bluuebunny/crossref_metadata_2025 huggingface dataset:
Connexin 43 Function in the Cranial Neural Crest
Authors: Jourdeuil Karyn , Taneyhill Lisa A.
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 either of homomeric or heteromeric connexin proteins, although only certain combinations of connexins are possible. Connexin 43 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 Connexin 43, in the neural crest. Immunohistochemical analyses revealed that Connexin 43 is expressed in premigratory cranial neural crest cells as well as during the epithelial‐to‐mesenchymal transition (EMT), migration, and coalescence of neural crest cells with placodal neurons as the trigeminal ganglion assembles. Interestingly, Connexin 43 is also observed in the cranial mesenchyme, but placodal neurons lack Connexin 43. Given the robust expression of Connexin 43 in the neural crest, we sought to elucidate the role of Connexin 43, and gap junctions in particular, during neural crest cell EMT and migration. To this end, we are assessing the presence of gap junctions in the neural crest and performing molecular and chemical perturbation experiments to alter Connexin 43 and general gap junction function. Our initial results reveal that gap junctions form between migratory neural crest cells generated from explants of premigratory neural crest tissue in an ex vivo culture assay. In future experiments, we will inhibit gap junctions using pharmacological reagents and modulate levels of Connexin 43 through knockdown and overexpression experiments, evaluating effects on neural crest cell EMT and migration in all approaches. Importantly, these assays will confirm that Connexin 43 is critical for gap junction function 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

This research was funded by ACS RSG‐1502301‐CSM and NIH R01DE024217 (L.A.T.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TMED2 is Required in both the Chorion and Placenta for Placental Labyrinth Layer Development
Authors: Jerome‐Majewska Loydie , Hou Wenyang
Published: June 2021

The transmembrane emp24 domain/p24 (TMED) family are essential components of the vesicular transport machinery. TMED family members serve as cargo receptors important for selection and packaging of endoplasmic reticulum (ER) luminal proteins into coatomer (COP) II coated vesicles for anterograde transport to the Golgi. Until recently the role of TMED proteins in development and disease remained a mystery. We postulated that members of the TMED family regulate transport of cargo proteins essential for normal development and homeostasis. Tmed2, the sole member of the vertebrate Tmed β‐subfamily, shows tissue and temporal specific patterns of expression during embryogenesis, but is ubiquitously expressed in all adult organs. We previously showed that mouse embryos homozygous mutant for a single point mutation, the 99J mutation, in the signal sequence of Tmed2 fail to form a functional placenta, and that heterozygous mice carrying this mutation developed non‐alcoholic liver disease. Using explants of chorion and allantois, the precursors of the developing placenta, we identified several secreted and membrane‐bound proteins that were abnormally expressed in Tmed2 mutant tissues, indicating a role for TMED2 in transport of secreted and membrane bound cargoes. In addition, explants between wild type chorion and Tmed2 homozygous mutant allantois, and vice‐versa, revealed both cell‐non‐autonomous and cell‐autonomous requirements for TMED2 during placental development. Our studies indicate that TMED2 is required for organogenesis of the placenta, which, is essential for survival of the growing fetus.

Support or Funding Information

Natural Sciences and Engineering Research Council

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does Stature Really Matter? The Effect of Short Stature on Adult Survivorship
Authors: Minsky‐Rowland Jocelyn D.
Published: June 2021

Stature is a multifactorial growth process, influenced by genetics, health and nutrition. Short stature estimates from skeletal remains have traditionally been used as an indicator of a disruption in physiological growth processes within past populations, which may decrease adult survivorship. This project questions whether this non‐specific indicator of stress, affected the survivorship of individuals associated with the historic Arikara Native Americans of South Dakota (AD 1600–1832). Previous research suggests that short stature decreased survivorship among adult individuals from one archaeological site. In that study, the two sexes were pooled and survivorship was estimated relative to different time periods. In the current project, survivorship differences between the sexes were estimated, while time period was pooled. It is hypothesized that individuals with below average stature will exhibit a decrease in survivorship compared to their taller peers. One‐hundred and eighty‐four adult individuals, (79 females, 105 males), across four archaeological sites, were analyzed. Survivorship was estimated using Kaplan‐Meier estimates with log‐rank tests in SPSS version 22. Two analytical steps were taken. First, differences in survivorship between tall and short individuals were estimated per sex. Next, survivorship of tall females and tall males was compared and survivorship of short females and short males was compared. The results were not statistically significant. Therefore, short stature does not affect survivorship relative to sex. Broadly, these results suggest that the significance of short stature is context specific and cannot always be used as an indicator of decreased survivorship. It is recommended that researchers utilize multiple indicators of stress to infer survivorship and health in past populations. This project contributes to research focused on human growth, the disruption of physiological processes and their effects on adult survivorship.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Barrels of Monkeys: Effects of Long‐term Ethanol Storage on Volar Fat Pad Morphology
Authors: Kingston Amanda Kathryn
Published: June 2021

