bluuebunny/crossref_metadata_2025 huggingface dataset:
Utilizing Social Networking Sites and Educational Videos in Public Outreach and the Classroom
Authors: Pena Angela , Brown Kirsten M. , DeVeau Kathryn M.
Published: June 2021

A central priority for educators is to clearly convey information to their students and the public. However, it is often difficult to provide information in a concise and engaging way. This can be especially problematic when engaging with the general public. While people may have a thirst for knowledge, messages can get lost in translation when scientists focus on minutiae and/or jargon. To help communicate scientific concepts to students and the public in a more relatable way, teaching strategies such as creating infographics, narratives, and short videos are being widely utilized by the scientific community. With technology linking people together more readily than ever, scientists have a wealth of options to disseminate these materials. In the the last 20 years, social networking sites (SNS) have exploded both for personal and professional use. Due to their widespread reach, SNS are ideal platforms for communicating science to students and the public.

The main objective of this project is to communicate anatomy concepts to students and the public in an engaging, easily digestible way by delivering the information through familiar media and SNS. We have created several short videos covering introductory anatomy concepts in order to provide students quality resources and study aids and to bridge the gap between scientists and the public. Based on format of the most popular scientific educators, we utilized the following criteria for our anatomy videos: 1. Videos should be under three minutes. 2. Videos should be visually pleasing. 3. Videos should focus on one or two concepts. Videos will be disbursed in class and advertised via SNS (e.g, Instagram and YouTube). We will also plan to disseminate the videos to schools in the DC area for students that may not have access to internet. To assess the efficacy of our videos, we will use social media metrics (i.e., reach, views, etc.) from the SNS, as well as data from student surveys. These data will be analyzed and used to tailor future videos and better guide us on how to communicate science more effectively.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Longitudinal Changes in Facial and Basicranial Integration in the Mediolateral Axis
Authors: Miller Steven F. , Welling Andrew , Holton Nathan E.
Published: June 2021

Objectives

Many studies over the past decade have employed morphological integration as a framework for understanding the interaction of the facial and basicranial skeleton in humans, the great apes, and our fossil lineage. Fewer studies, however, have examined morphological integration in the facial and basicranial portions of the skull in a true longitudinal sample. Most of these longitudinal studies have focused primarily on lateral cephalograms. While useful, these datasets only examine shape and morphological integration from the lateral aspect, limiting the types of questions that can be explored, primarily in the mediolateral axis. To rectify this gap in the data, we set out to study aspects of both symmetric and asymmetric shape variation in a longitudinal postero‐anterior (PA) celphalometric dataset in order to ascertain how patterns of morphological integration change in the mediolateral plane as a function of growth.

Methods

PA cephalograms from a total of 55 individuals from the longitudinal Iowa Facial Growth Study were digitized using 13 coordinate landmarks across the mandible, face, and cranial base to capture patterns of integration and modularity. This data was collected at three time points per patient: age 4, age 11, and in adulthood. Data were submitted to MorphoJ for a Two‐Block Partial Least Squares (2‐B PLS) analysis of both the symmetric and asymmetric shape components in order to examine morphological integration between the face and cranial base.

Results

2‐B PLS results for the symmetric shape component show that integration between the face and cranial base decrease (becoming more modularized) from age 4 (RV=0.41, p<0.001), to age 11 (RV=0.25, p<0.001) before increasing again at the adult time point (RV=0.35, p<0.001). For the asymmetric component, however, levels of integration between the face and basicranium decrease consistently from age 4 (RV=0.59, p<0.001) to adult (RV=0.47, p<0.001). These results indicate that while levels of cranial integration tend to decrease throughout growth, this decrease is only consistent in the asymmetric shape component, potentially indicating that modularity of the face and cranial base has a strong mediolateral (side‐to‐side) component.

Conclusion

These results show that while overall trajectories of facial/cranial base integration show a similar trend toward modularity as individuals grow, this pattern of modularity is only a linear when examining the asymmetric shape component. This aspect of shape has been largely overlooked in the integration literature to date and warrants further study.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Role For The Transcription Factor SNAIL In Alternative Splicing
Authors: Rudraraju Smruthi , Kumar Janani , Krueger Shawn , Qureshi Humaira , Scheidegger Adam , Dhasarathy Archana
Published: June 2021

The transcription factor SNAIL is one of the key regulators of an early developmental process called the Epithelial to Mesenchymal Transition. This process is characterized by a cell‐state change from a highly differentiated, epithelial phenotype to a less differentiated, migratory mesenchymal phenotype. SNAIL binds to E‐box motifs in promoter regions and recruits chromatin remodelers to repress or activate gene expression. To identify proteins associated with SNAIL, we used co‐immunoprecipitation of SNAIL‐bound proteins followed by mass spectrometry. A large number of splicing proteins associated with SNAIL, including SRSF1, a regulator of alternative splicing.

