bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural and Functional Characterization of the Adult Giant Danio Heart
Authors: Hester Kamil J , Carrillo Casandra , Kyaw Pyi T , Washam Cassie , Patel Purva , Abouelkheir Mahmoud , Hasegawa Ryoji , Moreno Rodriguez Ricardo A. , Reddy Anilkumar , Lafontant Pascal J
Published: June 2021

Cardiovascular diseases remain that number one cause of death in the northern hemisphere and are increasing globally in developing economies. In spite of evolutionary separation between fishes and mammalian, the use of non‐mammalian model species has significantly increased our understanding of cardiac diseases. We have recently described the growth and cardiovascular development of the Devario malabaricus, a giant danio (GD) species closely related to the zebrafish. We hypothesize that the adult GD heart can serve as a robust non‐mammalian model for in vivo studies of cardiac biology. Using immunostaining, transmission electron microscopy, scanning block‐face electron microscopy, and Doppler, we have characterized the anatomical and functional characteristics of the adult GD heart. First, we found that the adult giant danio possesses a thick and highly vascularized compact heart. Second, the GD heart possesses a junctional region populated with a fibroblasts network similar to that observed in zebrafish. Third, multiple large coronary vessels investing the compact myocardium are connected to and continuous with atrioventricular (AV) canal lumen. In addition, the AV canal and bulbus are highly innervated, suggesting complex regulation in heart function. Moreover, we demonstrate that their cardiac function can be measured using Doppler flow velocity. These studies along with previous findings support the GD as a robust model for the study of cardiac biology.

Support or Funding Information

1R15HD084262‐01, Buehler Family Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Size and Shape of the C4 Segment of the Intracavernous Carotid Artery Intermediate to the Petrolingual Ligament and the Abducens Nerve: Implications for Neurosurgery
Authors: Nestor Nicholas , Hunter Dawn , Ritz Brianna , Zdilla Matthew
Published: June 2021

Neurosurgical management of the neurovascular structures encompassed within the cavernous sinus may be aided by improved understanding of neurovascular anatomical relationships. Therefore, this study assessed the morphometry of the C4 segment of the intracavernous carotid artery as it relates to the petrolingual ligament and abducens nerve in cadavers. A total of 40 sides were assessed (18 female and 22 male sides). The area of the carotid artery intermediate to the petrolingual ligament and the abducens averaged 17.56±10.86mm2 (Mean±SD). The anterior and posterior distances from the petrolingual ligament to the abducens nerve averaged 3.12±1.72mm and 3.17±1.58mm, respectively. Further, unpaired T‐tests revealed no significant differences between sexes with regard to the area (p = 0.82), anterior distance (p = 0.96), and posterior distance (p=0.94). Further, three morphotypes for the window between the petrolingual ligament and abducencs were identified; namely, quadrilateral (n=30), triangular (n=8), and atretic (n=2). The results of this study will help improve the approach to lesions including C4 aneurysms and local tumors including those of the abducens.

Support or Funding Information

Funding: WV Research Challenge Fund [HEPC.dsr.17.06]

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Predictive model of cranial growth in infants with cranial deformation
Authors: Aldridge Kristina , Shock Leslie A , Panchal Sarjukumar , Martin Andrea L , Mukherjee Anish , Chakraborty Sounak , Muzaffar Arshad R
Published: June 2021

Differences in cranial growth trajectories define morphological variation in both normal and pathological conditions. Predicting divergent trajectories is key to understanding how and when morphological variation is produced. The incidence of cranial deformation in human infants, specifically deformational plagiocephaly and brachycephaly, has increased dramatically with the initiation of the Back to Sleep campaign in the 1990s. Treatment often requires several months of helmet therapy. However, practitioners have no objective means for reliably estimating the necessary duration of therapy. The goal of this study was to develop a model to provide individualized predictions of duration of helmet therapy using cranial growth trajectories.

We obtained STARscanner® 3D laser scan images of 541 children seen in clinic for abnormal head shape at their first visit and again at completion of helmet therapy with IRB approval. Measurements obtained from images at both visits included head circumference, cephalic ratio, oblique diameter difference, cranial vault asymmetry index, sex, and age. A Multivariate Random Forest Model was used to fit the model with 200 trees and 441 patients in the training set. A five‐fold cross‐validation was performed to assess accuracy of the model's prediction of cranial measurements with respect to the duration of helmet therapy. Data from the remaining 100 patients were used to validate the resulting model.

Our Multivariate Random Forest model predicts accurately cranial growth trajectories during helmet therapy in infants with cranial deformation. The five‐fold cross validation error of our model is more than 22% lower than standard linear models, with average mean squared error of 0.2568. These results demonstrate that necessary duration of helmet therapy can be accurately predicted using our smart machine learning method.

