bluuebunny/crossref_metadata_2025 huggingface dataset:
Exon Sequencing for the Presence of Late Onset Tay‐Sachs
Authors: Slostad Joshua Nathan , Goldberg Jodi
Published: June 2021

Tay‐Sachs is a disease that causes rapid developmental stagnation and death in infants. It primarily affects people of French Canadian, Cajun, and Ashkenazi Jewish descent. In recent years an alternate form of the disease, called late onset Tay‐Sachs (LOTS), has been described. LOTS is caused by the inheritance of one null allele and a pseudodeficiency allele of the hexosaminidase A (HEXA) gene. The mutation rate of the pseudodeficiency is not well known. The aim of this experiment was to examine the DNA of the descendants of an Ashkenazi Jewish individual who was believed to have undiagnosed LOTS. The purpose of this case study is to attempt to confirm this suspected diagnosis by identifying potential mutations in the family members. DNA of the HEXA gene from three family members of the suspected LOTS patient (wife and 2 daughters) and one control set (unrelated female) was sequenced. The sequences were aligned and compared both by hand and using the NCBI Blast database and the Benchling tool. No known mutations or confirmed novel mutations have been found in all fourteen exons sequenced. Currently re‐sequencing of some regions that showed potential single nucleotide polymorphisms (SNPs) as well as sequencing the promoter region of the gene is underway. If a mutation is confirmed this research could help pave the way for further genetic testing in the broader population in order to fully grasp the prevalence of LOTS pseudodeficiency alleles within the Ashkenazi population.

Support or Funding Information

Hamline Undergraduate Collaborative Research

Ridgeway Forum Fund

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermo‐responsive Estrogen Receptor‐fusions for Spatiotemporal Control of Signaling and Gene Regulation
Authors: Truong Anh T. , Li Zhe , Okamoto Curtis T. , MacKay J. Andrew
Published: June 2021

Many diseases are caused by aberrant cell signaling, leading to a dysregulation of proteins responsible for cell maintenance and repair. Understanding these events require a precise manipulation of the cells' machinery, but many technologies used to study signaling and transcription lack the temporal control to rapidly modulate these biologic processes. This limitation could be addressed by the introduction of elastin‐like polypeptides (ELPs) which are synthetic, thermally responsive biopolymers that self‐assemble into microdomains of ELP‐fusions within cells. Our research group recently discovered that these fusion proteins can modulate precise processes involved in cell trafficking. They are now being investigated to modulate estrogen receptor (ER) signaling and gene regulation. ER has been implicated in a number of diseases such as breast cancer and Sjögren's Syndrome and although this nuclear hormone receptor has been well studied, the lack of drug therapy and resistance, requires attention to develop a deeper understanding of the underlying mechanisms. Thus, ELP‐ERɑ fusions have been generated and successfully expressed in CHO, HeLa and 293T cell lines. They coalesce in the nucleus at physiological temperatures and rapidly solubilize at room temperature. These preliminary data indicate that these fusions retain ELPs self‐assembling properties and may control ER signaling and gene regulation. The ability to rapidly and reversibly, with spatiotemporal control and precision, may allow this fusion to serve as a powerful tool to study ER‐mediated biological processes.

Support or Funding Information

This funding is supported by the National Institutes of Health (NIGMS 1R01GM114839‐02) and the American College of Clinical Pharmacy Research Institute Futures 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.
Depletion of CRBN leads to proliferative cellular senescence by altering the level of cell cycle proteins
Authors: Jeon Seung‐je , Yoon Yi‐seul , Yang Seung‐joo , Park Chul‐Seung
Published: June 2021

Cereblon (CRBN) is the direct target of the immunomodulatory drugs (IMiD), such as lenalidomide, and the treatment of IMiDs is known to inhibit the proliferative activities of cancerous cells such as multiple myeloma. The expression level of CRBN protein is varied in different cancer cells and many somatic mutations in CRBN gene are found in numerous cancer patients. The mouse embryonic fibroblast (MEF) lacking Crbn was previously shown to be resistant to various cellular stresses (K.M. Lee et al. / Biochemical and Biophysical Research Communications 458 (2015) 34–39). However, the role of CRBN in cell proliferation and its regulation is still poorly understood. We observed that the Crbn‐knockout (KO) MEFs showed accelerated cell proliferation, and the increased level of replicative and premature senescence. We thus evaluated the level of cell cycle proteins such as cyclins, cyclin‐dependent kinases (CDK), and CDK inhibitors. We found that the expression of several cell cycle proteins was specifically altered by depletion of CRBN protein. In addition, the flow cytometric analyses showed the increased level of cell arrest at G2/M phase for CRBN KO cells by. Furthermore, the lifespan of Crbn KO mice was found to decrease compared to that of WT mice. Thus, CRBN deficiency may induce proliferative senescence via modulating the level of cell cycle proteins such as cyclins and CDK inhibitors. We are currently investigating the underlying mechanism for the specific alteration of cell cycle proteins induced by CRBN depletion.