Co‐immunofluorescence and co‐immunoprecipitation data from our lab indicate that SNAIL associates with splicing protein SRSF1. Further, SNAIL co‐immunoprecipitated with SRSF1 in the presence of DNase, but not in the presence of RNase, indicating that the SNAIL‐SRSF1 association is RNA‐dependent. Knockdown of SNAIL with siRNA resulted in loss of mesenchymal splice isoforms and reversion to epithelial specific isoforms. RNA Immunoprecipitation of SNAIL‐ and SRSF1‐ associated RNAs showed increased enrichment for mesenchymal specific isoforms of mRNAs, suggesting that SNAIL and SRSF1 influence alternative splicing.

We hypothesize that SNAIL regulates alternative splicing by binding to RNA and recruiting SRSF1. We are currently investigating genome‐wide changes in alternative splicing regulated by SNAIL, and working to understand the mechanism by which SNAIL interaction with the spliceosome leads to alternative splicing. As transcription and splicing are closely coupled, we predict that transcription factor regulation of splicing through RNA binding is universally utilized by other biological systems to alter phenotype in response to external stimuli.

Support or Funding Information

University of North Dakota, NIH Grant #1P30GM103329 and NIH COBRE #P20GM104360‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resolving Protein Interactions in Live Bacterial Cells through 3D Single‐Molecule Localization Microscopy
Authors: Richardson Charles Joseph , Rocha Julian , Gahlmann Andreas
Published: June 2021

In living cells, proteins form dynamic complexes essential for the functional regulation of cellular activities. While in vitro assays provide key insights into protein binding partners, interactions may be artificially reduced or enhanced once the proteins are removed from their native context. Single‐molecule localization microscopy is ideally suited to measure spatial locations of fluorescently‐labeled molecules in living cells and thus enables single‐particle tracking of the entire distribution of molecular behaviors. Here, we apply high‐throughput 3D single‐molecule localization microscopy to measure the diffusion behaviors of over 100,000 individual intracellular bacterial proteins. Quantitative analysis of the single‐molecule trajectories using Monte Carlo simulations of confined diffusion resolves the co‐existence of multiple diffusive states in the bacterial cytoplasm. Molecular and genetic perturbations show that these diffusive states consist of distinct hetero‐ and homo‐oligomeric complexes. These findings provide new molecular‐level insights into how dynamic molecular complexes function in their native environment.

Support or Funding Information

Research was supported by a Hartwell Postdoctoral Fellowship to CR and startup funds from the University of Virginia to AG.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metal binding specificity in a putative nickel solute binding protein.
Authors: Amato Matthew , Yukl Erik
Published: June 2021

Researchers are pressured to identify novel antibiotic targets in response to the epidemic of bacterial antibiotic resistance. Transition metal homeostasis is related to pathogenicity in gram‐negative bacteria. In particular, metal transporters of the ATP binding cassette (ABC) transporter family are essential for acquisition of specific metals in highly limited environments such as the human host. These systems depend upon a periplasmic solute binding protein (SBP) to bind the appropriate metal with high affinity and specificity. Transcriptional analysis in our model gram‐negative organism Paracoccus denitrificans showed three operons of note encoding ABC transporters to be upregulated under zinc deprivation. Two of these, aztABCD and znuABC, belong to the cluster 1‐A family, which is predicted to transport zinc. Further, the solute binding proteins (AztC and ZnuA) have been shown to bind extracellular zinc with high affinity and specificity in vitro. The third operon pden1341‐1345 encodes a cluster 5 ABC transporter system predicted to be a Nickel/di‐peptide transporter. Thus, it is perplexing that this operon should be significantly upregulated under conditions of zinc deprivation. To determine the possible metal substrate for the Pden1341‐1345 operon, the SBP Pden1341 was cloned, expressed, purified and binding to various metals was determined. Traditional cloning techniques failed by what is thought to be a complication of its large gene size. Gibson cloning proved an effective alternative to cloning full‐length pden1341. Anion exchange and size exclusion chromatography were used to purify Pden1341, and ICP‐OES analysis suggested Pden1341 purifies without bound metal. Binding to various metals was determined by a competitive fluorescence assay as well as isothermal titration calorimetry. The results of these experiments suggest alternate roles for the cluster 5 ABC transports beside the transport of nickel or dipeptides. Thus, cluster 5 ABC transporters may play unexpected functions in metal homeostasis among gram‐negative organisms that may be relevant to the development of metal transport inhibitors as novel antibiotics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Control of the rate limiting step by active site compactness: Reaction of UDPgalactopyranose mutase with UDP‐arabinopyranose
Authors: Sobrado Pablo , Pierdominici‐Sottile Gustavo , Tanner John J.
Published: June 2021