Museum fluid collections preserve tissues from endangered and protected species for study. Tissues are often fixed in 10% buffered formalin to halt metabolic activities and transferred to a solution of ethanol for long term storage. This process, however, forces water from the tissues and has been shown to alter the morphology of preserved specimens in ways that may influence the biological interpretation of results. The degree to which fluid preservation alters morphology is linked to multiple biological and methodological factors and should therefore be examined prior to functional analysis. This study was conducted as part of an evaluation of mammalian volar pad morphology and comprises two analyses. In the first, a sample of rats (Rattus norvegicus) is utilized to evaluate longitudinal changes in the dimensions of the volar pads across fixation in 10% buffered formalin and preservation in 70% ethanol for one year. In the second, a cross‐sectional sample of fresh primate volar pads from varying species is compared to a corresponding sample from museum specimens fixed in 10% buffered formalin and preserved in 70% ethanol. No significant changes to the measured dimensions of the volar pads are present across stages of fixation and preservation, nor are significant differences evident between fresh and preserved specimen groups. These findings are likely related to the anhydrous nature of the volar fat pads and their confined position between major joints.

Support or Funding Information

This work was funded by NSF Doctoral Dissertation Improvement Grant #1128861.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neurovasculature of the Upper Extremity in the Aye‐aye, Daubentonia madagascariensis
Authors: Avakian Melody , Townsend K.E. Beth , Smith Heather F.
Published: June 2021

The aye‐aye, Daubentonia madagascariensis, is an arboreal lemuroid primate and is the sole representative of its genus. The aye‐aye has unique anatomical characteristics including a thin, long, flexible third digit, unique dental anatomy, a single pair of inguinal mammary glands, greater brain size than those of other strepsirrhines, and large mobile ears that separate it from other lemuroids and make it an interesting organism to study from an anatomical, developmental, and evolutionary standpoint. The aye‐aye also has a distinctive method of obtaining its food from trees known as percussive foraging, which entails tapping on wood with its long middle finger, listening to the resulting sounds to determine the locations of cavities that may contain larvae, and then scooping out the larvae with its specialized third digit. The bony and muscular anatomy of the brachium, antebrachium, and manus of the aye‐aye has been previously studied and described in detail. However, there have been no studies that have described and depicted the complete neurovasculature of the forelimb of the aye‐aye, including the important brachial plexus. Given the unique anatomy and behavioral forelimb adaptations of this species, understanding the various nerves and blood vessels in relation to the musculature will provide a more complete picture of aye‐aye functional morphology and comparative anatomy.

This study identifies and describes the neurovasculature of the forelimb of the aye‐aye with emphasis placed on the musculature and neurovasculature on the hand. The forelimbs from one female Daubentonia madagascariensis were dissected, photographed, and described in detail. In this specimen, we confirmed that the anatomy of the forelimb muscles was generally consistent with previous descriptions, although we observed a few minor variations. The brachial plexus was found to originate from the C5‐T2 roots, a feature that has also been observed in other lemurs and rhesus monkeys. The large deltoid and brachioradialis muscles observed in this specimen may serve to support the frequent use of head‐first descent, which requires a robust shoulder girdle and specific alterations in hand and body position that allow the forelimbs to undergo increased compressive forces. Future studies will analyze the development of the muscles of the arm and hand, along with the vasculature that migrated with them, and also compare them to the developmental patterns of related primates which can give insight into the aye‐aye's specialized adaptations.

Support or Funding Information

This research was supported by a Midwestern University Kenneth A. Suarez summer research fellowship to MA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NGF and VEGF expressions on crushed sciatic nerve treated with laser therapy and latex protein
Authors: Dias Fernando Jose , Fazan Valéria Paula Sassoli , Iyomasa Mamie Mizusaki , Borie Eduardo , Coutinho‐Netto Joaquim , Watanabe Ii‐sei
Published: June 2021

Lesions that affect peripheral nerves still is a challenge in medical sciences. In this context, previous studies employing low‐level laser therapy (LLLT) and natural latex protein (Hevea brasiliensis, F1) revealed promising results in nerve regeneration.

The aim of this study was to analyze the effects of LLLT associated with F1 protein applied in sciatic nerve crush injury (15 kgf, 10 min.) in male rats. The animals (♂, 250 g) were allocated in 6 groups (n=5): C ‐ Control; E ‐ exposed nerve without injury; I ‐ injured nerve without treatment; L ‐ injured nerve treated with LLLT (15 J/cm2, 780 nm); F1 ‐ injured nerve treated with F1 application (0.1%); and L&F1 ‐ injured nerve treated with the LLLT and F1 protein. After 4 and 8 weeks of nerve injury the animals were euthanized by anesthetic overdose and cardiac perfusion (glutaraldehyde 2.5%, PBS 0.1, pH 7.4), the sciatic nerve samples were collected and processed. Quantitative morphological parameters (myelin and capillary density) and immunohistochemical reactions (peroxidase) for nerve growth factor (NGF) and vascular endothelium growth factor (VEGF) were performed and analyzed.