Thousands of infants per year are treated for cranial deformation. Using machine learning, we have developed a model that predicts individualized, evidence‐based treatment strategies accurately. The model is incorporated into a web‐based interface that can be used by practitioners in real time. Used in combination with practitioners' expertise, families will be informed about the likely length of treatment and degree of improvement that may be expected for their individual child. Importantly, while the model developed here is specific to infant cranial deformation, the approach is easily translatable to other craniofacial anomalies, as well as to normal patterns of cranial growth.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Airway management and anatomical development of the pediatric aerodigestive tract (ADT): current knowledge and clinical applications
Authors: Curcio Daniella Francon , Barros Mirna Duarte , Liquidato Bianca Maria , Mendes Carlos Jose Lazzarini , Cabral Julia Chaeves , Morad Helena Machado , Laitman Jeffrey T.
Published: June 2021

Pediatric airway management can be difficult and especially hazardous due to a number of anatomical features of the developing aerodigestive tract (ADT), which is so different from the adult. Such differences have been presented annually since 2013 at EB meetings (Micham and Laitman, 2013; Curcio et al., 2014; Curcio and Laitman, 2015; Meaike et al., 2016; Gupta et al., 2016; Curcio et al., 2017, Curcio and Laitman, 2018). Particularly in regards to clinical applications involved in airway management procedures, our attention has been drawn to bedside tests that have been used to predict difficult intubation and difficult laryngoscopy in infants and young children. In stage 1 of this study we compiled a multi‐field database review of the mostly used bedside tests that predict difficult intubation of infants under 5 months of post‐natal life. We verified that ease of tracheal tube passage depends on the size of the cricoid ‐ the circular or near circular cartilage located at the inferior most portion of the funnel‐shaped infant larynx. We also selected the Laryngoscore (Piazza et al. 2014; 2018; Incandela et al, 2018), the frontal plane to chin distance, and the FPCD/weight ratio (Mansano et al., 2016) to be included in the next stages of this study. Our hypothesis is that it might be possible to adapt and increase the accuracy of such bedside tests and protocols that predict difficult intubation and laryngoscopy in infants and young children. This study is part of a larger project designed by our Brazilian‐US joint research team that seeks to explore possibilities and optimize airway management in elective procedures, critical and emergency care of pediatric patients under 5 months of age.

Support or Funding Information

In conjunction with the Building Bridges initiatives. Funded in part by CNPq ‐ Brazilian National Counsel of Technological Development.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Connexin 43 Function in the Cranial Neural Crest
Authors: Jourdeuil Karyn , Taneyhill Lisa Anne
Published: June 2021

Connexins are transmembrane proteins that cluster to form intercellular channels called gap junctions, which function to allow direct diffusion of small molecules and ions between adjacent cells. Gap junctions are composed of two connexons (one on each cell which are docked head‐to‐head) consisting of either homomeric or heteromeric connexin proteins. Connexin 43 (Cx43) is one of the predominant components of gap junctions and is expressed in multiple organisms and their tissues. To date, however, very little is known about the role of gap junctions, and specifically Cx43, in the neural crest. Our prior immunohistochemical analyses revealed that Cx43 is expressed in premigratory cranial neural crest cells both prior to and during their epithelial‐to‐mesenchymal transition (EMT), and in migratory neural crest cells. Given the robust expression of Cx43 in premigratory neural crest cells, we sought to elucidate the role of Cx43, and gap junctions in particular, during neural crest cell EMT. To this end, we evaluated the presence of gap junctions in the neural crest and performed molecular and chemical perturbation experiments to alter Cx43 and general gap junction function. Our results reveal that gap junctions form between migratory neural crest cells, demonstrated in the embryo and in explants of premigratory neural crest tissue cultured ex vivo. Live‐imaging of embryos initially possessing Calcein‐AM (a dye that only traverses gap junctions) in a small number of premigratory neural crest cells demonstrates passage of dye to adjacent cells over time. Additionally, depletion of Cx43 from premigratory neural crest cells reveals changes in some neural crest cell molecular markers. In ex vivo explants, Cx43 knockdown in migratory neural crest cells precludes transfer of Calcein‐AM dye, and abrogation of gap junction function through chemical inhibition hinders neural crest cell EMT and/or migration. In future experiments, we will ascertain the effects of Cx43 knockdown and overexpression on neural crest cell EMT and migration in vivo by examining passage of Calcein‐AM dye to assess gap junction function and through immunohistochemistry to evaluate changes in neural crest cells. These data will provide insight into how neural crest cells interact and communicate during EMT and migration to allow for proper embryonic patterning throughout vertebrate development.

Support or Funding Information

The work was supported by grants to L.A.T. (NIH R01DE024217, American Cancer Society RSG‐15‐023‐01‐CSM).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developmental localization of syntaxin 1 and synaptosomal‐associated protein 25 in rat palate
Authors: Hakami Afaf , Shafei Ahlam , Alhakami Khalid , Hakami Zaki
Published: June 2021

Introduction

The palate, which plays an important role in oral cavity function, including the mastication and swallowing of food, obviously differs structurally with feeding habits. Nevertheless, the palate is considered the most common location of both benign and malignant minor salivary gland tumors. Soluble N‐ethylmaleimide‐sensitive fusion protein attachment protein receptor (SNARE) proteins are important in the control of tumorigenesis through the regulation of multiple signaling and transportation pathways. However, the developmental distribution of SNARE proteins on the palatal structures has been scarcely reported. Therefore, this study aimed to investigate SNARE proteins, syntaxin‐1, synaptosomal‐associated protein of 25 kDa (SNAP‐25) during the development of rat palates.