Galactose is a common monosaccharide exclusively found in mammals as galactopyranose (Galp), the six‐membered ring form of this sugar. In Aspergillus fumigatus and mycobacteria, galactose in the cell wall is found as galactofuranose (Galf), the five membered ring form of this sugar. In these microorganisms, Galf biosynthesis begins with UDP‐Galp being converted to UDP‐Galf by UDP‐galactopyranose mutase (UGM). UGM is critical for the survival and proliferation of several prokaryotic and eukaryotic pathogens. Kinetic analyses have shown that bacterial UGM can also catalyze the isomerization of UDP‐arabinopyranose (UDP‐Arap). The possibility of using this sugar as a substrate for eukaryotic UGM has not been evaluated. Here, we probe the Galp‐Galf and Arap‐Araf isomerization catalyzed by Aspergillus fumigatus UGM using viscosity effects, solvent kinetic isotope effects, site directed mutagenesis, and computational biology. Kinetic studies show that eukaryotic UGM use UDP‐Arap/f as a substrate but with a much lower catalytic efficiency than UDP‐Galp/f. The data is consistent with a switch mechanism controlling the rate limiting steps, from the chemical step for UDP‐Galp to product release with UDP‐Arap or the Trp315Ala mutant. Computational studies suggest that tightness in the active site is responsible for this distinct behavior when either the substrate is changed or the side chain of Trp315 is deleted. These results provide important insights into understanding general enzyme catalysis and the UGM reaction mechanism, and will facilitate the development of inhibitors.

Support or Funding Information

Research reported in this publication was supported by grants from the National Institute of General Medical Sciences (NIGMS) award R01GM094469, the National Science Foundation award CHE‐1506206, and PICT 2436 préstamo BID. The authors are also very grateful to CONICET for financial support.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutational analysis of the Clu2593c homing endonuclease reveals specific residues required for DNA binding and cleavage.
Authors: Davis Lorraine , Takushi Brittany , Kaiser Brett K.
Published: June 2021

Homing endonucleases (HEGs) are selfish genetic elements that recognize and cleave long DNA target sequences (18–22 basepairs) within an organism's genome. This cleavage induces homology‐directed repair using the HEG‐encoding gene as a template, facilitating its lateral transmission. The I‐CreI family of HEGs is present within organellar genomes of diverse single‐celled algae species and is incorporated at identical positions within the large ribosomal subunit gene. Homologs of this family, which share 35–70% pairwise amino acid identity, have evolved to recognize nearly identical target sites while tolerating sequence polymorphisms along the DNA target site. Although the I‐CreI and I‐CvuI enzymes have been well characterized, the mechanisms by which other homologs of this family achieve target sequence recognition are not well understood. In order to explore the structure‐function relationship of the I‐CreI family, we carried out a detailed molecular‐level analysis of the I‐CreI homolog Clu2593c (57% sequence identity). Based on a co‐crystal structure of WT Clu2593c bound to DNA, we generated 12 single‐residue mutations at positions in the enzyme‐DNA interface. We then tested the functionality of the bacterially‐expressed and purified mutant proteins using in vitro binding and cleavage assays. Additionally, we solved co‐crystal structures of a subset of the mutants bound to DNA. Our analyses revealed that several of the mutations behaved in a similar manner to analogous mutations in I‐CreI, whereas others indicated divergent mechanisms of DNA recognition. These results highlight that homologs of the I‐CreI family have evolved distinct solutions to recognizing a shared DNA target.

Support or Funding Information

Murdock Charitable Trust

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antitumor Activity of Simarouba tulae Extracts in a Panel of Cancer Cell Lines
Authors: Del Moral Isabel Conde , Reyes Jeyshka , Vivas‐Mejia Pablo , Ospina Claudia
Published: June 2021

Cancer is among the principal causes of death globally. Statistics from the American Cancer Society project an increase of 50% in new cases and 60% in worldwide deaths by 2030. Although several chemotherapeutic agents are currently being used for the treatment of most cancer types, recurrence, drug resistance, and severe side effects demand the need for alternative novel drugs. Even with the evolution of synthetic chemistry, nature remains as a fundamental source of molecular diversity and structural innovation. Of the 140 anti‐cancer agents approved since 1940, over 60% could be traced to a natural product. One of the most popular bioactive molecules found in natural extracts are quassinoids which are highly oxygenated triterpenes found in the Simaroubaceae family. Simalikalactone D is a quassinoid isolated from Simarouba tulae, an endemic tree from Puerto Rico. Our objective was to evaluate the anticancer effect of this Simalikalactone D in a panel of human cancer cell lines. The cytotoxicity was evaluated in vitro through dose‐response assays in A2780CP20 cisplatin‐resistant ovarian cancer and in MDA‐MB‐231 breast cancer cell lines. Our results showed that Simalikalactone D exhibits significant cytotoxicity against both cell lines. Partial organic extracts of Simarouba also were toxic to these cells lines. The IC50 values were around 50–100 nM which indicate that Simalikalactone D is a potential anticancer drug. Ongoing experiments evaluate the cytotoxicity of Simalikalactone D in other cancer cell types including colon and prostate cancer cells, and in normal cells. To assess the mechanism of action of this compound, future experiments will measure the effect of Simalikalactone D on activation of apoptosis, cell cycle progression arrest and in proteins associated – with these biological effects.

