bluuebunny/crossref_metadata_2025 huggingface dataset:
Collecting duct NOS1 activation is necessary for increased GFR in response to high salt diet
Authors: Allan John Miller , Pollock David M. , Hydnman Kelly A. , Pollock Jennifer S.
Published: June 2021

We previously reported that nitric oxide synthase‐1 (NOS1) in the collecting duct (CD) is critical for maintaining fluid‐electrolyte balance during high salt diet. Lack of NOS1 in the CD (CDNOS1KO) results in reduced sodium excretion, urine output, and urinary nitrite/nitrate excretion compared to control mice after 1 day of a high salt diet. By day 7 of a high salt diet, CDNOS1KO mice display significantly increased systolic blood pressure with normalized sodium excretion. We also previously reported that after 7 days of high salt diet there is inappropriate suppression of plasma and urinary aldosterone excretion, renal angiotensin II levels, and CD ENaC activity. This inability to appropriately suppress renal angiotensin II and systemic aldosterone with high salt diet in CDNOS1KO mice lead us to hypothesize that the loss of activation of CD NOS1 during high salt diet challenges blunts the physiological renal hemodynamic response to high salt diet. The purpose of the current study was to determine if CDNOS1KO mice on high salt diets have attenuated glomerular filtration rate (GFR) response. To test this, GFR was measured by transdermal FITC‐sinistrin clearance in conscious male control and CDNOS1KO mice on sodium deficient (LS, n=4), normal salt (NS, n=11) or after 1 day and 7 days of high salt (HS1, n=4 and HS7, n=10) diets. On LS and NS, GFR was similar between control (LS: 263 ± 14 ml/min, NS: 224 ± 6 ml/min) and CDNOS1KO mice (LS: 237.2 ± 10 ml/min, NS: 218 ± 10 ml/min) (Pgenotype = 0.4, Pdiet = 0.7, Pgenotypexdiet = 0.3). HS1 control mice had a significant increase in GFR (333 ± 10 ml/min, P=0.008), while GFR remained unchanged in CDNOS1KO mice (255 ± 23 ml/min, P=0.2) compared with LS. HS7 control and CDNOS1KO mice had similar levels of GFR (251 ± 16 and 291 ± 27 ml/min, respectively). Taken together these data indicate, that CD NOS1 is critical for the adaptation to a high salt diet, such that CDNOS1KO mice are volume expanded, have inappropriately high RAAS activation, and fail to increase GFR. After a few days of high salt diet, blood pressure is increased resulting in a rightward shift in the pressure natriuresis curve, with GFR and sodium excretion normalized to control levels. In conclusion, CD NOS1 activation is critical for regulation of tubular and hemodynamic pathways during consumption of HS diets.

Support or Funding Information

K01‐DK‐105038 KAH, HL136936 JSP and DMP.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alterations in the intestinal microbiota contribute to the gastrointestinal toxicity of the anti‐rejection drug mycophenolate mofetil
Authors: Flannigan Kyle L. , Pereira Sheldon K. , Taylor Michael R. , Alston Laurie , Wang Xuemei , Rioux Kevin P. , Galipeau Heather J. , Workentine Matthew L. , Greenway Steven C. , Hirota Simon A.
Published: June 2021