After 4 weeks in the injured groups (I, L, F1 and L&F1) was observed a significant reduction in myelin density. After 8 weeks the treated groups showed improvements compared to the previous period, the F1 and L&F1 groups presented values similar to the non‐injured groups (C and E). Capillary density was significantly higher in the F1 and L&F1 groups after 8 weeks of nerve damage. NGF was present in the injured groups (I, L, F1, L&F1) after 4 weeks, the group I showed the highest and the F1 group revealed the lowest positive expressions. VEGF was present in all study groups, showing more intense expressions in the injured groups, especially after 4 weeks of lesion in the F1 and L&F1 groups. After 8 weeks all the groups reduced the marking intensity.

It was concluded that the nerve injury crushing results in a reduction in myelin density. NGF is mainly expressed when nerve tissue is injured. The treatments were effective in the improvements of the morphological parameters analyzed. The F1 protein presented the best quantitative results and an increase in vascular density when applied alone or in association with LLLT. In addition, the less intense NGF expression after 4 weeks associated with morphological improvement in group F1 suggests the acceleration of sciatic nerve recovery.

Support or Funding Information

Funding: FAPESP 2014/07253‐4

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Resolution X‐ray Micro‐Tomography of the Posterior Ampullary Nerve
Authors: LABROUSSE Marc , BLANCHOT Delphine , DUBERNARD Xavier , SETTEMBRE Nicla , AVISSE Claude , DEVY Jerôme , RENARD Yohann
Published: June 2021

Introduction

The posterior ampullary (singular) nerve runs from the posterior ampulla to the saccular nerve (forming the inferior vestibular nerve) through the singular canal of Morgagni in temporal bone. Posterior ampullary neurectomy can be proposed in a few chronically disabling benign paroxysmal positional vertigo. The purpose of this study is to investigate the surgical anatomy of the posterior ampullary nerve using 3D reconstructions high resolution X‐ray micro‐tomography.

Materials and Methods

10 human petrous parts of temporal bones were fixed with Winckler's solution. CT images were acquired with a CT scanner (Skyscan 1076, Bruker, Kontich, Belgium). The camera was continuously rotated by 180° and the following parameters were used for acquisition: 50 kV, 0.5 mm Al filter, 200 A source current, 35 m isotropic resolution, 180 ms exposure time, and 0.7° rotation step. The projections were reconstructed with a filtered backprojection algorithm in Skyscan software (NRecon, Skyscan). Reconstructed images were analyzed with ORS Visual software (v1.5, Montreal) which allowed multiple display and post‐processing possibilities (3D VR; orthogonal/curved multiplanar reformation).

Results and Discussion

Three‐dimensional and multiplanar studies of the singular canal of Morgagni morphology were possible with appropriate thresholding. The topographical correlations between the posterior ampullary nerve, posterior semicircular canal and the round window were evaluated. The level of the nerve in comparison with the round window may vary. Three types of positions are described. Type 1 is the lowest position: the nerve is located under the inferior part of the scala tympani. Type 3 is the highest position: the nerve is located higher than the superior part of the round window (this type goes straight from the internal auditory canal to the posterior ampulla). Type 2 is an intermediate form. Difficulty level in singular neurectomy depends on the course of the singular nerve. Type 1 is the easiest to operate on with the lowest risk, whereas type 2 and particularly type 3 are riskier in terms of damage to the round window membrane.

Conclusion

3D reconstructions using high resolution X‐ray micro‐tomography aid comprehension of posterior ampullary nerve complex anatomy. Transtympanal posterior ampullary neurectomy can be performed only in selective cases.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Role of α3‐containing Nicotinic Acetylcholine Receptors (nAChRs) in Anxiety Behavior
Authors: Smith Ciearra B. , Tapper Andrew R. , Gardner Paul D.
Published: June 2021

More than 260 million people suffer from anxiety disorder, thus it is important to understand the molecular mechanisms underlying anxiety disorder in order to develop more treatments. Studies showed that nicotinic acetylcholine receptors (nAChRs) play a role in anxiety‐related behaviors. nAChRs are ligand‐gated cation channels that bind the endogenous neurotransmitter acetylcholine for activation. There are eleven mammalian neuronal nAChR subunits, α2‐α7, α9, α10 and β2‐β4, which are encoded by the CHRNA2‐CHRNA7, CHRNA9, CHRNA10, and CHRNB2‐B4 genes. These subunits co‐assemble to form functional homopentamers or heteropentamers consisting of various combinations of alpha and beta subunits. nAChRs are highly expressed in the habenulo‐interpeduncular pathway, a brain circuit that is involved with negative emotional state. Although knockout studies identified nAChRs as key players in anxiety‐related behaviors, little is known regarding the physiological relevance of α3‐containing nAChRs in anxiogenic behaviors due to α3 nAChR subunit knockout mice being inviable shortly after birth. I plan to test the physiological role of α3‐containing nAChRs in anxiety‐related behaviors by knocking down α3 nAChR subunits in the medial habenula (MHb) or interpeduncular nucleus (IPN) of 6‐week old mice and subsequently testing for anxiogenic behaviors. Here, I present approximately 45% and 80% knockdown of Chrna3 mRNA expression in Neuro‐2A (N2A) cells by lentiCRISPRv2‐sgRNA20 and shRNA 8843, respectively. Chrna3 mRNA levels were measured by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Thus, both CRISPR and shRNA approaches produced significant reduction of Chrna3 mRNA expression and can be applied in vivo to knockdown α3 nAChR subunit expression. My findings may identify α3‐containing nAChRs as novel targets for anxiety disorder therapies.