Support or Funding Information

Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103475.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis of Fluorescein‐linked, Zinc‐based Metal Organic Frameworks as Carriers of Targeted Treatments for Retinopathies
Authors: Fralish Zachary , Bromfield‐Lee Deborah , Eubank Jarrod , Shelby Shameka
Published: June 2021

Metal‐Organic Frameworks (MOFs) are compounds composed of metallic ions linked by organic ligands that form porous units with highly specialized shapes and sizes. Beyond this, MOFs have intrinsic biodegradability, chemical diversity, and a high loading capacity. These characteristics make MOFs optimal candidates as vehicles for drug delivery. More specifically, these programmable, nano‐like particles have the potential to hold specific materials and dissolve in particular environments which makes entrapment and targeted release of various treatments possible. These qualities could be advantageous when treating diseases of the retinal pigment epithelium (RPE). Current treatment involves subretinal injection, which requires a skilled surgeon and often results in retinal detachment and occasional proliferative vitreoretinopathy which leads to acute deterioration of visual facility. MOFs with entrapped treatments can potentially be injected into the vitreous of the eye and targeted to release their payload in the RPE, removing the risk of retinal tearing and the need of a skilled surgeon. To pursue this potential, we modified 4,5‐imidazoledicarboxylic acid (ImDC) ligands with fluorescein via an esterification reaction, while synthesizing MOFs with ImDC. The inclusion of fluorescein will allow for observation of MOF incorporation and processing within the cell. Analysis of the esterified ligand through Gas Chromatography‐Mass Spectroscopy and Nuclear Magnetic Resonance, including homonuclear correlation spectroscopy, indicated that the ligand had been modified. Zinc‐based metal‐organic frameworks with ImDC ligands were synthesized and analyzed by Powder X‐Ray Diffraction to confirm the crystalline structure of the framework. The MOFs were also shown to be partially soluble in potassium chloride and sodium chloride following sonication. Preliminary studies of zinc‐based MOFs in HEK‐293 cells have indicated that the MOFs will likely enter the cells by a phagocytic mechanism. Taken together, these studies will lead to the creation of a system to assess the functionality of MOFs in drug delivery. Future studies will investigate the use of various metals to direct the cellular processing of MOFs.

Support or Funding Information

Florida Southern College Faculty‐Student Collaborative Research Grant

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 Role of Fruits on the Cancer Incidence in Cusco Peru
Authors: Jones Janae , Munoz Crystabel , Ledesma Litza , Faustino Vroniqa , Nogaj Luiza A. , Roberts Lia , Deprele Sylvine
Published: June 2021

Breast cancer remains as one of the most prevalent types of cancer in the world. Although Western industrialized nations rely on a contemporary approach to modern medicine, many developing countries, such as Peru, rely on traditional medicine and alternative natural remedies. Therefore, the focus of this study was to determine the extent to which natural remedies and nutrition, contribute to the prevention and incidence of cancer. Here, we focus on the use of certain fruits, like papaya and the noni fruit, as natural remedies for illness. First, survey data was collected in Cusco, Peru regarding which fruits are consumed as natural remedies for illness. Then, molecular techniques such as MTT assay and LDH Cytotoxicity assay were used to test the effect these fruits have on HeLa cells. The survey results showed, papaya and noni as the most commonly used fruits for illness. Noni powder and papaya fruit were extracted with ethyl acetate and ethanol and used in MTT and cytotoxicity assays. Preliminary results in mammalian cells showed no effect of the ethyl acetate extracts on the viability of HeLa cells. However, MTT assay results for the ethanol extraction of papaya skin showed a decrease in mammalian cell viability at high concentrations. Further experiments will be conducted to test fresh noni and papaya as well as other commonly eaten fruits in Peru for their anticancer properties.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adoption of DNA Aptamer‐Based Biosensors into Nanoparticle‐Based Complexes for Raman Detection
Authors: Clark Derek , Bonham Andrew J.
Published: June 2021

DNA aptamers have been engineered that bind a variety of targets with high specificity. However, turning that binding into a measurable change to enable detection of the target can be challenging, and has inspired multiple detection approaches. Here, we present a sensitive assay of DNA aptamer‐based detection for biological targets using surface enhanced Raman scattering (SERS) by gold nanoparticles long range plasmonic coupling. Gold nanoparticles have unique electrical and optical properties, including the promotion of SERS, wherein closely spaced nanoparticles dramatically increase the sensitivity of Raman spectroscopy, a molecular fingerprinting technique. We have synthesized 60nm gold nanoparticles (AuNP) that produce increased SERS. These AuNP are directly functionalized with thiolated DNA aptamers that contain a terminally conjugated Raman reporting agent (methylene blue), complementary DNA is conjugated to separate AuNP to allow controlled annealing of functionalized AuNP into multiple‐particle complexes (multiplexes). Controlled introduction of silver nitrate further increases SERS by silver coating AuNP multiplexes, causing roughened surfaces (localized surface plasmon resonance) and reducing multiplex gaps. These silver coated multiplexes provide SERS amplification and minimize signal‐quenching geometries. We demonstrate that the binding of target proteins (including Ricin A chain (RTA) and the Myc/Max complex) to the DNA aptamers cause a change in the conformational dynamics of the aptamer, by taking a signal‐OFF approach for target protein detection. Aptamer conformational changes effectively disassembles previously annealed multiplexes, that emit an intense dye “fingerprint”. Aptamer based conformational changes are favorable in the presence of the target protein. Introduction of target protein effectively breaks hybrid silver coated multiplexed AuNP in a concentration dependent fashion, releasing functionalized silver coated AuNP. Bound target protein effectively extinguishes the dye “fingerprint” as target concentration increases. Released silver coated AuNP reduce long‐range plasmonic coupling and signal‐quenching geometries reform as multiplexes disassemble, creating a reduction in signal. Ultimately, such assemblies should offer a stable optical platform for the creation of highly sensitive biosensors for a range of biological targets.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolomic Analysis of HER2‐positive Breast Cancer Cells
Authors: Bush Jason A. , Sahni Malika K. , Ravindran Resmi K. , Khan Imran H. , Krishnan Viswanathan V.
Published: June 2021