Materials and Methods

Immunohistochemistry for protein gene product 9.5 (PGP 9.5), a pan‐neuronal marker, syntaxin‐1 and SNAP‐25 was performed on frozen sections of rat palatal tissues at different stages.

Results

PGP 9.5 immunoreactive fibers (IRF) were present in developing palate. They were observed along the palatal sub‐epithelial layer and the underlying connective tissues in association with developing acini, ducts, blood vessels. Nerve fibers containing syntaxin‐1 and SNAP‐25 showed similar distribution to PGP 9.5 and thick bundles of nerve fibers were observed with smooth muscles and varicoses. With maturation after postnatal day 7 (PN7), thin nerve fibers containing PGP 9.5 and SNAP‐25, but less syntaxin‐1, were detected penetrating into the epithelium, reaching beneath the cornified layer. They ramified out from subepithelial thick bundles into connective tissues until encircling the secretory units observed (PN21). In adults, the number of syntaxin‐1‐IRF was low in comparison with SNAP‐25 and PGP 5 fibers.

Conclusion

The findings indicated the importance of SNARE proteins during the maturation of the palatal tissues.

Support or Funding Information

None

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
G‐Triplex DNA Formation Determined by SPR and Native Agarose Gel Electrophoresis
Authors: Al‐Der Carina C , Smith Sydney E , Tippayasak Krishna , Tuesuwan Bodin , Kerwin Sean M , David Wendi M
Published: June 2021

G‐quadruplex DNA structures (G4 DNA), such as those found in human telomeres, may play important roles in cellular replication and gene regulation. Dysregulation of processes involving G4 DNA is associated with cancer and some neurodegenerative diseases. G4 DNA, composed of stacked G‐tetrads based on hydrogen bonding between guanine nucleotides, has been characterized by a number of techniques, including the gel electrophoresis and surface plasmon resonance (SPR) methods used in the current study. G‐triplex DNA structures (G3 DNA) are folding intermediates of some quadruplexes, but their role in vivo is less clear. To further characterize G3 DNA we adapted a native agarose gel electrophoresis method for separating small oligonucleotides and also used SPR to probe the extent of folded and unfolded structures. We have previously used SPR to calculate the extent of intramolecular folding in the human telomeric G4 sequence under varying buffer conditions. G3‐forming oligonucleotides from a truncated human telomeric sequence, truncated thrombin binding aptamer sequence, and other similar G‐rich sequences were subjected to the same SPR method. The extent of folding was determined by calculating % hybridization values for a C‐rich complementary duplex binding probe to the immobilized G3‐forming sequence. Under most buffer conditions, hybridization to the truncated thrombin binding aptamer was >50%, in agreement with the lower thermal stability of this G3‐folded structure. Differences in kinetic profiles for the hybridization of the probe in the presence of different cations (Li+, Na+, K+, Ca2+) were apparent. In contrast, almost no hybridization was observed for a G3TG3TG3 sequence under similar buffer conditions. Analysis by native agarose gel electrophoresis indicates the persistence of folded structures for the truncated human telomeric sequence (G3), similar truncated telomeric G‐rich (G3) sequences, and a G4‐forming sequence from c‐MYC, but not for the truncated thrombin binding aptamer sequence.

Support or Funding Information

Cancer Prevention and Research Institute of Texas

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sequence and Environmental Effect on the Formation of G‐Triplex DNA
Authors: Bracey Harrison , Baqdounes Yasmin , Navapan Nattinee , Tuesuwan Bodin , David Wendi , Kerwin Sean
Published: June 2021

It has recently been shown that certain G‐quadruplex DNA (G4) structures fold via a “G‐triplex” intermediate, and that the corresponding truncated G‐quadruplex‐forming oligonucleotides also adopt G‐triplex structures. However, it is not clear how general G‐triplex formation is, or how sequence and buffer conditions affect G‐triplex formation. Our approach to investigate G‐triplex formation by a series of truncated G4‐DNA sequences used CD spectroscopy, UV thermal‐difference spectroscopy, CD derived and UV derived melting temperature measurements, and native PAGE gels to characterize the topology and thermal stability of these structures in Li+, Na+, K+, Mg2+ and Ca2+ containing buffers.

G‐triplex formation is a general phenomenon for a wide range of truncated G4 DNA sequences. We examined 16 different variants of the G≥2T1–4G≥2T1–4G≥2 sequence as well as truncated versions of the TBA and human telomeric G4 DNA in both forward and reverse permutations. All of these sequences can adopt G‐triplex structures. However, we note that the number of nucleotides in the loop and sequence direction affect G‐triplex topology. Sequences with longer G‐tracks tends to form parallel G‐triplex, as do sequences with fewer loop residues. Permutation of the direction of the truncated G4 DNA sequence also affect G‐triplex folding topology. Environmental effects on topology were also noted, with divalent metal ions (Mg2+ and Ca2+) favoring parallel topologies. Using thermal‐difference spectroscopy, we have identified two families of spectra that correlate to G‐triplexes with a parallel topology. Analysis by native PAGE indicates that some sequences may form multiple G‐triplex topologies under certain environmental conditions.