Support or Funding Information

This work was supported by GIST Research Institute(GRI) grant funded by the GIST in 2017.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caveolin‐1 Phosphorylation is Essential for iPSC‐derived Human Neuronal Axonal Growth during Early Stage of Differentiation
Authors: wang Shanshan , Zhang Zheng , Leem Joseph , Patel Hemal , Head Brian
Published: June 2021

Proper axonal growth and guidance is essential for functional neuronal connections. Formation of aberrant neuronal connections due to genetic or epigenetic influences can contribute to neurological and mental disorders. Identification and targeting of the molecular events that promote normal neuronal growth and differentiation may promote structural and functional neuroplasticity and improve behavior in neurodegenerative diseases such as Alzheimer's disease (AD), Down syndrome, schizophrenia, and depression. Using human neurons derived from induced pluripotent stem cells (iPSCs), we show that neuron‐specific targeted (synapsin promoted, Syn) caveolin‐1 (Cav‐1) overexpression (SynCav1) significantly augmented axonal growth (SMI 31), findings akin to effects from SynCav1 murine neurons in vitro and in vivo. In contrast, neurons infected with a Cav‐1 phosphorylation mutant (SynCav1(Y14A)) exhibited stunted axonal growth. In addition, SynCav1(Y14A) significantly impaired Rac1/Cdc42CA‐mediated axonal growth. These results suggest that 1) P‐Cav‐1 is necessary for proper axonal growth during its early differentiation stage, 2) SynCav1‐mediated axonal growth is dependent upon Cav‐1 phosphorylation, 3) Rac1/Cdc42CA‐mediated neuronal growth may in part be dependent upon P‐Cav‐1. In conclusion, phosphorylation of Cav‐1 is crucial for axonal growth during early stage of differentiation in human neurons.

Support or Funding Information

NS073653 (B. P. Head); Veteran Affairs Merit Award from the Department of Veterans Affairs BX003671

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
c‐JUN N‐terminal kinase (JNKα1) binds eNOS and phosphorylates at S116, differentiating it from p38 and ERK phosphorylation
Authors: Solone Xzaviar K. , Caldara Amber L. , McMurry Jonathan L. , Chrestensen Carol
Published: June 2021

We have previously shown that the mitogen activated protein kinases (MAPK), p38 and ERK bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via interactions with a pentabasic non‐canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind MAPK. In the present study, eNOS binding by c‐Jun N‐terminal kinase (JNKα1) was examined using optical biosensing. Similar to p38 and ERK, JNK evinced binding to eNOS (31 nM KD) but not nNOS as measured by fit to a one‐state global model. Rate constants were determined (kon = 4125 M−1s−1 and koff = 1.3 × 10−4 s−1). In vitro kinase assays and immunoblotting identified phosphorylation by JNK at S116, contrasting with ERK, which phosphorylated S602, and p38, which phosphorylated both sites. Phosphorylation by JNK at S116 did not alter the conformational manifold, where phosphorylation at S602 by ERK and p38 does. In HMEC1 cells we observe that phosphorylation at these MAPK sites happens concurrently with activating sites (S1177), creating antagonistically phosphorylated eNOS. Further, we examine how phosphorylation of eNOS alters the cellular interactions of the MAP kinases. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.

Support or Funding Information

NIH 1 R15 GM110634‐01A1 | R25GM111565

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of Gap Junctional Coupling by Secreted Factor(s) from Mesothelial Cells is Required for the Invasiveness of Endometriosis
Authors: Nicholson Bruce J. , Chavez Jeffery B. , Dorsey Emma K. , Olivas Taryn , Wang Chiou‐Miin , Lucio Nicholas , Gillette Laura , Burney Richard O. , Kirma Nameer
Published: June 2021

Intercellular interactions, including gap junction intercellular coupling (GJIC), have been implicated in many invasive processes (e.g. extravasation, metastasis and oocyte implantation). Yet the role of gap junctions in endometriosis, a major invasive medical problem that affects 6–10% of women worldwide, has not been investigated. Comparisons of the expression profile of endometrial cells (ECs) from endometriosis patients and normal subjects revealed a consistent reduction in the expression levels of connexins (the protein components of gap junctions), including the most abundant isoform, Cx43. This was most notable in stromal cells as the disease progressed, but was also evident in epithelial cells. GJIC of ECs from endometriosis patients was also reduced compared to normal subjects, again most notably in stromal cells. In mimicking the endometriosis process, heterotypic interactions with mesothelial cells induced an increase in GJIC, with stromal cells from endometriosis patients showing the largest response. This enhanced coupling correlated with enhanced trafficking of Cx43 to the cell surface, and enhanced invasiveness through a mesothelial monolayer. Blocking GJIC between endometrial and mesothelial cells using either siRNA or a Cx43 specific peptide blocker dramatically reduced this enhanced invasiveness of endometriosis derived ECs, whether epithelial or stromal in origin. Finally, GJIC induction by mesothelial cells was shown to be mediated by a secreted factor, which is heat resistant and approximately 5–10K is size. This presents a unique opportunity for developing novel therapy for treating endometriosis that would prevent the formation of lesions that cause the pain and incidence of infertility associated with this widespread disease. Currently the only therapies are based on hormonal treatments that only further compromise fertility problems associated with endometriosis (representing 35% of the cases on infertility in the nation).