Purpose

HER2‐positive breast cancer accounts for more than 20% of the diagnosed cases and is characterized by aggressive growth, increased disease recurrence, and poor prognosis. While much of the signaling mechanism(s) have been elucidated, the impact of HER2 positivity on cellular metabolism is not well understood. We examined (1) gene expression profiles of mammalian breast cancer cell lines with increasing HER2 expression levels (MCF10A, MDA‐MB‐468, MDA‐MB‐453, HCC1954, HCC2218) and correlated changes to metabolic expression profiles and (2) the effect of serum starvation and pervanadate treatment on the metabolic profiles of these HER2‐expressing cell lines.

Methods

Our study used a combination of 1H‐NMR and 2D‐NMR (TOCSY), multivariate analyses, and protein expression validation in human breast cancer cell lines (untreated, serum‐starved, serum‐starved with pervanadate treatment).

Results

We found that both gene and metabolic expression profiles of HER2‐positive cell lines correlated with leucine and isoleucine biosynthesis (p‐value < 0.05). Comparative analyses of profiles from untreated, serum‐starved, and serum‐starved with pervanadate‐treated cell samples revealed that lactate concentration (p‐value <0.05) dropped significantly with serum starvation and pervanadate treatment consistent with reduced glycolytic metabolism and potentially mediated through suppression of tyrosine phosphatase activity.

Conclusion

This study highlights important metabolites that are potential biomarkers of HER2‐positive breast cancer and suggests alternate/novel pathways to consider for future understanding and treatment.

Support or Funding Information

This work was funded in part through intramural programs at Fresno State to MKS and JAB, and through the NIGMS 1SC3GM122630‐01 to JAB.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Urine Odor Profiling for Diagnosis of Interstitial Cystitis
Authors: Park Rebekah Julie , Shahid Muhammad , Yeon Austin , Kim Jayoung
Published: June 2021

Interstitial cystitis (IC) is a clinical condition that manifests as a sensory hypersensitivity of unknown cause and is characterized by frequent urination, bladder discomfort, and pelvic pain. Chronic inflammation is a potential systemic risk factor for IC. However, it remains unknown the underlying mechanism through which a healthy bladder protects itself from inflammatory triggers. In this study, we profiled urinary odor, which consists of various volatile organic chemical compounds in urine obtained from IC patients and healthy controls. Using comprehensive solid phase micro extraction‐gas chromatography‐time‐of‐flight mass spectrometry profiling combined with comprehensive bioinformatics analyses, levels of urinary volatile metabolites in healthy individuals were compared to those in IC patients. Among the several metabolites whose levels were significantly altered, menthol showed significantly reduced levels in IC. In an attempt to understand the mechanistic meaning of IC‐associated volatile metabolite, menthol, we performed cytokine profiling and DNA microarray. Our findings suggest that inflammatory events and the activation of signaling networks were suppressed by menthol treatment. Further validation with biochemical experiments and Western blot analysis confirmed that menthol reduced the expression of cytokines and signaling factors that are closely associated with inflammation. These findings will provide potentially new strategies to alleviating both the odor and inflammation associated with IC.

Support or Funding Information

This work was supported by the National Institutes of Health grants (1U01DK103260, 1R01DK100974, U24 DK097154, NIH NCATS UCLA CTSI UL1TR000124), Department of Defense grants (W81XWH‐15‐1‐0415), Centers for Disease Controls and Prevention (1U01DP006079), IMAGINE NO IC Research Grant, the Steven Spielberg Discovery Fund in Prostate Cancer Research Career Development Award, and the U.S. ‐ Egypt Science and Technology (S&T) Joint Fund, funded by the National Academies of Sciences, Engineering, and Medicine and USAID (all to J.K.). Any opinions, findings, conclusions, or recommendations expressed in this article are those of the authors alone, and do not necessarily reflect the views of any of the previously mentioned sponsors. J.K. is former recipient of Interstitial Cystitis Association Pilot Grant, a Fishbein Family IC Research Grant, New York Academy of Medicine, and Boston Children's Hospital Faculty 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.
Comparing the Solution Conformation and Activin‐binding of Follistatin Isoforms
Authors: Bhenderu Lokeshwar Sai Santosh , Murray Kyle , Stepurko Natalija , Wang Xuelu , Hyvönen Marko , D'Arcy Sheena
Published: June 2021