Temperature melts have provided information regarding sequence composition, sequence direction, and environmental conditions on the thermal stability of G‐triplex DNA. Sequences with longer G‐runs form G‐triplex structures that are more thermally stable (GGGG > GGG > GG). Shorter T‐loops form G‐triplex structures that are more thermally stable (TT >TTT > TTTT). Direction of sequence also effects the stability. Environmental conditions such as the presence of different cations have an effect of stability (Ca2+ > K+ > Mg2+ ≥ Na+).

The biological relevance of G‐triplex DNA is still not entirely understood. Understanding how sequence effects and environmental conditions affect G‐triplex structure and stability is essential for better studying G‐quadruplex DNA and the biological relevance of G‐triplex DNA.

Support or Funding Information

CPRIT HIHR Grant RP160852 and Texas State University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing Basal‐like Triple Negative Breast Cancer using Gene Expression Analysis: A Data Mining Approach
Authors: Alsabi Qamar , Zadeh Amir Hassan , Ramirez Jaime , Nosoudi Nasim
Published: June 2021

Triple‐negative breast cancer (TNBC) constitutes approximately 20%–25% of all breast cancer cases with poor prognosis. It is unresponsive to targeted hormonal therapies, which limits treatment options to nonselective chemotherapeutic agents. Recently, gene expression analysis has shown promise to characterize triple negative breast cancers (TNBC). Although TNBCs have many basal‐like breast cancer characteristics, the relationship between clinically defined triple‐negative breast cancer and the gene expression profile of basal‐like breast cancer (BLBC) is not fully examined. The purpose of this study is to assemble publicly‐available TNBC gene expression datasets generated on Affymetrix gene chips and define a set of genes, or gene signature, that can classify BLBC and Non‐BLBC subtypes under TNBC type. We compiled over 3,500 breast cancer gene expression profiles from several individual publicly available datasets and extracted Affymetrix gene expression data for 580 TNBC cases. Several popular data mining along with variable reduction and feature selection techniques were applied to the resultant data sets to build predictive models to understand molecular characteristics and mechanisms associated with BLBCs and classify them more accurately according to important features extracted from Microarray data analysis of BLBC and Non‐BLBC cases. Our result can lead to proper identification and diagnosis of BLBCs, which can potentially direct clinical implications by dictating the most effective therapy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening for Novel Regulators of Wingless Signaling
Authors: Marsh‐Armstrong Gwynn Isabel , Kennell Jennifer
Published: June 2021

The Wingless (Wg) pathway within Drosophila melanogaster is a well characterized intercellular signaling network that has been implicated in tissue specific cellular differentiation. However, the mechanism by which Wg signaling contributes to tissue specific changes in gene expression is still unclear. Previously, the Kennell Lab conducted a series of misexpression screens in order to identify novel regulators of Wg signaling. Wg signaling alters gene expression in various tissues through the action of the transcriptional coactivator Armadillo (Arm). As such, the Kennell lab was able to identify potential regulators of Wg signaling during eye development by measuring whether the alteration of a gene's expression using UAS enhancers had any effect on the development of an Arm‐dependent small‐eye phenotype. Once genes known to cause apoptosis independent of Wg signaling were discarded from the list of genes that caused suppression of the small‐eye phenotype, fifteen promising candidates remained. Of these identified genes, siz and CG5902 were selected for further in vivo characterization. The gene CG5902 has never before been characterized and the gene siz has been implicated in actin cytoskeleton organization and central nervous system development. The gene CG5902 was successfully subcloned into expression vectors and transfected into Kc167 cells in order to test Arm target gene expression using reporter gene assays. Additionally, siz and CG5902 knockdown strains of Drosophila melanogaster are currently being created in order to further characterize the effect of these genes on Arm target gene expression and eye development.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SV40 virion formation functions as a novel epigenetic switch controlling early and late transcription
Authors: Milavetz Barry , Balakrishnan Lata , Ajeeth‐Kumar Meera
Published: June 2021

Simian Virus 40 (SV40) is a small double‐strand DNA virus, which exists in the form of chromatin throughout its lifecycle. Because of its chromatin structure, the virus is expected to undergo the typical forms of epigenetic regulation, including differential nucleosome location and histone modifications. In order to characterize the epigenetic changes occurring during the formation of virions from late‐stage SV40 minichromosomes (encapsidation), we have compared the location of nucleosomes containing acetylated or methylated lysines from the histone tails of H3 and H4 present in the chromatin from late‐stage minichromosomes, encapsidation intermediates, and disrupted virions. In late‐stage minichromosomes, we found that nucleosomes carrying histone modifications were primarily found in the SV40 regulatory region within the enhancer and at the early and late transcription start sites. Significantly, the nucleosome containing modified histones within the enhancer would be expected to repress early transcription by overlying regulatory sequences in the SP1 and enhancer regions required for early transcription while still allowing for late transcription. In contrast, in chromatin from virions the nucleosome found in the enhancer has been shifted approximately 70 base‐pairs toward the late region uncovering the regulatory sequences necessary for early transcription and likely repressing late transcription. This encapsidation‐dependent epigenetic switch was associated with the final stages of virion formation.

