is catalyzed by lipoyl synthase (LipA), a member of the radical S‐adenosylmethionine (SAM) superfamily. In addition to its [4Fe–4S] radical SAM cluster, LipA contains a second [4Fe–4S] ‘auxiliary cluster’ which is sacrificed during catalysis to supply the requisite sulfur atoms. The destruction of its auxiliary cluster renders LipA inactive in the absence of a system to restore it. Further studies have identified that Escherichia coli (E. coli) has an additional protein, NfuA, an iron‐sulfur cluster carrier protein, that can target LipA and regenerate its auxiliary cluster to reactivate it for catalysis. However, while E. coli LipA has been extensively studied, the S. aureus LipA homolog has not been fully characterized. Interestingly, in vitro studies have shown that the activity of S. aureus LipA is unaffected by S. aureus Nfu alone, indicating that a second factor may be involved. Recent in vivo studies have reported that an S. aureus protein, SufT, is involved in iron‐sulfur cluster assembly and is implicated in lipoic acid biosynthesis; however, the role of SufT is unknown. We will, therefore, use a combination of genetic, biochemical, and spectroscopic approaches to functionally characterize Staphylococcus aureus LipA and understand the potential roles of Staphylococcus aureus Nfu and SufT in the regeneration of LipA's auxiliary cluster. Ultimately, this information could be used to design antibiotics that target this pathway and obstruct the essential biosynthesis of lipoic acid, inducing death to pathogenic bacteria.

Support or Funding Information

I acknowledge support from the HHMI EXROP program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulating Activity of ABC Transporters in Blood Brain Barrier Cells in Culture
Authors: Holcomb‐Webb Kasi L. , Nanayakkara Amila K. , Aljowni Maha , Lippert Alexander R. , Vogel Pia D. , Wise John G.
Published: June 2021

The blood‐brain barrier (BBB) is a physical and dynamic barrier that separates the central nervous system from the circulatory system in order to protect the brain from harmful endogenous and exogenous chemicals. The capillary endothelial cells that form the BBB are firmly sealed via tight junctions and express relatively high amounts of ATP‐binding cassette efflux transporters like P‐glycoprotein (P‐gp) and the breast cancer resistant protein (BCRP) to protect the brain from entry of potentially harmful chemicals. While protective to the brain under normal circumstances, this high level of expression of these proteins at the BBB are problematic when attempting to treat diseases of the brain like neurodegenerative diseases or brain cancers since P‐gp and BCRP impede entry of therapeutic drugs into the brain. Previously, we have computationally identified several drug‐like molecules that inhibit or modulate P‐gp and/or BCRP activities. The cell line, hCMEC/d3, was used in our studies as a model for the BBB. Using qPCR, we confirmed a higher level of expression of both P‐gp and BCRP in hCMEC/d3 cells when compared to non‐cancerous HFL‐1 cells. When hCMEC/d3 cells were treated with the P‐gp and BCRP inhibitors, accumulation of the P‐gp substrate rhodamine‐123 as well as the P‐gp and BCRP substrates, mitoxantrone and daunorubicin, was observed. We conclude that our newly discovered P‐gp and BCRP inhibitors may make good leads for the development of co‐therapeutics to inhibit and/or modulate the activity of P‐gp and BCRP in the BBB and facilitate therapeutic drug uptake through the BBB and into the brain.

Support or Funding Information

This work was supported by NIH NIGMS [R15GM094771‐02] to PVD and JGW, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas as well as the Hamilton Undergraduate Research Scholars Program, the SMU Summer Research and Undergraduate Research Assistantship Programs as well as the SMU Engaged Learning Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pitavastatin Selectively Kills PTEN Knock Out Cells and Cancer Organoids in Mouse Model via the Mevalonate Pathway
Authors: Jiao Zhihua , Cai Huaqing , Long Yu , Sirka Orit Katarina , Ewald Andrew , Devreotes Peter N
Published: June 2021