Activins are members of the Transforming Growth Factor‐β superfamily. This superfamily of secreted growth factors activates transmembrane serine‐threonine kinase receptors, thereby stimulating Smad‐mediated intracellular signaling cascades. These cascades alter the gene expression profile of the target cells. Follistatin (FST) is a protein antagonist that directly binds to activins and other TGF‐β‐like proteins, preventing their interaction with cell‐surface receptors. Alternative splicing and proteolysis result in three isoforms of FST which differ in their C‐terminal regions. The three FST isoforms have different affinities for activins, as well as the extracellular matrix component heparan sulfate. We are focused on two FST isoforms, FST315 and FST288. FST315 contains an acidic C‐terminal region that is absent in FST288. Crystal structures of FST315 and FST288 with activin A are unable to explain the basis for isoform‐specific effects. The structures are similar and the acidic C‐terminal region of FST315 is not observed in the electron density. To identify differences between FST315 and FST288, we have used hydrogen/deuterium exchange coupled to mass spectrometry. We compare exchange of FST315 and FST288, both alone and in complex with activin A. The results will improve our molecular understanding of how FST isoforms regulate signaling by activin A.

Support or Funding Information

Startup funds to S. D'Arcy from The University of Texas at Dallas.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beta‐cell‐specific loss of the inhibitory G protein, Gαz, prevents development of Type 1 Diabetes in NOD mice.
Authors: Fenske Rachel , Wienkes Haley , Peter Darby , Kimple Michelle
Published: June 2021

The mechanisms that underlie the beta‐cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our lab has discovered a role for the unique inhibitory G‐protein, Gαz, in beta‐cell function, survival, and replication in both normal conditions and those of pathological stress. Gαz, when acting as a canonical inhibitory G‐protein, reduces production of cyclic adenosine monophosphate (cAMP) through inhibition of adenylate cyclase. cAMP acts as essential player in the amplification of glucose‐stimulated insulin secretion, while also mediating pathways that regulate beta‐cell death and replication. We've shown previously that removal of this inhibitory protein is sufficient to promote insulin secretion, beta‐cell growth and survival using chemically‐induced beta‐cell death and diabetes in C57BL/6J mice. Previous work in our lab has also shown that non‐obese diabetic (NOD) mice with global loss of Gαz are protected from developing T1D‐like hyperglycemia through a multi‐faceted maintenance of beta‐cell health. Whole body Gαznull animals exhibited a significant increase in beta‐cell function and replication and decreased beta‐cell death. Most interestingly, islets from Gαz‐null NOD animals exhibited a marked decrease in immune infiltration. In order to determine if Gαz in the beta‐cell was responsible for the protected phenotype, we generated a beta‐cell‐specific Gαz knockout by breeding Gz Flox/Flox mice with Rat Insulin Promoter (RIP) driven CreHerr mice, on the NOD background. After 10 generations of backcrossing, we began by determining if loss of beta cell Gαz was sufficient to protect NOD animals from developing T1D‐like hyperglycemia. Weekly random‐fed blood glucose measurements were taken beginning at weaning. In the initial cohort, we saw a 12% penetrance of hyperglycemia in male Cre‐positive NOD mice and a 23% penetrance of hyperglycemia in female Cre‐positive NOD mice by 30 weeks of age. In contrast, Gαzβ‐cell‐KO males and females were entirely protected from developing hyperglycemia during this period. Despite this stark contrast, fasting blood glucose was no different between genotypes, grouped by sex, at either 12 or 28 weeks of age. Glucose tolerance was also not different between Cre‐positive NOD controls and Gαzβ‐cell‐KO animals. Experiments are ongoing to further investigate other facets of beta‐cell health, including function, replication and survival. Additionally, it will be crucial to evaluate the presence and extent of insulitis in our Gαzβ‐cell‐KO animals. Thus far, our results show a critical role for beta‐cell Gαz in promoting T1D pathophysiology, although the precise mechanism remains to be determined.

Support or Funding Information

NIH F31 DK109698 to RF

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 PKC inhibitor BimI functions as a bi‐topic ligand that toggles a conserved allosteric switch to drive potent kinase inhibition
Authors: ma ning , Lee Sangbae , Devamani Titu , Sandhu Manbir , Sommese Ruth , Sivaramakrishnan Sivaraj , Vaidehi Nagarajan
Published: June 2021