Support or Funding Information

NIH Award Number: DA035371

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Alaskan low bush cranberry on alpha‐synuclein overexpression in a transgenic model of C. elegans
Authors: Lu Brianna , Maulik Malabika , Bult‐Ito Abel
Published: June 2021

Parkinson's Disease is the second most prevalent neurodegenerative disorder affecting more than 10 million worldwide (Tanner and Goldman 1996). The disease is characterized by a loss of dopaminergic neurons and aggregation of α‐synuclein proteins in the brain, resulting in impaired motor function and cognition that worsens with age. This study aims to observes the effects of Alaska low‐bush cranberry (Vaccinium vitis‐idaea) extracts on the α‐synuclein overexpression in a popular transgenic model, Caenorhabditis elegans. We tested the hypothesis that the low‐bush cranberry extracts would aid in reducing protein aggregation, and increase overall motility and lifespan of the OW13 C. elegans expressing human α‐synuclein proteins by using fluorescent imaging and microscopy. Preliminary results from our study finds that the certain doses of this botanical extract reduces the protein expression significantly (p<0.05). This study encourages further research on such endemic natural compounds as potential treatment for Parkinson's disease and identifying associated molecular mechanisms for the same.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neuroprotective effects of estrogen on Alzheimer induced SH‐SY5Y neuroblastoma cells and N38 hypothalamic neuronal cells
Authors: Almathhur Rayan , Yakubov Rakshim , Donkor Issac , Suh Catherina , Chakraborty Sanjoy , Chakraborty Tandra R.
Published: June 2021

Alzheimer's disease is a neurodegenerative disease characterized by neuronal cell damage and death, neurofibrillary tangle formation and cerebral plaques formed by beta amyloid peptides. Previous studies have shown that the hippocampus, a region of brain related to cognition and memory is mainly affected by Alzheimer's disease. This study is designed to induce Alzheimer's disease by treating SH‐SY5Y neuroblastoma cells and N38 hypothalamic neuronal cells with synthetic beta‐amyloid plaques under in vitro conditions. In the current study, we wanted to determine whether hypothalamus; a region involved in feeding, sleep, reproduction and energy balance is affected similarly as well. Hypothalamic cells were treated with beta‐amyloid1‐42 to induce neurofibrillary tangles and plaque formation. The cell viability, toxicity and proliferation were determined using different bioassays like cell count, MTT and lactate dehydrogenase assay. Preliminary results indicated a significant loss in mitochondrial function when exposed to beta‐amyloid. Cell damage and cellular toxicity were further analyzed by lactate dehydrogenase (LDH). Further, western blotting was done to determine the changes associated with biomarkers like P53 and Tau protein. Taking together the biochemical and molecular data the neuroprotective effect of estrogen on beta amyloid plaque formation will be analyzed.

Support or Funding Information

Adelphi 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.
Radial Glia and Cell Heterogeneity Across The Lizard Central Nervous System: Distal Spinal Cord Rupture Does Not Induce a System‐Wide Response
Authors: McDonald Rebecca Paula , Donato Sarah Vita , Austin Laura , Vickaryous Matthew Kenneth
Published: June 2021

Radial glia are resident neural stem/progenitor cells of the central nervous system of many non‐mammalian species. Radial glia are capable of spontaneously generating new neurons, and are considered to be crucial for both brain development and injury‐mediated regeneration. Recently, radial glia have also been identified in the tail spinal cord of the lizard Eublepharis macularius (the leopard gecko), a species capable of tail spinal cord regeneration. Here, we investigated whether similar populations of radial glia were present within the body spinal cord (bSC) and brain. Using a bromodeoxyuridine pulse‐chase strategy, we determined that label‐retaining cells were found throughout the bSC and the forebrain following a 20‐week chase. Next, we established that populations of cells lining the central canal (of the bSC) and ventricular system (of the brain) express a panel of radial glia markers, including the transcription factor SOX2 and the intermediate filaments GFAP and Vimentin. However, Vimentin expression was conspicuously absent from the diencephalon (except for the hypothalamic eminence), as well as the midbrain and hindbrain. Further, cell populations lining the central canal of the bSC include both radial glia and cells with a neuronal phenotype (as evidenced by expression of the neuronal marker HuCD). An identical diversity of cell types is also reported for in the regeneration‐competent tail spinal cord. Finally, we determined that constitutive proliferation by cells lining the central canal (of the bSC) and the ventricular system (of the forebrain) are essentially unaltered in response to distal central nervous system injury (tail spinal cord rupture as a result of tail loss). Combined, our data demonstrates that radial glia are present throughout the central nervous system of the gecko, that the bSC is essentially identical to that of the tail, and that the bSC and forebrain are resilient to distal spinal cord injuries.