Support or Funding Information

This work was funded by grants from the National Institutes of Health, AI094441 (to B.M.) and GM098328 (to L.B.) and P20 GM113123 (to Epigenetics Core)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“The Flexible Nucleosome: A Cross‐Kingdom Perspective”
Authors: Mohamed Farzanna A. , Melters Daniël P. , Dalal Yamini
Published: June 2021

In order to maintain genomic integrity, chromosomes must be segregated faithfully. The centromere is the region of the chromosome that seeds the kinetochore and is epigenetically marked by the centromere‐specific histone H3 variant CENP‐A/CENH3. Although centromeres are functionally essential, their underlying DNA sequences and associated proteins are rapidly evolving. This is known as the centromere paradox. CENP‐A's sequence is highly diverged across species, yet its function to seed the kinetochore remains intact. One possibility is that specific structural features of CENP‐A nucleosomes are conserved across the species. Indeed, in silico, in vivo, and in vitro studies have focused on the characterization of the dynamic structural behaviors of human and yeast CENP‐A nucleosomes. This dynamic behavior is thought to be predominantly driven by its αN‐helix, loop L1, and four‐helix bundle. Recently, we developed a technique to directly assess CENP‐A structural plasticity by using single‐molecule nano‐indentation force spectroscopy. We observed that human CENP‐A nucleosomes are twice as elastic compared to canonical H3 nucleosomes. In order to determine whether this observation is unique to human CENP‐A or represents a universal feature that defines a rapidly evolving CENP‐A nucleosome, we set out to investigate the elasticity of CENP‐As derived from 17 distinct species, including those that have holocentric chromosomes, spanning the three main eukaryotic kingdoms. Here, we present a progress report on our efforts to elucidate structural features underpinning the evolution of centromeric histones and its associated chromatin, which in turn serves as a model for the study of the evolution of epigenetic systems.

Support or Funding Information

Intramural Research Program of CCR

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining if Mating Type Proteins Play a Role in Immaturity of the Ciliate Tetrahymena thermophila
Authors: Enerson Rebecca Leigh , Cervantes Marcella
Published: June 2021

Tetrahymena thermophila is free‐living ciliate, commonly found in freshwater ponds, and is used as a model research organism. Mating type gene expression is induced in mature cells after they have been starved and put in the presence of cells of a different mating type (Tetrahymena Genome Database, November 10, 2018). T. thermophila are known to have developmental stages of maturity, and immature cells are unable to mate, although the scientific processes that prevent mating are unknown. It is known that mating type gene expression increases one‐hundred fold from logarithmic phase growth to three hours of starvation (Tetrahymena Genome Database, November 10, 2018). It is currently unknown if the mating type genes are expressed in immature progeny since immature cells do not mate.

The goal of this study was to determine if immature progeny cells expressed mating type proteins under a variety of conditions. We hypothesized that mating type genes would not be expressed in immature progeny because T. thermophila will not mate until they reach an level of maturity, and the expression of the mating type genes is essential for mating (Rogers and Karrer, 1985). We plan to quantify the mating type gene expression using q‐RT PCR and RNA Seq transcriptase analysis.

This study mated two strains, one mating type VI and II, of T. thermophila and isolated the progeny based on drug sensitivity differences from parental cells. For the q‐RT PCR experiment, mating type gene expression for the parental strain was tested at log phase, thirty minutes of starvation, and two hours of starvation. The mating type gene expression for the progeny strain was tested at the same three conditions as the parental strain. RNA samples from each of the six conditions were isolated and used to create cDNA. The cDNA was confirmed through a PCR containing a genomic control, mating type primers, starvation versus log primers, and negative control primers. Then qPCR was utilized to quantify the expression of mating type genes in the six previously described conditions. RNA Seq was utilized to quantify the expression of all genes for triplicate samples of parental cells and progeny, each starved for 24 hours.

Mating type gene expression will be validated through the cross referencing of quantified results provided by RNA Seq and qPCR for T. thermophila and are hypothesized to be lower or absent in progeny samples. There may also be differences in expression levels of transcription factors between the progeny and mature cells. However, if there is no difference in either mating type gene or transcription factors, then prevention of mating in immature cells occurs after transcriptional processes and needs further investigation.