Support or Funding Information

Women's Health Initiative at UTHSA School of Medicine and CPRIT grants RP150600 and RP160844, and NIH grant UL1 TR001120 for Cores

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ANXA5 Suppresses COX‐2 Expression by Downregulating the PKC‐ζ–NF‐κB Signaling Pathway in Prostate Cancer Cells
Authors: Baek Hyoung Seok , Kwon Yeo‐Jung , Ye Dong‐Jin , Chun Young‐Jin
Published: June 2021

Annexin A5 (ANXA5) is a member of the annexin family of proteins. Previous studies showed that ANXA5 is involved in anti‐inflammation and apoptosis. However, detailed mechanisms of the role of ANXA5 in cancer cells are less well explored. In this study, we aimed to identify the cyclooxygenase‐2 (COX‐2) suppressive function of ANXA5 in prostate cancer cells. TNF‐α‐induced COX‐2 expression was suppressed by ANXA5 overexpression, and suppression of COX‐2 expression by auranofin, which may induce ANXA5 expression, was recovered by ANXA5 knockdown. In addition, ANXA5 knockdown induced phosphorylation of NF‐κB p65 in prostate cancer cells, indicating that ANXA5 causes COX‐2 downregulation via suppression of p65 activation. We also found that protein kinase C (PKC)‐ζ protein levels were upregulated by inhibition of ANXA5, although the mRNA levels were unaffected. We confirmed that upregulated COX‐2 expression caused by the inhibition of ANXA5 was suppressed by PKC‐ζ siRNA. In summary, our study demonstrates that downregulation of PKC‐ζ‐NF‐κB signaling by ANXA5 may suppress COX‐2 expression in prostate cancer cells.

Support or Funding Information

NRF‐2015R1A5A1008958

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CYP1B1 induces tumorigenic metastasis through promotion of uPAR and subsequently activation of uPAR signaling via regulation of MDM2‐uPAR system
Authors: Kwon Yeo‐Jung , Ye Dong‐Jin , Baek Hyoung‐Seok , Chun Young‐Jin
Published: June 2021

Human CYP1B1 is known as a major metabolizer for estrogen and shows tumor‐specific hyper‐expression. To explore the role of CYP1B1 on metastasis of human cancer cells, we studied the effects of CYP1B1 inhibition and activation in MCF‐10A, MCF‐7, MDA‐MB‐231, and HeLa cells. CYP1B1 significantly induced expression of urokinase‐type plasminogen activator receptor (uPAR) as well as uPA. Treatment with DMBA, a well‐known CYP1B1 inducer, enhanced uPAR expression while CYP1B1 siRNA and TMS (tetramethoxystilbene), a specific CYP1B1 inhibitor, suppressed uPAR. We also found that CYP1B1 activated uPAR signaling through regulation of related factors including integrin β1 and α5. Interestingly, uPAR overexpression only caused a significant increase of integrin β1 and α5 in protein levels, indicating protein degradation may play an important role in regulating integrin protein level. Surprisingly, CYP1B1 down‐regulated p53 expression through MDM2 activation and nutlin‐3a, an inhibitor for MDM2, blocked the promoting effects of DMBA on uPAR, which explains that CYP1B1 induced uPAR expression through regulation of MDM2‐p53 system. Taken together, our data suggest that CYP1B1 promotes cancer cell metastasis via activating uPAR pathway which is one of the targets of p53 signaling.

Support or Funding Information

This research was supported by a National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (NRF‐2015R1A5A1008958).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GPR56/ADGRG1 activation induces IL‐6 production in melanoma cell via the Gα12/13‐ RhoA‐ROCK pathway
Authors: Huang Kuan‐Yeh , Chiang Nien‐Yi , Lin Hsi‐Hsien
Published: June 2021