Support or Funding Information

Natural Sciences and Engineering Research Council Discovery Grant 400358 (MKV), Canadian Foundation for Innovation Leaders Opportunity Fund 25371(MKV)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defects in base excision repair and homologous recombination, but not nucleotide excision repair, lead to altered cell cycle phase distributions in Saccharomyces cerevisiae
Authors: Oviedo Alejandro , Valencia Sarah , DeForge Taylor R. , Fitzgerald O'taveon R. , Lewis L. Kevin
Published: June 2021

The DNA within cells is constantly subjected to chemical modifications resulting from exposure to endogenous and exogenous DNA damaging agents. Damaged or oxidized bases are primarily repaired by the base excision repair (BER) pathway, crosslinks induced by ultraviolet (UV) light and many bulky lesions are repaired by nucleotide excision repair (NER), mismatches are resolved by the mismatch repair system (MMR), and double‐strand breaks in DNA are repaired by either the homologous recombination (HR) pathway or the nonhomologous end‐joining (NHEJ) pathway. Inactivation of NER genes such as RAD2, RAD7 or RAD14 leads to extreme UV sensitivity in yeast cells. Mutants with defects in BER genes OGG1 or UNG1 are defective in repair of oxidized guanines and the removal of uracil bases from DNA, respectively. Yeast rad52 mutants are deficient in HR and are hypersensitive to ionizing radiation and chemicals that induce double‐strand breaks.

Our laboratory has previously observed that rad52 haploid and diploid mutant cell cultures have high levels of distended large‐budded G2 phase cells and long cell cycle transit times (see poster by C. England et al.). This work also demonstrated that the high G2 phase cell phenotype of rad52 cells was abolished when any of seven known DNA damage checkpoint genes were co‐inactivated. The current project has extended these studies and asked whether defects in other DNA repair pathways also lead to accumulation of G2 phase cells and increases in doubling times. Initial studies revealed that log phase cultures of mutants deficient in NER (rad2, rad7 and rad14) or MMR (msh2, msh3 and msh6) do not have high levels of large‐budded cells. By contrast, such cells were strongly elevated in mutant cultures defective in the BER pathway (ogg1 and ung1 cells). Recent experiments are attempting to determine the fraction of these large‐budded cells that are G2 vs. M phase cells via DAPI staining and whether they are specifically associated with activation of the DNA damage checkpoint system. Cell cultures containing mutants defective in other DNA repair processes, such as Rad6‐mediated postreplication repair, translesion synthesis and nuclease processing of DNA ends, are also being analyzed using phase contrast and fluorescence microscopy to identify unusual cell morphology and cell cycle phase characteristics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heterochromatin proteins influence the choice of DNA repair in euchromatin domains
Authors: Forsburg Susan , Jensen Amanda , Jones Chance , Li Ting‐Ting , Yuan Ji‐Ping
Published: June 2021

In most eukaryotes including the fission yeast, constitutive heterochromatin in pericentromere and telomere domains is marked by methylation of histone H3K9, which provides a binding platform for specialized proteins including heterochromatin protein 1 (HP1). These proteins contribute to transcriptional silencing, cohesion, and kinetochore recruitment. We showed previously that loss of heterochromatin leads to genome rearrangement in the pericentromere repeats that is exacerbated by replication fork destabilization (e.g., Li et al (2013) Cell Rep. 3:638). This indicates that heterochromatin and fork stability make unique contributions to maintain a repetitive domain and resist recombination.

Evidence from mammalian systems has implicated heterochromatin proteins in transient response to induced DNA double strand breaks in euchromatin domains. Unexpectedly, we observed that mutants lacking the histone methyltransferase Clr4, or chromodomain proteins Chp1 or Swi6 show evidence of genome instability in euchromatin even in the absence of exogenous damage. Using molecular and cytological approaches, we have investigated the role of these proteins in maintaining euchromatin genome stability in otherwise unperturbed cells, as well as in response to distinct forms of genome stress. We determined that a subset of heterochromatin‐associated proteins contribute to the choice of repair pathway in euchromatin domains by modulating resection of DNA ends. Thus, H3K9 histone methylation and its associated proteins are not limited to heterochromatin domains and influence chromosome stability broadly throughout the genome.