Support or Funding Information

Albion College Foundation for Undergraduate Research, Scholarship, and Creative Activity

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of lncRNA CRNDE in obesity‐associated endothelial dysfunction
Authors: Nguyen Nghi Minh , Moran Matthew , Sun Xinghui
Published: June 2021

Obesity, a risk factor for type II diabetes and cardiovascular disease affects 35.7% of the U.S. population and costs approximately $210 billion per year. Endothelial cells (ECs) are cells that line blood vessels, are important in signaling and transport and have been implicated in obesity. The role of long non‐coding RNA (lncRNA) in regulating endothelial function, especially under disease conditions, has been understudied. In an RNA‐seq analysis of palmitate‐treated ECs, the lncRNA Colorectal Neoplasia Differentially Expressed (CRNDE) was determined to be upregulated. The purpose of this study is to determine the role of CRNDE in EC function through examination of various signaling pathways and exploring how obesogenic stimuli induce CRNDE expression. To determine the effect of obesogenic stimuli on CRNDE expression, HUVECs were treated with albumin‐conjugated palmitic acids or albumin for 4 or 12 hours and CRNDE expression was determined by qPCR. CRNDE is significantly increased in palmitic acid‐treated HUVECs after 12 hours compared with cells treated with albumin. Bioinformatic analysis of CRNDE and its promoter reveals the presence of p53 binding sites at CRNDE promoter which mediate the induction of CRNDE. To test this, CRNDE expression will be examined in HUVECs in the presence or absence of p53 knockdown. Chromatin immunoprecipitation will be used to determine the binding of p53 at the CRNDE promoter in HUVECs. Loss‐of‐ and gain‐of‐function studies will be performed to examine the signaling pathway that is regulated by CRNDE. Understanding the role of CRNDE in obesity‐associated endothelial dysfunction could provide new targets to reduce vascular complications associated with obesity and diabetes.

Support or Funding Information

UCARE

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of miR‐21‐5p and ‐3p expression and function during pathological neovascularization
Authors: Tripodi Giuseppe Daniele , Thounaojam Menaka C , Bartoli Manuela
Published: June 2021

Retinal neovascularization (RNV) is a sight compromising complication of diabetes and prematurity. Previous studies from our laboratory have shown that the small noncoding microRNA, microRNA‐21‐5p (miR‐21‐5p), is an inducer of RNV. Emerging new evidence has shown that miRNA passenger strands, the 3p forms, which were thought to be degraded, possess biological functions. In this study, we have investigated the role of miR‐21‐3p in RNV. In particular, we have assessed the effect of miR‐21‐3p on the same molecular targets of miR‐21‐5p: the tissue inhibitor of matrix metalloproteinases 3 (TIMP3), peroxisome proliferator‐activated receptor alpha (PPAR‐α) and pigment epithelium‐derived factor (PEDF). Real‐time PCR (RT‐PCR) showed that the expression of miR‐21‐3p is significantly upregulated in retinas and blood spot samples of mice subjected to oxygen‐induced retinopathy (OIR), a model of RNV. Furthermore, we noted a significant upregulation of miR‐21‐3p levels, measured by RT‐PCR, in human retinal endothelial cells (HuREC) and pigment epithelial cells (ARPE‐19) when exposed to angiogenic stimuli such as 20ng/mL of vascular endothelial growth factor (VEGF) or to hypoxic conditions (pO2= 2+/−0.5) for different times (1, 3, 6, and 12 hours). Transfection of HuREC and ARPE‐19 with miR‐21‐3p mimic (50nM) for 24 and 48 hours had only marginal inhibition on the miR‐21‐5p target genes (TIMP3, PPARα, and PEDF). Additionally, blockade of miR‐21‐3p achieved by transfecting cells with a specific miR‐21‐3p inhibitor (50nM) did not rescue VEGF‐changes in TIMP3, PPARα, and PEDF expression. Our data demonstrated that the passenger strand, miR‐21‐3p, is co‐expressed with miR‐21‐5p in ischemic conditions, but targets different genes. Although further studies will be necessary to clarify miR‐21‐3p biological function, our studies suggest that, together with miR‐21‐5p, miR‐21‐3p may hold potential diagnostic and therapeutic value for treatment of RNV in ischemic retinopathies.

Support or Funding Information

All support for this research came from the Bartoli laboratory of the Ophthalmology Department of the Medical College of Georgia at Augusta University and the funding sources (EY0246, Knights Templar –GA Chapter).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MicroRNA‐92b is a potential therapeutic target for Glioblastoma treatment
Authors: Grafals‐Ruiz Nilmary , Lozada‐Delgado Eunice , Santana‐Rivera Yasmarie , Rivera‐Diaz Monica , Valiyeva Fatma , Vivas‐Mejía Pablo
Published: June 2021