GPR56/ADGRG1, a member of the adhesion G protein‐coupled receptor family, is important for tumor progression. The role of GPR56 in melanoma development is unresolved. Here, we showed that GPR56 receptor ligation by immobilized CG4 mAb in melanoma cell enhanced the production of IL‐6, which promoted melanoma cell migration and invasion. siRNA‐mediated Gα12/13 knockdown significantly reduced CG4‐induced IL‐6 production in melanoma cell. Likewise, the IL‐6 level was decreased in melanoma cells expressing the dominant negative RhoA N19, treated with exoenzyme C3, or the ROCK inhibitors (Y27632 and H1152). We conclude that GPR56 activation‐induced IL‐6 production is mediated through the Gα12/13‐RhoA‐ROCK signaling pathway. IL‐6 in turn promotes melanoma cell migration and invasion. Our results indicate a critical role of GPR56 in melanoma progression.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Basis of Glucocorticoid Receptor Recognition of Both Unmodified and Methylated Ancient Binding Sites, Precursors for a Modern Recognition Element
Authors: Liu Xu , Weikum Emily , Ortlund Eric
Published: June 2021

DNA methylation plays crucial roles in gene silencing and development in eukaryotic cells. The most common DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine‐phosphate‐guanine (CpG) dinucleotide. Ancient genomes were enriched in CpG sites that may have served as transcription factor (TF) binding sites. These sites, through the process of methylation, deamination and subsequent mutation to TpG sites, may have maintained equal or enhanced TF binding. Recently, this has been described as a “mutator” system to produce genetic diversity during evolution. Here we test feasibility of this hypothesis by using an ancestral glucocorticoid receptor (GR) binding site (aGBS) that contains a CpG dinucleotide. We examine the binding of GR to ancestral unmodified, methylated and extant GBS and find two‐fold affinity increase upon the methylation and deamination. Additionally, this binding specificity is maintained from ancient steroid receptor AncSR2 to extant GR during the evolution. We also present the first structural studies of GR binding to unmodified and methylated aGBS. A methyl‐specific van der Walls contact with a Arg residue in the GR recognition helix is the molecular determinant of the binding specificity. Our study provides the first biochemical and structural evidence of high affinity binding for ancestral GR binding site containing methylated CpG, which could evolve to the extant, TpG‐containing site. This also indicates GR recognition of methylated CpG‐containing site is ancestral and offers new insight into evolution of steroid receptors and their binding sites.

Support or Funding Information

These studies were supported by National Institutes of Health [R01DK095750] and W.M. Keck Foundation Medical Research Grant to E. O. X.L was supported by an American Heart Association postdoctoral fellowship [17POST33660110].

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glycine betaine accumulation in Avicennia germinans propagules and seedlings as a function of abiotic stress
Authors: Warren Jasmine N. , Day Zachary , Shor Audrey C.
Published: June 2021

The black mangrove (Avicennia germinans) is native to the subtropical coasts of Florida and can tolerate moderate to high salinity environments. Propagules, germinated seeds that remain attached to the maternal plants until mature, are dispersed in shallow waters and transported through currents until washing ashore, and establishing roots on an appropriate substrate to initiate growth. Recently, the poleward expansion of A. germinans has been observed. The literature and findings in our laboratory suggest that A. germinans gains its salt and temperature tolerance in part by the modified amino acid, glycine betaine (GB). We sought to investigate the expression of GB in A. germinans as a function of abiotic stressors: high saline and low temperatures. The role of endogenous GB accumulation as a function of substrate condition and temperature was investigated in propagules. The accumulation of exogenous GB was measured in seedlings exposed to various saline and temperature environments. Propagules exposed to colder temperatures accumulated more GB (100–200 ug/umol) than moderate temperatures (50 ug/umol). Propagules exposed to 18 ppt saline accumulated more GB than those exposed to 36 ppt at 0°C. Propagules exposed to saline environments accumulated more GB at 0°C compared to sand and air samples. Significantly more propagules treated at 36 ppt developed into seedlings and demonstrated faster growth rates. Additionally, an exogenous application of 10 μM GB improved seedling survival after exposure to −2°C and high saline stress (60 ppt and 100 ppt). Preliminary findings demonstrate that APX and SOD enzymes are both present in the leaf samples after these stressors. These results further characterize the role GB contributes to the ability of A. germinans to acclimate following environmental stressors. Propagules express endogenous GB in response to abiotic stress, while exogenous application of GB may confer additional protection from continued stress. Subsequent investigations will further elucidate the roles APX and SOD may influence in the GB mechanism. The significance of these findings further characterize the mechanisms by which A. germinans survive high saline environments and colder temperatures.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein tyrosine phosphatase, receptor type B (PTPRB) inhibits brown adipocyte differentiation through regulation of VEGFR2 phosphorylation
Authors: Kim Won‐Kon , Han Baek‐Soo
Published: June 2021

Brown adipocytes have an important role in the regulation of energy balance through uncoupling protein‐1 (UCP‐1)‐mediated nonshivering thermogenesis. Although brown adipocytes have been highlighted as a new therapeutic target for the treatment of metabolic diseases, such as obesity and type 2 diabetes in adult humans, the molecular mechanism underlying brown adipogenesis is not fully understood. We recently found that protein tyrosine phosphatase receptor type B (PTPRB) expression dramatically decreased during brown adipogenic differentiation. In this study, we investigated the functional roles of PTPRB and its regulatory mechanism during brown adipocyte differentiation. Ectopic expression of PTPRB led to a reduced brown adipocyte differentiation by suppressing the tyrosine phosphorylation of VEGFR2, whereas a catalytic inactive PTPRB mutant showed no effects on differentiation and phosphorylation. Consistently, the expression of brown adipocyte‐related genes, such as UCP‐1, PGC‐1α, PRDM16, PPAR‐γ and CIDEA, were significantly inhibited by PTPRB overexpression. Overall, these results suggest that PTPRB functions as a negative regulator of brown adipocyte differentiation through its phosphatase activity‐dependent mechanism and may be used as a target protein for the regulation of obesity and diabetes.