Support or Funding Information

Support by NIH R35 GM118109

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reprogramming Cancer Cell Metabolism through the Combinatorial Action of PK11195 and 5‐Azacytidine
Authors: Duran Daniel , Steinsaltz Matthew , Anstett Sarah , Custance Michaeal , O'Donnell Robert
Published: June 2021

Deregulated cellular energetics is a widely observed phenotype across numerous cancer cell lines, making it a logical target for cancer therapies. This aberrant metabolism commonly manifests as the “Warburg Effect,” wherein fractional pyruvate oxidation results from a tumor cell's over‐reliance on glycolysis. One of the simplest molecular explanations for this effect is the loss of a multisubunit mitochondrial pyruvate carrier (MPC) that shuttles pyruvate into the mitochondria. The MPC‐1 and MPC‐2 genes code for distinct subunits of this carrier protein and are commonly silenced in cancer cell genomes. In this investigation, we use a DNA methyltransferase inhibitor, 5‐Azacytidine, in combination with a constitutive androstane receptor (CAR) inhibitor, PK‐11195, to re‐express the coregulated MPC subunit genes. Through reverse transcription PCR, we demonstrate that the combinatorial action of these two drugs in the HTB‐4 cell line results in a 6.36 and 3.54 fold increase for the MPC‐1 and MPC‐2 genes, respectively. Additionally, in the MDA‐MB‐435 cell line, the MPC‐1 and MPC‐2 genes were upregulated by a factor of 3.54 and 3.18, respectively. Preliminary western blot analysis on whole cell lysates reveals no distinction between the levels of the 12 kDa MPC‐1 subunit from treated and untreated samples. Currently, experiments are underway to test whether the treatment results in a more oxidative phenotype that is indicative of pyruvate oxidation within the mitochondria. Finally, PK‐11195 also binds a peripheral benzodiazepine receptor (PBR) on the surface of the outer mitochondrial membrane, subsequently limiting the rate of glycolysis through inhibition of hexokinase. Cytotoxicity assays reveal that while PK‐11195 introduces a glycolytic block on both cell lines, supplementation with fatty acids rescues them from the glycolytic stress. Collectively, our work demonstrates the significance of both restoring the mitochondrial pyruvate carrier, and working around other metabolic pathways, as a means of repressing the Warburg effect.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNMT3A mutations in AML patients cause loss and gain of function and differential regulation by protein partners
Authors: Sandoval jonathan Enrique
Published: June 2021

Eukaryotic DNA methylation, an integral component of the epigenetic machinery, prevents genomic instability by regulating the expression of oncogenes and tumor suppressor genes. The importance of dysregulated DNA methylation in diverse blood cancers including Acute Myeloid Leukemia (AML) pathogenesis is highlighted by the strong correlation between mutations in the de novo DNA methyltransferase gene, DNMT3A, and adult patients with poor prognoses. We show that clinically observed DNMT3A mutations result in dramatic changes in enzyme activity, including mutations that lead to 6‐fold hypermethylation and 3‐fold hypomethylation on the p15 human promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN‐AML) constitutes 40–50% of all AML cases, is the most epigenetically diverse AML subtype and has pronounced changes in DNA methylation in non‐CpG regions. We identified a subset of mutations in DNMT3A that lead to a 2–8 fold enhancement in the enzyme's ability to perform non‐CpG methylation. Many of the mutations studied map to regions on the protein that are well known to interact with partner proteins, which themselves contribute to AML, such as the DNA base excision repair (BER) enzyme Thymine DNA glycosylase (TDG). Here we show that DNMT3A mutations cause significant and diverse changes in the ability of these regulatory partner proteins to affect DNMT3A function. Our results present a link between DNMT3A mutations and the disruption of the epigenetic landscape in AML.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ACE and AGTR1 are differentially methylated in a hypertensive versus normotensive cohort of Kenyans
Authors: Shelton Dimond A. , Rice Nancy A.
Published: June 2021

Cardiovascular diseases (CVD) are emerging as the primary cause of global mortality and are disproportionately affecting low and middle‐income countries (LMICs). The leading risk factor for CVD is hypertension (HTN), a multifactorial disease with no single genetic cause. Epigenetic control, i.e. methylation, of blood pressure regulatory genes likely contributes to the etiology of this disease. From a rural population of Kenyans previously identified to have a high incidence of HTN, we investigated the cytosine‐phosphate‐guanine dinucleotide (CpG) methylation pattern of two promoter regions that regulate renin‐angiotensin aldosterone system (RAAS) gene expression. Specifically, angiotensin II receptor type 1 (AGTR1) and angiotensin‐converting enzyme (ACE) genes, that function in maintaining salt and water homeostasis, were analyzed. The promoter regions of AGTR‐1 and ACE contain 11 and 19 CpG islands, respectively, making them a prime target for methylation driven regulation. Briefly, genomic DNA was isolated from buccal cells isolated from either hypertensive or normotensive individuals using standard alkaline lysis. DNA was quantified, and subsequently unmethylated cytosines were converted to uracils by demethylation using bisulfite treatment. Gene‐specific CpG islands were amplified by PCR and sequenced. From preliminary results, ACE promoter methylation is increased by 10 % (50.4 ± 3 vs 60.1 ± 4; p <0.05) and AGTR1 promoter methylation is decreased by 8 % (61.0 ± 8.2 vs 53.8 ± 4.0; p, 0.05) in hypertensive individuals versus normotensive, respectively. Specifically, the 18th CpG dinucleotide in ACE was unmethylated in all samples isolated from normotensive or prehypertensive individuals, yet was methylated in the majority of samples isolated from participants with elevated blood pressure. These results may suggest a role for this dinucleotide in regulating ACE gene expression in endothelial dysfunction.