Glioblastoma is the most aggressive type of primary brain tumor with an overall survival of 14.6 months with standard care treatment (surgery, radiotherapy, and Temozolomide chemotherapy). About 90% of patients develop recurrent tumors due to acquired resistance to temozolomide. Therefore, new multimodal treatments that target more than one molecular pathway are needed. MicroRNAs (miRNAs) are small (18–22 nucleotides) non‐coding RNAs that regulate more than 60% of protein coding genes post‐transcriptionally. Their dysregulation is implicated in many diseases including diabetes, heart disease, and cancer. MicroRNA‐92b (miR92b) is highly upregulated in glioblastoma tumors and its expression has an inverse correlation with patient overall survival. This study aims to determine the biological effect of targeting miR92b in glioblastoma. We hypothesized that its inhibition would lead to cell death and decreased proliferation by allowing the expression of messenger RNA (mRNA) targets needed for normal cell function. To determine miR92b targeting effect in vitro, we transfected glioblastoma cell lines with an anti‐miR92b and evaluated cellular apoptosis, clonogenicity, and proliferation. Results showed that by inhibiting miR92b there was an increase in apoptotic cells and reduction of cell proliferation. We next evaluated the miR92b targeting effect in vivo, administering intraperitoneal injections of anti‐miR92b encapsulated liposomes to glioblastoma‐xenograft mice. Inhibiting miR92b in mice xenografts led to a significant decrease in tumor volume and tumor weight. Furthermore, we performed bioinformatic analysis to identify mRNA potential targets of miR92b. We both inhibited and overexpressed miR92b in glioblastoma cells (U87 and CRL1620 respectively) and determined the expression of more than 90 potential mRNA targets by quantitative PCR. This analysis lead to the identification of five potential miR92b mRNA targets‐ASB5, TEF, KIAA1024, ZNF776, FBXW7 and BAZ2‐ which are currently under further validation using Western blot analysis. Our results show that miR92b is a good therapeutic target, since its inhibition leads to increased cell death and decreased cellular proliferation both in vitro and in vivo. MiR92b is a potential target for glioblastoma patients and its targeting could lead to the regulation of tumor suppressive protein coding genes.

Support or Funding Information

NIGMS‐RISE Grant Number R25‐GM061838, RCMI grant U54 MD007600 (National Institute of Health and Health Disparities), and UPR Comprehensive Center Seed Funds.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
lncRNA Chronos Exacerbates Pathological Cardiac Dysfunction and Fibrosis
Authors: Neppl Ronald L
Published: June 2021

Long non‐coding RNAs (lncRNAs) are a recently discovered class of genes involved in epigenetic modifications, the regulation of transcription and translation, as well as RNA metabolism. Importantly, the expression of many lncRNAs are known to be developmentally regulated with cell and tissue type specificity and to be dysregulated under pathological conditions. Our previous work identified Chronos as a muscle enriched and aging‐induced, Akt1‐inhibited lncRNA that negatively regulates myogenesis. Testing the hypothesis that Chronos expression is deleterious, we generated Chronos deficient mice and subjected them to transverse aortic constriction (TAC) in order to study the role of Chronos in cardiac hypertrophy and myocardial remodeling. Preliminary data indicate that at early timepoints, Chronos deficient mice have less interstitial fibrosis, as indicated by Masson's Trichrome staining and qPCR analysis of extracellular matrix genes, as compared to WT controls, while still exhibiting hypertrophic growth. Pilot studies at late timepoints indicate no statistical difference between WT and Chronos deficient mice with regard to cardiac hypertrophy or interstitial fibrosis. These data suggest that lncRNA Chronos may play a role in the promoting the initial phases of interstitial fibrosis in response to cellular stress, rather than replacement fibrosis following cardiomyocyte apoptosis. Future studies will examine the role of Chronos in both the early and late stages of myocardial remodeling and compensatory cardiac hypertrophy.

Support or Funding Information

This work is supported by the Brigham and Women's Hospital Department of Orthopedic Surgery.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single Amino Acid Substitutions Dramatically Shift Equilibria of Physiologically Relevant Alternate Protein Assemblies
Authors: Jaffe Eileen K , Saphire Erica Ollmann , Arturo Emilia C
Published: June 2021

Increasing numbers of proteins are being revealed to “shape shift” in physiologically relevant ways that do not dramatically alter secondary structure. As each has been discovered they have been called morpheeins, transformers, metamorphic polymorphs, and others. In each case the alternate assemblies have different functions ranging from “on vs. off” to “moonlighting”. These examples continue to surprise their discoverers because they defy the “one sequence, one structure, one function” paradigm in an unexpected way. These alternate assemblies are likely physiologically relevant as their interconversion is thermodynamically feasible and the equilibrium of alternate assemblies is responsive to biological signals (e.g. pH, ligands). Experience now teaches that single amino acid substitutions can also dramatically shift an equilibrium of alternate assemblies. In some cases this shift is reasonably hypothesized, or established, to contribute to diseases such as inborn errors of metabolism and cancer.

We present examples culled both from our work and the literature of such shape‐shifting proteins whose equilibria respond to single amino acid substitutions. Prime examples come from diverse branches of life, including viral proteins, bacterial proteins, and human metabolic enzymes. The growing number of such examples suggests that alternate assemblies, which have been called the fifth level of protein structure, may be a common factor in protein structure‐function relationships. Experience teaches that this fifth level of structure, for a given protein, may be revealed by the unexpected biophysical behavior of single amino acid variants.