Support or Funding Information

This work was supported by grants from the KRIBB and from the Research Program (grants 2016R1C1B2013430, 2015M3A9B5030308, 2015M3A7B6027948, and 2015M3A9D7029882) through the National Research Foundation of Korea.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acid Phosphatase in Soil
Authors: Ayers Katherine Elizabeth , Fogle Emily J.
Published: June 2021

Plants require phosphorus for growth, which they acquire from soil. This process is facilitated by the presence of enzymes, including acid phosphatases (AP). This enzyme catalyzes the cleavage of phosphate bonds, releasing the phosphate through hydrolysis. The degree of adsorption and activity of AP when adsorbed to soil is analyzed in order to determine how this environment effects the enzyme's action. A Bradford assay was used to determine concentration of AP and an activity assay utilizing fluorescent MUB to create a baseline characterization of AP in the presence of a clay, montmorillonite. Initial studies show that AP adsorbs to the clay and retains 34% activity. Future studies will look at the effect of time, concentration and temperature on activity levels.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tumor Endothelial Marker 1 (TEM1/endosialin/CD248) Enhances Wound Healing Process by Cooperation with Platelet‐Derived Growth Factor (PDGF) Receptor
Authors: Hong Yi‐Kai
Published: June 2021

Tumor endothelial marker 1 (TEM1), also named endosialin and CD248, is a type I transmembrane glycoprotein containing a C‐type lectin‐like domain. TEM1 is highly expressed on tumor‐associated fibroblasts, stromal cells, pericytes, and dermal fibroblasts. Dermal fibroblasts play a pivotal role in cutaneous wound healing which occurs in three dynamic and overlapping but distinct stages, including inflammatory phase, proliferation phase, and remodeling phase. However, the physiological function of TEM1 in wound healing is still unknown. In wound healing process both TEM1 and platelet‐derived growth factor receptor α (PDGFRα) expressions were highly upregulated in myofibroblasts. The cell activation, proliferation, and collagen deposition in granulation tissues were decreased and slower wound healing was found in TEM1‐deleted mice. In addition, the cell migration, adhesion and proliferation in NIH3T3 cells were decreased when TEM1 expression was knockdown by shRNA. The downstream signaling, mitogenic, and chemoattractive effects of platelet‐derived growth factor‐BB‐treated NIH3T3 cells were inhibited by knockdown of TEM1. TEM1 and PDGFRα were co‐localized in sub‐cellular organelles in fibroblasts and could be co‐immunoprecepitated. Based on these results we proposed that TEM1 in combination with PDGFRα plays a critical role in wound healing by enhancing the mitogenic and chemoattractive effects of platelet‐derived growth factor‐BB and collagen deposition in myofibroblasts.

Support or Funding Information

This work was supported by a grant from the Ministry of Science and Technology, Executive Yuan, Taiwan. (MOST 105‐2320‐B‐006 ‐039 ‐MY3 to Hua‐Lin Wu)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiac Nav1.5 Channel is Regulated by LITAF
Authors: Turan Nilufer Nermin , Moshal Karni S. , Roder Karim , Xie An , Lu Yichun , Werdich Andreas , MacRae Calum , Koren Gideon
Published: June 2021

Background

Voltage‐gated Na+ channels (Nav1.5) are highly expressed in the heart, trigger electrical impulses and contribute to the action potential (AP). Genome‐wide association studies suggest that lipopolysaccharide‐induced tumor necrosis factor‐alpha factor (LITAF) is a likely modifier of the QT interval. LITAF is an adapter molecule for members of the Nedd4‐like family of E3 ubiquitin ligases and is involved in endosomal trafficking. Nedd4‐2 is responsible for ubiquitination of Nav1.5 resulting in its degradation.

Goal

We postulate that LITAF inhibits the ubiquitination activity of Nedd4‐2 or restricts the access of Nedd4‐2 to the Nav1.5 complex on the plasma membrane, thus inhibiting channel ubiquitination and degradation via the lysosome pathway. Hence, a loss‐of‐function of LITAF (as seen in heart failure) will lead to increased degradation of voltage‐gated sodium channels and impair conduction in the heart.