Support or Funding Information

This work was funded by NIH‐NCRR P20 RR16481 (NAR) as well as support from the Kentucky Academy of Sciences (NAR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA Methylation of Multiple Genes involved in Bladder Cancer among Saudi Population
Authors: Siddiqui Amna , Yaqinuddin Ahmed , Alshehri Faizah A.
Published: June 2021

Objective

To identify bladder cancer specific methylated DNA sequences for Saudi population in order to detect and predict bladder cancer progression.

Methods

In this study, we analyzed DNA methylation levels of 94 tumor suppressor genes loci in 24 bladder tissues (19 bladder cancer samples and 5 control samples taken from histologically normal bladders). DNA Methylation analysis was done using Human Tumor Suppressor Genes EpiTect Methyl II Complete PCR Array from Qiagen TM.

Results

We identified significant difference in DNA hypermethylation levels at APC, BRCA1, CDH1, CDH13, CDKN2A, DAPK1, ESR1, FHIT, MGMT, RASSF1, SOCS1, TIMP3, TP73, VHL, WIF1, E2F1, ERBB2, H1C1, OPCML, SFN, SFRP1, SFRP2, SPARC, and TERT between controls and cancerous samples. It was also observed that CADM1, DKK3 and SCGB3A1 were deferentially methylated in non‐muscle invasive versus muscle invasive bladder cancer samples. Additionally, DNA hypermethylation of ESR1 was notified as the novel tumor suppressor gene specific for Saudi population in bladder cancer.

Conclusion

Our findings suggest that these aberrant DNA methylation patterns in bladder cancer are disease and population specific and have a potential to develop as distinct DNA methylation based bio markers in future.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cross‐drug gene expression overlaps reveal potential targets for pharmaco‐epigenetic interventions in drug addiction
Authors: Padilla Adriana Mercedes , Ghezzi Alfredo Mercedes
Published: June 2021

Sustained exposure to drugs leads to homeostatic adaptations in the brain brought on by responsive changes in gene regulation mechanics at synapses which modify neuronal circuitry. This genetic plasticity is involved in the persistence of symptoms of drug addiction even after extended periods of abstinence and give rise to behavioral phenotypes such as tolerance and dependence. After exposure to sedatives such as alcohol we have observed epigenetic modulations induced by ethanol exposure that activate the expression of genes producing an excitative state which opposes the drug's inhibitory effects. Congruently, addiction to opiate drugs such as heroin and fentanyl, or the effects of the noncompetitive NMDA antagonist ketamine, have also been found to be mediated by an epigenetic machinery. Although genes and possible mechanisms involved in the development of drug addiction have been uncovered, the use of an epigenetic reversible system as a potential pharmacologic treatment is still unknown. Here we use a combination of behavioral assays with the drosophila model system and genome‐wide analyses of gene expression to investigate the common molecular responses to the different drugs. We exposed groups of wild‐type flies to single sedating doses of vaporized ethanol, morphine or ketamine, and perform genome‐wide analyses of gene expression using RNA‐seq. Results from RNA‐sequencing experiments allow for cross‐analyses of the gene expression profiles of each addiction‐inducing drug. Identifying and mapping genetic overlaps specify regions in the genome that are potentially regulated during the adaptations underlying drug addiction. Commonalities in drug‐responsive genetic expression propose novel targets for pharmaco‐epigenetic interventions at molecularly modulated sites. Findings might further elucidate the intricacies that are at work within the flow of genetic plasticity indispensable for the development of addiction to drugs.

Support or Funding Information

Department of Biology and NeuroID Program, University of Puerto Rico ‐ Rio Piedras, San Juan, PR 00931

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monitoring Cytotoxic Effects In Non‐Cancerous Cells via Docetaxel
Authors: Couvertier Iyonna , Dhimolea Eugen , Mitsiades Constantine
Published: June 2021