Support or Funding Information

NIH R01 NS100081 (EKJ)NIH R01 AI118016 (EOS)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In silico Analyses of the Binding of Experimentally Tested or Potentially Anti‐Prion Drugs to the PrPC Structures of Several Species of Mammals
Authors: Martinez Nelson
Published: June 2021

Transmissible Spongiform Encephalopathies (TSE's) or prion diseases are a group of rare neurodegenerative diseases that include Creutzfeldt–Jacob Syndrome in humans and Bovine Spongiform Encephalopathy (BSE) in cattle, and which are inevitably fatal. We have made in silico analyses on the docking of several drugs with either experimentally determined or potential anti‐prion activity, to the experimentally determined structures of the cellular isoform of the prion protein (PrPC) from several species of mammals, including humans.

Objectives

The objectives of our study are, in the first place to identify differences, if any, between the predicted affinities and/or binding regions of the drugs tested to the PrPC structures from several species of mammals including humans, cattle, mouse, rabbit, and the Bank vole and, secondly, to determine whether these differences, if found, have any correlation with the different susceptibilities to infection with scrapie (PrPSc) prion protein from other species that different mammals exhibit.

Methods

We used free web–based software in our analyses, especially AUTODOCK VINA to model the binding of the drugs tested to experimentally determined crystal or NMR structures that are already available in the Protein Data Bank (PDB). We also used the web–based PRODRG platform to generate the structures of the drugs tested in PDB format, the NCBI Blast software for sequence alignments, and the Statdisk software to perform statistical analyses.

Results

So far, we have not found yet any significant differences in the predicted affinities and/or binding regions for the drugs tested between binding to the Bank vole prion protein structure and binding to PrPC structures from other mammals which are less susceptible to infection with PrPSc from other species.

Conclusions

We have successfully modeled the binding of several drugs with potential anti‐prion activity to the experimentally determined NMR or crystal structures of the cellular prion protein of several mammals, including humans. Our results suggest that the differential susceptibilities to infection between the Bank vole and other species of mammals that have been found by experiments cannot be explained, at least alone, by structural differences between their PrPC isoforms.

Support or Funding Information

The author is a PhD candidate at the Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, and has received financial support for his studies from the grants NIMHD‐G12MD007600 and 8U54MD007587 to the University of Puerto Rico, and also from grant NIGMS‐RISE‐R25GM061838 and NIGMS‐INBRE.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Macromolecular crowding modulates actin bundle formation induced by actin crosslinking proteins
Authors: Park Jinho , Lee Myeongsang , Kang Hyeran
Published: June 2021

Bundles of filamentous actin generate high ordered cellular structures including filopodia and lamellipodia with the help of actin binding proteins. α‐actinin and fascin are essential actin crosslinking proteins that affect the mechanical properties of bundles. While the roles of both actin binding proteins in bundling have been well studied in dilute buffer conditions, how they affect bundle formation in crowded environments is not established. Here, we investigate the effects of macromolecular crowding on actin bundles crosslinked by α‐actinin and fascin in vitro and in silico. Time‐dependent bundling processes by both proteins were monitored in the presence of the polymeric crowding agent polyethylene glycol (PEG) using total internal reflection fluorescence (TIRF) microscopy. Biophysical analysis show that PEG reduces the amount and average lengths of α‐actinin‐induced bundles but promotes the formation of thicker bundles induced by fascin. Utilizing molecular dynamics (MD) simulations, we evaluated the interaction between actin filaments and bundling proteins by measuring the contact area and the number of hydrogen bonds. The simulation results demonstrate that PEG increases the binding affinities between actin and binding domain of fascin, while lowering the affinities between actin and α‐actinin. Taken together, our work suggests that crowded environments may modulate actin bundling induced by actin binding proteins in vivo.

Support or Funding Information

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

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards revealing the molecular mechanism of STF
Authors: Pathak Prabhat Kumar
Published: June 2021

Leaf is a primary organ for the plants to sustain their life and support life on earth by assimilating solar energy and carbon dioxide into chemical energy. STENOFOLIA (STF), a WUSCHEL (WUS)‐like homeobox transcriptional regulator, plays a key role in embryonic patterning, blade outgrowth, and leaf vascular patterning. STF has also been shown to help in increasing biomass and solubilized sugar yield. STF does not affect leaf outgrowth directly but is critical for their development. It is known that transcriptional repressor activity mediated by the WUSbox of STF acts to promote blade outgrowth, and STF executes its functions with its interactions with other protein and DNA. STF binds to GA rich DNA sequence and there is specific DNA binding domain present in the sequence. Although the DNA binding domain (HD) of the protein is conserved across the species, the WOX family HD seems to be unique and cannot be replaced by another HD protein from the plant. WOX HD domain is longer than usual HD domains and has additional protein interaction sites to achieve stable repressive activity. The molecular mechanism of STF function and how WOX genes have adopted the WUS in evolutionary development are unknown. Here we are working towards determining the atomic structure of STF in complex with DNA by using x‐ray crystallography.

Support or Funding Information

NIH

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