Methods

Cultured 3‐week old rabbit cardiomyocytes (3wRbCM) were infected with adenovirus (MOI 10) expressing HA‐tagged LITAF or GFP. HEK cells stably expressing Nav1.5 were transfected with expression plasmids for HA‐LITAF, its PXY double mutant (HA‐LITAF‐Y23A‐Y61A) and/or Nedd4‐2. Patch clamping was performed to measure INa. Western blotting and co‐immunoprecipitation were used to measure protein levels and detect protein‐protein interactions, respectively.

Results

In 3wRbCM, Nav1.5 channel expression and Nav1.5 current were significantly increased by LITAF. These effects could be recapitulated in HEK cells stably expressing Nav1.5. However, the PXY mutations in LITAF abolished the LITAF‐dependent increase in Nav1.5 protein and encoded current. Immunoprecipitations demonstrated a physical interaction between LITAF and Nav1.5 or NEDD4‐2. Additionally, LITAF suppressed the expression of both endogenous and co‐expressed NEDD4‐2.

Conclusions

LITAF is a novel regulator of cardiac Nav1.5 and action potential duration, likely by affecting Nedd4‐2 levels via increasing its autoubiquitination activity in the cell.

Support or Funding Information

International Postdoctoral Research Scholarship Programme (2219) by The Scientific and Technological Research Council of Turkey.

The National Institutes of Health [R01‐HL‐096669, T32HL094300, R01HL110791, R01HL134706].

The Rhode Island Foundation [701‐7131778].

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Early growth response protein‐1 (Egr‐1) expression by hydrogen peroxide (H2O2) is mediated via c‐Src and PKB‐dependent CREB signaling events in vascular smooth muscle cells (VSMC)
Authors: Truong Vanessa , Rondeau Vincent , Jain Ashish , Srivastava Ashok K.
Published: June 2021

Increased generation of reactive oxygen species (ROS) is believed to play a key role in the pathophysiology of cardiovascular diseases. Excessive growth and proliferation of vascular smooth muscle cells (VSMC) have been suggested to be major contributors to vascular dysfunction. A potential involvement of early growth response protein‐1 (Egr‐1), a zinc‐finger transcription factor, in the development of vascular injury has been suggested. Recent studies have shown that H2O2, one of the most stable ROS, increases Egr‐1 expression in VSMC, however, signaling events leading to H2O2‐induced Egr‐1 expression are not fully understood. Here, we report that H2O2 increased Egr‐1 expression in a time and dose‐dependent fashion in A10 VSMC and pharmacological blockade of insulin‐like growth factor‐1 receptor (IGF‐1R) and c‐Src, by AG1024 and PP2 respectively, abolished H2O2‐induced Egr‐1 expression while AG1478, an inhibitor of the epidermal growth factor receptor (EGFR), and PP3, the inactive analog of PP2, had no effect. H2O2 also stimulated the phosphorylation of PKB, and siRNA‐induced silencing of c‐Src resulted in a significant reduction in PKB phosphorylation as well Egr‐1 expression. In addition, phosphorylation of cyclic AMP response element binding protein (CREB) was also enhanced by H2O2, however, inhibition of PI3K/PKB by wortmannin and SC‐66 attenuated this phosphorylation, as well as suppressed protein and mRNA levels of Egr‐1 induced by H2O. In summary, our data suggests that H2O2 provokes Egr‐1 expression through the activation of upstream tyrosine kinases IGF‐1R and c‐Src as well as PKB and CREB‐dependent signaling events in VSMC.

Support or Funding Information

(Supported by CIHR)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Revealing Subcellular Redox Dynamics with Multiplex Imaging of Compartment‐Specific Redox Probes
Authors: Tantama Mathew , Norley Jacob , Radhakrishnan Saranya , Rajendran Megha
Published: June 2021

Reactive oxygen species (ROS), redox enzymes, and redox buffers are central to the propagation of redox signals and oxidative stress between cells in the tissue environment and between organelles in the cellular environment. For example, hydrogen peroxide generated in mitochondria can alter the thiol oxidation states on proteins to alter their function, and subsequently redox active biomolecules such as glutathione can act as reducing equivalents in the face of oxidative stress. In some cases, it has become apparent that ROS mediate physiologically important signaling across compartments, such as in mitonuclear retrograde signaling. However, it has remained technically challenging to quantitatively correlate compartment‐specific redox dynamics with subcellular resolution within live single cells. In order to address this challenge, we are developing genetically‐encoded biosensors that enable the visualization of redox dynamics simultaneously within multiple compartments within the same cell. Our approach utilizes Förster‐type resonance energy transfer in a spectral relay strategy to extend the fluorescence emission of the redox‐sensitive green fluorescent protein (roGFP) into orange and red emission wavelengths. Furthermore, we are developing multiplexed, multiphoton imaging methods to study redox coupling between organelles and compartment‐specific dynamics in thick neural tissues.