Therapeutic chemotherapy drugs are known to induce cytotoxicity in both non‐cancerous cells and in malignant cells. However, it is still unknown if the chemotherapy drug Docetaxel induces cytotoxic events in non‐cancerous cells. Our approach to measuring cytotoxic events was H&E staining (hematoxylin and eosin) followed by immunohistochemistry (IHC) with Estrogen Receptor α (ERα), Androgen Receptor α (ARα) or Ki‐67 antibodies to determine if the cytotoxicity occurred in cancerous or non‐cancerous cells. In the MCF7 breast cancer cell line, chemotherapy drug Docetaxel induced cytotoxic effects in 44.20% of malignant breast cancer cells and 34.86% in non‐malignant cells as classified according to their response to the ERα antibody with a standard deviation of 26.89%. Patient derived prostate cancer cells grown in mice generated from the MDA‐MB‐231 cell line were also treated with Docetaxel. An average of 28.3% of prostate cancer cells exhibited ARα monoclonal antibody sensitivity, and 28.3% of prostate cancer cells displayed cytotoxic effects after administration of Docetaxel with a standard deviation of 34.3%. In conclusion, Docetaxel was shown to induce cytotoxic effects in both in prostate cancer cells and non‐malignant cells however it induced a greater toxic effect in nonmalignant breast cancer cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Gene Expression Analysis of Brain Tissue RNA From Mucolipidosis IV Knockout Mice
Authors: Chacon Jonathan , Rosas Lauren , Cuajungco Math P.
Published: June 2021

Mucolipidosis IV (MLIV) is an autosomal recessive disorder that is characterized by progressive neuronal and retinal degeneration. MLIV is caused by loss of function mutations in the human Mucolipin‐1 (MCOLN1) gene, whose protein product, Transient Receptor Potential Mucolipin‐1 (TRPML1), serves as a lysosomal non‐selective cation channel. We have previously reported abnormal zinc levels in MLIV patient fibroblasts, human MCOLN1‐knockdown cells, and Mcoln1‐knockout (KO) mouse brain tissues. Collectively, these observations suggest that the loss of cellular zinc homeostasis may play an underlying role in MLIV pathogenesis. To investigate whether these irregularities are reflected in the gene expression patterns of zinc buffering and transport proteins, we performed RNA sequencing (RNA‐seq) to analyze the brain transcriptome of three Mcoln1 KO mice and three wild‐type (WT) littermate controls. Gene ontology data revealed significant differential gene expression in the category of “Zinc Ion Transmembrane Transporter Activity.” Gene cluster analyses of zinc transport and zinc buffering genes revealed a distinct reduction in brain transcript levels of solute carrier 30a3 (Slc30a3; also known as the efflux zinc transporter, ZnT3) and transmembrane (Tmem)‐163 genes in KO mice, both of which facilitate zinc transport within neuronal synaptic vesicles. Comparison of RNA‐seq data between individual assembly and combined assembly of KO and WT sequencing reads showed distinct transcript‐level variability that could be explained by inter‐subject differences. Validation using real‐time quantitative polymerase chain reaction (qPCR) confirmed this effect. Nevertheless, both RNA‐seq and qPCR results consistently showed a decrease in ZnT3 transcript levels in KO mice compared to WT littermate control mice. These data imply that abnormal ZnT3 expression could be a mitigating factor for the observed zinc dyshomeostasis in MLIV cells. Overall, our findings suggest that the loss of human TRPML1 protein function perturbs the expression of specific genes necessary for intracellular zinc homeostasis. Future studies will investigate whether the reduction of ZnT3 transcript levels also reflect decreased protein levels in brain tissues of MLIV KO versus control mice.

Support or Funding Information

This work was funded by NIH R15 NS101594 and NIH R25 MD010397.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Membrane‐anchored E‐cadherin/AGO2 complex promote non‐canonical miRNA biogenesis of miR‐451a
Authors: Li Jie‐Ning , Kuo Yao‐Lung , Ku Wen‐Hui , Lu Yu‐Jhan , Wang Ming‐Yang , Chen Pai‐Sheng
Published: June 2021

Micro‐RNAs are small non‐coding RNAs which regulate target mRNAs expression. Canonical miRNA biogenesis requires two‐step processing conducted by Drosha/DGCR8 and Dicer/AGO2 complexes in the nucleus and cytoplasm, respectively. Aberrant miRNA biogenesis have been reported in malignant human cancers. E‐cadherin is a junctional protein which localizes on plasma membrane to maintain cell‐cell adhesion. Downregulation of E‐cadherin is widely observed in cancers and correlates with epithelial–mesenchymal transition (EMT), it maintains cell‐cell contact and epithelial‐like phenotype to suppress migratory and invasive abilities of cancer cells. Although abnormal regulation of miRNA biogenesis has been reported in cancer, the integrated spatial and temporal regulatory pathways remains unclear. Here, we study the physical interaction between E‐cadherin and AGO2, and the effects on miRNA biogenesis. We found that E‐cadherin enhance the accumulation of AGO2 protein on cell membrane. Membrane‐anchored E‐cadherin/AGO2 complex promotes AGO2‐mediated specific miRNA, miR‐451a, biogenesis in a Dicer‐independent manner. Furthermore, the induced miR‐451 targeted EPH receptor A6 (EphA6) 3′UTR and downregulated EphA6, a metastasis‐promoting gene family member in breast cancer. Our findings demonstrate the spatial and temporal regulation of an AGO2‐dependent miRNA biogenesis pathway modulated by E‐cadherin, which provides new insight into the reciprocal interaction between EMT and miRNA biogenesis.

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