PTEN is one of the most frequently mutated tumor suppressors in human cancer. Loss of PTEN leads to hyperactivation of the PI3K–AKT signaling pathway, cell migration, and cell proliferation. Aiming to identify specific drugs that could target cancer cells, we screened 2650 FDA approved compounds and discovered that certain statins selectively kill Dictyostelium pten‐cells. This result carries over to mammalian cells: MCF10A cells lacking PTEN are more sensitive to statins than MCF10A wild type (WT) cells. By testing the differential sensitivity between MCF10A WT and pten−/− cells with multiple statins, we found that pitavastatin showed the best performance. Mevalonic acid and geranylgeranyl pyrophosphate (GGPP), but not farnesyl phyrophosphate or squalene, rescued pitavastatin treated MCF10A pten−/− cells. Pitavastatin induced an increase in RhoA expression suggesting that the requirement for geranylgeranylation of this GTPase may mediate the sensitivity of MCF10A pten−/− cells. The different sensitivity between MCF10A WT and pten−/− cells also carried over to three‐dimensional culture model (3D). Moreover, we found pitavastatin significantly decreased the growth and Twist1‐induced cell dissemination in 3D culture of murine mammary gland organoids. Finally, we tested the effects of pitavastatin on growth and survival of WT(FVB) and tumor organoids (MMTV‐PyMT (luminal breast cancer model), C31Tag (basal breast cancer model)) in 3D culture. Both MMTV‐PyMT and C31Tag were killed by 100 nM pitavastatin, while WT organoids survived. These findings implicate pitavastatin as a potential therapeutic candidate for tumors.

Support or Funding Information

This work was supported by NIH Grant R35 GM118177.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical evaluation of drug inhibition of human MDR1 P‐glycoprotein
Authors: Oliveira Maisa C. , Okwuone Dakota D.D. , Farokhnia Roxana , Jensen Madeline K. , Edgar Jennifer M. , Wise John G. , Vogel Pia D.
Published: June 2021

P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer. To search for P‐gp inhibitors as co‐therapeutics to combat MDR, we had previously used high‐throughput in silico ligand docking studies to identify drug‐like compounds and their effects on P‐gp ATPase hydrolysis activity, followed by assays of MDR‐reversal of a prostate and ovarian cancer cell lines. More recently, additional computationally identified compounds were shown to reverse MDR in cancer cells. Biochemical assays using purified P‐gp provide important information regarding the mechanism of inhibition and have been used as primary screens in our drug finding process. Some of these newly found compounds were observed to be inhibitors of ATP hydrolysis activity of a cysteine‐less mouse MDR3 homolog of the human MDR1 P‐glycoprotein. Even though human and mouse P‐gp share about 87% identity in amino acid sequence, there may still be sufficient differences between the two proteins that may prevent MDR3 from faithfully reporting on inhibitor action on the human protein. High expression of human P‐gp in Pichia pastoris in a biologically active form was previously reported and the purified P‐gp was used on our recent assays. Lipid activation of ABC transporters, as shown by others, is a requirement to achieve optimal enzyme activity, but in our hands has proven to be more challenging than expected. The best P‐gp activation was achieved using L‐α‐phosphatidyl choline. The resulting lipid‐activated P‐gp showed low basal activity (~8.5 nmol NADH/min/mg P‐gp), but more than 30‐fold activation by the P‐gp transport substrate, verapamil (~306 nmol NADH/min/mg P‐gp). Here we report our optimized strategy to lipid‐activate wild‐type human P‐gp as well as the evaluation of the potential of our previously discovered drug‐like compounds to serve as inhibitors of human P‐gp ATP hydrolysis activity.

Support or Funding Information

This work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Inhibitors of Glutaminyl‐tRNA synthetase from Pseudomonas aeruginosa
Authors: Escamilla Yaritza , Hughes Casey A , Bullard James M
Published: June 2021

Introduction

Antibiotic resistance is a growing global threat and infections caused by resistant bacteria have become increasingly difficult to treat with most current antibiotics. Pseudomonas aeruginosa is a Gram‐negative opportunistic pathogen and is the leading cause of mortality in cystic fibrosis patients. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. The glutaminyl‐tRNA synthetase (GlnRS) from P. aeruginosa was over‐expressed, enzymatically characterized and developed into a screening platform for the discovery of chemical compounds that inhibit protein synthesis.

Methods

The P. aeruginosa GlnRS was developed, using scintillation proximity assay (SPA) technology, as a platform to screen chemical compounds for detection of compounds that inhibited function. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with inhibitory activity.