Support or Funding Information

We gratefully acknowledge support from the Ralph W. and Grace M. Showalter Trust, the Purdue Research Foundation, and from National Institutes of Health grants R21 NS092010 and R21 EY026425.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Converting Nature's Switches Into Scientists' Tools: How Biophysical Insights Lay The Foundation For Artificial Control Of Protein Activity
Authors: Gardner Kevin H. , Dikiy Igor , Edupuganti Uthama , Orth Elizabeth , Abzalimov Rinat
Published: June 2021

Environmental cues regulate many biological processes, coordinating cellular pathways to respond to changing conditions. Such regulation is often initiated by sensory protein domains which expand their chemical repertoire by using small molecule ligands to convert environmentally‐triggered changes into altered protein/protein interactions. Several families of these domains have evolved with remarkable diversity in their inputs and outputs. Using a combination of biophysics, biochemistry and synthetic chemistry, we seek to gain insight into the mechanistic controls of such environmental sensing domains for both fundamental understanding and subsequent artificial control.

Here I will discuss examples of our work exploring how the Light‐Oxygen‐Voltage (LOV) protein domains convert changes in a common blue light stimulus into control of a wide range of output functions. Such photosensory domains utilize flavin chromophores and a photochemically‐generated protein/flavin adduct as a trigger for the allosteric control of effectors as diverse as kinases to DNA‐binding domains. While high‐resolution structures of inhibited LOV proteins have provided insights into certain regulatory principles, it has been difficult to apply similar techniques to spontaneously‐activated “dark noise” and photoactivated conformations that are essential for a full understanding of control. We have tackled this challenge by combining data from several biophysical approaches, including HDX‐MS and EPR spectroscopy, letting us observe and characterize these states. This information has shown several commonalities of LOV signaling in diverse classes of sensory proteins, and further, develop structure‐based mutations to shift these proteins among these states. Doing so, we have taken advantage of this mechanistic understanding to develop the artificial regulation of such systems, both in vitro and in living cells. Taken together, our work provides an integrated view of a fascinating class of natural switches and suggests routes by which these can be manipulated to achieve desired therapeutic and/or technological outcomes.

Support or Funding Information

Support from NIH R01 GM106239 is gratefully acknowledged.

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 Growth of Fusarium oxysporum Spore Isolates from Potato is Inhibited by 1,4‐dimethylnaphthalene
Authors: Adams Rachel , Campbell Michael
Published: June 2021

Prolongation of storage via temporary meristematic sprout inhibition has effectively been carried out in tubers after brief exposure to 1,4‐dimethylnaphthalene (DMN), a compound naturally abundant in the tissues of dormant potatoes. Not only does DMN suppress meristematic growth, but studies have shown that DMN greatly impacts the microbiome on the surface of potatoes, especially with regards to species diversity. Among the diverse microbiome population is Fusarium oxysporum, a particular strain of pathogenic fungus responsible for the plant disease fusarium wilt. F. oxysporum was successfully isolated from the surface of a tuber using the fusarium selective media Nash‐Snider, to which cultures were transferred and maintained on nutritionally complex potato dextrose agar plates. DNA sequencing using selective primers NL1, NL2, ITS1F, and ITS2R confirmed the fungus to be F. oxysporum. A single spore isolation technique was utilized to record the number and growth response of spores treated with DMN vs spores treated with water (control). Single spore isolate plates were contained in vacuum sealed chambers and incubated at a constant 22C while exposed to 7.5ul DMN via aerosol dispersion for four days. After a day of rest, the spores on each plate were measured and counted. The results concluded spores exposed to DMN showed slower diameter growth than those treated with water. Knowing the effect of DMN on F. oxysporum is critical in controlling and preventing harvest loss brought on by fusarium wilt. In an attempt to gain more insight on the specificities of the DMN‐fungal conundrum, further studies are still being conducted.

Support or Funding Information

Supported in part by a grant from the 1,4‐Group, Meridian, ID

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 a Six Week Aerobic Exercise Intervention on the Composition of Oral and Skin Microbiota: a Pilot Study
Authors: Mailing Lucy , Allen Jacob , Niemiro Grace , Cohrs Josh , Holscher Hannah , De Lisio Michael , Woods Jeffrey
Published: June 2021

Introduction

Exercise causes numerous physiological changes that may impact microbial communities at mucosal surfaces. Previously, our lab has shown that six weeks of aerobic exercise significantly alters the composition of the human gut microbiota. In the present study, we characterized the effects of an aerobic exercise on skin and oral microbiotas.

Methods

Previously sedentary but otherwise healthy adults (n= 10) were recruited to undergo a six‐week aerobic exercise intervention. Participants performed three exercise sessions per week on a bike or treadmill, starting with 30 min at 60% HRR and progressing to 60 min at 75% HRR by week six. Skin and oral swabs were collected at baseline and after cessation of exercise training. An additional skin swab was collected after the first exercise session to measure acute changes in skin microbiota. Skin swabs were moistened with sampling buffer before swabbing the antecubital fossa. Oral swabs were taken by swabbing the center of the tongue. DNA was isolated from samples and 16S rRNA gene sequencing was performed.