ATP‐competitive small‐molecule kinase inhibitors (SMKIs) are viewed primarily from the lens of tight and specific binding to the conserved catalytic domain. The allosteric effects of ATP‐competitive SMKIs are generally ignored, despite evidence that they can influence the potency and selectivity of kinase inhibition. We have recently showed that the ATP‐competitive PKC inhibitor BimI can displace substrate binding leading to potent inhibition of the kinase (Sommese and Sivaramakrishnan, JBC, 2016). In this study, we examine the substrate‐competitive behavior of a range of SMKIs derived from staurosporine and nucleotide analogs. We find that BimI functions like a bi‐topic ligand that acts on both the orthosteric ATP binding pocket and a remote allosteric site that determines overall kinase conformation. Combining novel FRET‐based sensors and multi‐scale MD simulations we identify a novel, conserved structural mechanism that regulates substrate binding in protein kinases. Bi‐topic ligands such as BimI disrupt a conserved ionic‐lock that drives global reorganization of the kinase domain through long‐range allosteric pathways that connect the N‐ and C‐terminal lobes. Together our studies identify a novel allosteric switch that regulates substrate binding in protein kinases that can be leveraged to develop highly selective kinase inhibitors.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rare Alzheimer's Disease‐Associated Protein Kinase C Variant Displays Altered Pharmacological Profile in a Cellular Environment
Authors: Yang Yimin , Callender Julia , Newton Alexandra
Published: June 2021

Ever since protein kinase C (PKC) was discovered as receptors for tumor‐promoting phorbol esters in the early 1980s, efforts have been made to target PKC in the treatment of disease. Here, we show that PKCα‐M489V, a rare Alzeheimer's Disease (AD)‐associated PKC variant with enhanced agonist‐evoked signaling output, displays an altered pharmacological profile compared to wild type PKCα in a cellular environment. Using a genetically encoded PKC activity reporter and a phospho‐serine PKC substrate antibody as readouts for PKC activity, we find that overexpressed PKCα‐M489V in COS7 cells is more sensitive to inhibition by the ATPcompetitive PKC inhibitor Gö6976, but not by the uncompetitive PKC inhibitor bisindolylmaleimide IV (BisIV). However, PKCα‐M489V is equally sensitive to inhibition by Gö6976 in vitro, suggesting that the difference may result from protein‐protein interactions within the cell. The altered pharmacology of the M489V variant in vivo may provide a novel method of targeting PKCs in the treatment of AD.

Support or Funding Information

This work was supported by N!H R35 GM122523 and NIH GM43154 (AN) and the Cure Alzheimer's Fund (AN). JC was supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deep Reading of the Literature and the Biosynthesis of Nucleotides
Authors: Anderson Ellen
Published: June 2021

Biosynthesis of Nucleotides is a Master's level elective course that was developed to advance students' comprehension of peer‐reviewed articles using a detailed study of the biosynthesis of purine and pyrimidine ribonucleotides and their conversion to deoxyribonucleotides. Nucleotides are ubiquitous molecules essential for nearly every biochemical pathway serving as substrates, regulatory molecules or monomers for RNA and DNA synthesis. Many medical conditions are the result of abnormalities in these biosynthetic pathways and their enzymes are targets for pharmaceutical intervention. Unfortunately, one‐semester biochemistry courses rarely have time to cover nucleotide metabolism. Coupling this fundamental topic in biochemistry with in‐depth reviews of the literature enabled graduate students to learn how to effectively read and analyze the literature while learning nucleotide metabolism. A scaffoldedset of assignments, adapted with permission from B.D. Spielgelberg (2014), was created to deepen students' understanding of experimental details, interpretation of the results and development of conclusions from these results. These assignments consist of a mix of individual assessments, group‐generated analyses of figures and tables of data, and online discussions based on a peer‐reviewed paper for each section of the course. A final project, completed individually, combines these elements and is used to evaluate student progress in literature evaluation and comprehension. Examples of the assignments will be presented, as well as preliminary data demonstrating the improved performance over the semester, especially on a comprehensive analysis of a set of data or a figure from the assigned paper. Likewise, data from student evaluations will be presented to demonstrate the value of these assignments.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell‐Free Protein Synthesis: A Platform Technology for Education
Authors: Kao Wesley Y. , Gregorio Nicole E. , Oza Javin P.
Published: June 2021

Hands‐on learning of transcription and translation remains limited due to a lack of classroom modules for early‐career students. Currently, classrooms implement animations, simulations, games, and bacterial transformation/protein expression to teach the genetic code. The cell wall poses the primary barrier for students to directly observe and manipulate the intracellular environment, limiting inquiry‐based learning of the genetic code. We have overcome this limit by levering transcription and translation in vitro. The open nature of cell‐free protein synthesis (CFPS) is achieved by removing the cell wall and genomic DNA, while isolating the cellular machinery involved in transcription and translation. This allows experimenter to harness the genetic code in a test tube and directly manipulate the reaction conditions for hands‐on learning. While reconstitution of cellular machinery in vitro for CFPS has been transformative for biotechnology, CFPS has not been accessible to early‐career students due to the complexity in reagent preparation and reaction set‐up. We have successfully adapted the CFPS biotechnology to develop a learning module accessible to early‐career STEM students focusing on learning objectives involving the genetic code. Here we report the reformulation of the CFPS reaction setup for simplicity and improved reagent shelf life in order to adapt CFPS for the classroom. First, our simplified platform requires the pipetting of only 3 pre‐mixed reagents, an 80% reduction in setup steps, making it more accessible to an early‐career student with little‐to‐no laboratory experience. Second, our simplified platform is shelf‐stable at −20°C rather than −80°C, making it accessible for storage and implementation in undergraduate and high school classrooms. Longer‐term experiments are in progress to validate shelf‐life at 4°C and room temperature. Our data show that these improvements are achieved without sacrificing CFPS performance in reaction kinetics or total protein yield. Third, our analysis of green fluorescent protein synthesis kinetics shows that 90% of maximum protein yields can be achieved within 3 hours at 37°C, making this platform compatible with most college laboratory courses. We believe that this educational technology will be transformative to the way the genetic code is taught in classrooms today. Toward this end, we have developed a CFPS classroom ‘kit’ for broad dissemination.