Results

From this screening campaign three compounds (BM02E04, BM04B05, and BM04H03) were identified and confirmed as inhibitors of the activity of P. aeruginosa GlnRS. BM02E04, BM04B05, and BM04H03 were observed to inhibit the activity of GlnRS with IC50 values of 1.7, 2.5, and 39.3 μM, respectively. The minimum inhibitory concentration (MICs) was determined against a panel of bacteria including; E. coli, E. coli tolC mutant, E. faecalis, H. influenzae, M. cattarrhalis, P. aeruginosa, P. aeruginosa PAO200 (efflux pump mutant), P. aeruginosa hypersensitive strain, S. aureus, and S. pneumoniae. There was no activity observed against the wild‐type strain of E. coli or P. aeruginosa, but broad spectrum activity was observed against other Gram‐negative and Gram‐positive bacteria. Time‐kill studies indicated a bacteriostatic mode of inhibition for each compound against cultures of S. aureus and M. catarrhalis. When tested in cultures of human embryonic kidney 293 (HEK‐293) cell lines using MTT cell proliferation assays, there was no toxicity observed by either BM02E04 or BM04B05 at any concentration tested up to 400 μg/ml.

Conclusion

P. aeruginosa GlnRS was cloned, over‐expressed, characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms. Three compounds were identified as having inhibitory activity against the aminoacylation activity of GlnRS. Two of the compounds were further characterized for inhibition of enzymatic activity, bacterial growth and toxic effects in human cell cultures.

Support or Funding Information

National Institutes of Health (grant number: 1SC3GM098173)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of caffeine on actin filament assembly
Authors: Zhai Arianna , Kang Hyeran
Published: June 2021

The essential protein actin polymerizes into double‐stranded helical filaments that play critical roles in many cellular processes including cell shape change and movement. Actin is ubiquitous in eukaryotic cells and is particularly abundant in muscle cells. Caffeine is a chemical that is used to create mental alertness. A previous study shows that caffeine relaxes the smooth muscle of the body. We predicted that this effect of caffeine on muscle cells may be coupled to actin assembly/disassembly dynamics. To test the main hypothesis, we explored how caffeine affects actin polymerization with purified rabbit skeletal muscle actin. Solution containing actin filaments with varying concentrations of caffeine were sedimented down using ultracentrifuge. Supernatants and pellets were run through a Sodium Dodecyl Sulfate‐PAGE using gel electrophoresis. UV gel imaging analysis allowed us to determine the amount of polymerized actin in the presence of caffeine. Our preliminary results show that the higher concentrations of caffeine cause actin to polymerize to a greater degree, suggesting that caffeine aids in actin polymerization. This study suggests that caffeine promotes actin filament assembly and may have a stimulating effect on muscle cells.

Support or Funding Information

This study was supported by the UCF start‐up fund for Hyeran Kang.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tracking and Detection of Bactericidal Quantum Dots
Authors: Untracht Zachary , Ozcan Ali , Santra Swadeshmukul , Kang Hyeran
Published: June 2021

Zinkicide is a bactericidal nanoparticle that combats Citrus Greening with exceptional field efficacy. Bactericidal nanoparticle detection is important for surpassing EPA criteria and bringing biocide products to market; however quantitative analysis on Zinkicide degradation in particular is currently lacking. Here, we have developed a protocol using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) to monitor and track bactericidal nanoparticles in varying solutions, including water and acidic buffer mimicking citrus pH. The nanoparticle detection was performed using UV fluorescent imaging of polyacrylamide gels. This assay is a cost‐effective and relatively simple approach for detecting and quantifying the intensity, concentration, and molecular weight change of nanoparticles. We observed intensity fluctuations of Zinkicide over time, indicating that the capping agent of the nanoparticle may be affected. Our results show that citric acid buffer induces the greatest degradation of Zinkicide evidenced by molecular weight changes. These findings are beneficial for general tracking and detection of bactericidal quantum dots.

Support or Funding Information

The authors would like thank the Kang Lab members for their continued support and assistance. This study was supported by the UCF start‐up fund (Kang). Authors acknowledge the UCF Materials Innovation and Sustainable Agriculture Center for facilities and technical support.

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