Results

Six weeks of chronic exercise caused a significant shift in the skin microbiota (unweighted PERMANOVA p = 0.017) that was not evident after a single acute exercise bout. Oral microbial communities were not significantly different from baseline after six weeks of chronic exercise training. For both skin and oral samples, there were no significant changes in alpha diversity or species evenness with exercise, and no significant differences in relative abundance on the genus level.

Conclusion

A six‐week aerobic exercise intervention is sufficient to alter the skin microbiota, but not the oral microbiota. This study should be replicated with a larger sample size to confirm these preliminary findings.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Meeting the Challenges of Minorities in Higher Education: A Review of the Underrepresentation of Hispanics in Medical School
Authors: Miller Alyson Jeanine , Ynalvez Ruby A.
Published: June 2021

Bridging the gap between Hispanics and the medical profession is critical to addressing the diversity disparities and in upholding quality healthcare to individuals of all ethnic backgrounds. The deficiency in Hispanic healthcare providers is attested by the low representation of Hispanics in medical schools across the nation. Despite current research, there is lack of research that would help develop a comprehensive approach to increase Hispanic representation in the medical profession. The objective of this study is to interpret, analyze, and integrate information about the challenges experienced by underrepresented minorities (URM) in their quest to medical school. This research seeks to answer the question: What individual‐level and sociocultural factors impede Hispanic participation and representation in medical school? To answer this question, a library research to review the literature using peer‐reviewed articles was conducted. The articles were read thoroughly, scrutinized, evaluated for their relevance, and interpreted with new understandings. The analysis of key factors contributing to low representation of minorities in higher education was done through synthesis of the pertinent information reported in previous studies. Our review showed that a key factor contributing to low representation of Hispanics in medical school was their low acceptance rate. While seemingly obvious and its proximal correlates equally obvious (i.e., low academic performance metrics such as grade point average (GPA), science GPA, and MCAT scores), the less visible and the more distal reasons relating to educational inequalities such as deficiency in resources, lower quality schools, working student status, and constrained financial means for test preparation courses and materials all conspired to deter and diminish competencies that are critical to maintaining high academic performance metrics. Other seemingly inconspicuous and distal reasons and yet adversely impacting academic performance metrics have to do with inadequate social capital such as lack of access to academic support networks, academic mentors, real‐life role models, and familial support. Combatting these hindrances requires special attention to develop outreach initiatives with primary and secondary schools to attract and better prepare underrepresented minority students. Implementation of summer programs, collaborative enrichment initiatives, and mentoring systems with the aid of medical schools as well as the primary and secondary schools are indeed needed to stimulate growth in Hispanic representation.

Support or Funding Information

Act on Ideas Texas A&M International University and College of Arts and Sciences Texas A&M International University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Creativity in biomedical science education
Authors: Macaulay Janet Olwyn , Kim Alice Minji , Speed Caroline Jane
Published: June 2021

Our students – who are our future workforce, need to develop the skills that will prepare them for the workplace of the future including creativity. Evidence of the growing recognition of the value of creativity is demonstrated by the revised cognitive category of Bloom's taxonomy in which “Create” replaced “Evaluate” as the pinnacle cognitive skill of the cognitive domain. In Australia, creativity has been incorporated into the policies of the Australian education sector. Creativity is now listed as a skill all Bachelor degree graduates should possess by the Australian Qualification Framework – supporting the importance of creative thinking. There is also published research highlighting the need to promote creative thinking in science education in particular the methods that can be used to achieve and measure creativity.

As scientist, we know that science is a creative endeavor with scientists routinely taking creative approaches to discovery, problem solving and inquiry. Although there is this increased acknowledgment of the value of creativity, we were concerned that our educational programs were not offering sufficient opportunities. Students need opportunities to develop their creative capabilities but these opportunities need to be explicit to the students. Students also need to understand what creativity is.

The aim of this research was to map the Bachelor of Biomedical Science degree for opportunities provided to students to develop their capacity as creative thinkers and to collect data from students of their perception of creativity in the curriculum. All units in the program were mapped for incorporation of learning activities that would develop/require creativity. This data was collected from the University unit guides and manuals. Student perspectives were collected from surveys completed by currently enrolled students across the three years of the program.

The data collected from the mapping exercise showed that most units in the course including assessment activities require creativity. In many examples, the instructions and marking criteria did not explicitly mention creativity ‐ hence it was not clear to the students. The survey data indicated that the students would like the inclusion of more activities/assessment that develop creativity skills and that this should be included in the marking schemes.

Based on this data we are now working to, make creativity more explicit to students and redesigning some learning and assessment activities to incorporate creativity and assessment that rewards students for their creative approaches.

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