Support or Funding Information

Our research is funded by the Center for Applications in Biotechnology @ Cal Poly SLO, Bill and Linda Frost Fund, and NSF‐1708919.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
14‐3‐3 Protein‐Dependent GATA Transcription Factor Control in Saccharomyces cerevisiae
Authors: Tate Jennifer J. , Rai Rajendra , Buford David , Cooper Terrance G.
Published: June 2021

14‐3‐3 proteins, Bmh1/2, are major participants in the control of most nutrient‐ and stress‐sensitive regulons in Saccharomyces cerevisiae. However, the Nitrogen Catabolite Repression (NCR)‐sensitive regulon and its Gln3 and Gat1 GATA transcription activators have not been reported to be Bmh1/2‐regulated. Observations from proteomic studies identify Gln3 as a candidate Bmh1/2 interacting protein (Biochem 46:7781; Genomics 94:287), and NCR‐sensitive GAP1 expression is about 1.5‐fold higher in bmh2 than bmh1 mutants treated with TorC1 inhibitor, rapamycin (Biosci. Rep. 34;art:e0009). Therefore, we determined whether Bmh1/2 participates directly in Gln3 and Gat1 control or more indirectly via Bmh1/2‐dependent TorC1 regulation. The question of Bmh1/2‐dependent GATA factor regulation can only be effectively investigated in a ∑1278b background because in most other strains the double mutant is lethal. In poor nitrogen conditions, Gln3 and Gat1 are nuclear and support high level NCR‐sensitive transcription in wild type cells. In contrast, when abundant nitrogen is available, Gln3 and Gat1 relocate to the cytoplasm and GATA factor‐dependent transcription is repressed. This wild type regulation is achieved via post‐translational Gln3 regulation by the TorC1 and Gcn2 kinases, Sit4 and PP2A phosphatases and downstream negative regulator Ure2. In a bmh1Δbmh2Δ double mutant, we find that: (i) NCR‐sensitive transcription of three reporters, GDH2, DAL80 and DAL5, is almost completely abolished. (ii) Gln3 and Gat1‐mediated transcriptional responses to rapamycin‐treatment are detectable but severely diminished. (iii) Loss of Bmh1/2 results in Gln3 dephosphorylation similar to the levels observed following rapamycin treatment. (iv) The response of Gln3 and Gat1 nuclear localization parallels that observed for GATA‐factor mediated transcription. (v) Cells form pseudohyphae‐like chains when provided with nitrogen sources whose utilization requires NCR‐sensitive transcription. Prior to the point when bmh1Δbmh2Δ mother and daughter cells separate, only one contains detectable GFP‐Gln3. Either Bmh1 or Bmh2 will fulfill the 14‐3‐3 requirement as nuclear Gln3 localization is normal in single bmh1 mutants. Using bmh1Δbmh2Δ and bmh1Δbmh2Δure2Δ mutants, we demonstrate that a bmh1Δbmh2Δ double deletion is epistatic to an ure2 deletion. We conclude Bmh1/2 are absolutely required for nuclear Gln3 localization and likely function downstream of Ure2, heretofore the most downstream regulator of the nitrogen‐sensitive regulon.

Support or Funding Information

National Institute of Health General Medical Sciences GM‐35642‐27.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of capsicoside G on lipolysis and fatty acid oxidation in 3T3‐L1 adipocytes
Authors: LEE JUNSOO , Kim Younghwa , Sung Jeehye
Published: June 2021

We have previously demonstrated that capsicoside G, a furostanol saponin, isolated from pepper seeds, inhibited adipogenesis in 3T3‐L1 adipocytes. However, the effects of capsicoside G on lipolysis and fatty acid oxidation remain unclear. The aim of this study was to extend the anti‐obesity investigation of capsicoside G by examining its effect on lipolysis and fatty acid oxidation in mature 3T3‐L1 adipocytes. Capsicoside G promoted lipolysis of adipocytes, which effectively decreased the intracellular triglyceride concentration of adipocytes and increased glycerol released into the medium. The increased lipolysis was accompanied by an augmentation in the expression of adipose triglyceride lipase and hormone‐sensitive lipase. Furthermore, capsicoside G also induced the expression of fatty acid oxidative genes such as peroxisome proliferator activated receptor α and carnitine palmitoyltransferase‐1 in mature 3T3‐L1 adipocytes. These results suggest that capsicoside G exerts anti‐obesity property by stimulating lipolysis and fatty acid oxidation in adipocytes.

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