Transplantation is curative for end‐stage organ failure but requires potent immunosuppressive medications to prevent rejection including the immunosuppressant mycophenolate mofetil (MMF). While MMF is effective as maintenance therapy, it is commonly associated with gastrointestinal (GI) side effects (e.g. diarrhea, rapid weight loss, colitis) which can necessitate its discontinuation, increasing the chances of graft failure and rejection. While the mechanism(s) contributing to MMF‐related GI toxicity are not understood, we recently reported a clinical case where MMF complications were associated with shifts in the composition of the intestinal microbiota. Thus, in the current study, we hypothesized that the GI toxicity associated with MMF was dependent on the intestinal microbiota. To test this, we fed C57BL/6 mice chow containing MMF and assessed a variety of outcomes. Mice consuming MMF exhibited significant weight loss (>20% after 9 days), which reflected a loss of body fat and lean muscle mass. This was accompanied by marked inflammation of the colon. MMF treatment also promoted changes in gut microbial composition, demonstrated by a loss of overall diversity, expansion of Proteobacteria and loss of beneficial bacterial genera. PICRUSt predicted gene counts identified several components of LPS biosynthesis that were significantly upregulated following MMF treatment. This was associated with an increase in fecal and serum levels of LPS in MMF‐treated mice. Treatment with broad‐spectrum antibiotics prevented and reversed MMFinduced weight loss and colonic inflammation. Importantly, MMF did not induce intestinal toxicity in germ free mice. Our results reveal that MMF triggers GI toxicity by altering the intestinal microbiota and highlights the microbiota as an important driver of the side‐effects associated with immunosuppressive anti‐rejection therapies.

Support or Funding Information

Alberta Innovates, Canadian Institutes of Health Research, Canadian Foundation for Innovation, Dr. Lloyd Sutherland Fund in IBD/GI Research, Cumming School of Medicine

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using a Zebrafish Model to Study the Gut Microbiota's Effect on the Brain's Neurochemistry and Behavior.
Authors: Ball Alexander G. , Bell Emily A. , Sienerth Karl , Uno Jennifer K.
Published: June 2021

Despite the vast amount of Autism Spectrum Condition (ASC) research, the cause of ASC remains unknown. Recently, several studies indicate a link between ASD and the gut microbiota, a complex bacterial ecosystem present in the digestive tract of animals. The gut microbiome is an important component of the gut‐brain axis, a bi‐directional communication pathway between the gastrointestinal tract and the brain. Additionally, the gut microbiome is a key regulator of the serotonin, which has been found in higher concentrations in the autistic individual's blood serum. Serotonin is involved in neuron growth and may be responsible for the alterations in brain structure seen in autistic individuals. This study employs a zebrafish model to examine how dysregulation of the gut microbiome influences serotonin levels in the brain and how that potentially impacts social interaction. We hypothesize that alterations in the gut microbiota will produce higher levels of serotonin in the brain and therefore cause alterations in behavioral interactions. Adult zebrafish were administered amoxicillin, or an ampicillin/clindamycin combination and the shoal area was measured to examine the zebrafish's social behavior. Intestinal bacterial‐DNA was extracted, and bacterial phyla were examined and quantified by qPCR. Serotonin levels in a zebrafish brain were measured directly though fluorescence spectroscopy and indirectly, through serotonin receptor expression. Results confirmed amoxicillin significantly decreased the quantity and diversity of the intestinal bacterial communities by 60% (p value < 0.05). Antibiotic treatment also correlated to behavioral changes in the fish, illustrated by the 50% larger shoal areas (p value <0.05). The results from this study will help shed light on a complex neurodevelopmental condition.

Support or Funding Information

This work was funded by Elon 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.
Four Weeks of Intermittent Fasting Improves Sleep Quality, Decreases Stress Levels, Improves Quality of Life, and Enhances Body Composition in a Middle Aged Male: A Case Study
Authors: Bains Gurinder , Silver Sasha , Wilson Christine , Garcia Henry , Lohman Everett , Berk Lee
Published: June 2021

Introduction

The obesity epidemic, affecting nearly 78 million adults and 13 million children in the United States, is linked to increased propensity for metabolic syndrome, Type 2 diabetes, heart disease, and stroke. Weight loss and improvement of body composition through physical activity and dietary modifications can help decrease the risk for these obesity‐related diseases. One alternative method of caloric restriction is Intermittent Fasting.

Purpose

Determine the effect of 4 weeks of Intermittent Fasting on sleep quality, stress, quality of life, and body composition.

Methods

A forty‐seven year old male completed a four‐week session of Intermittent Fasting. The subject is a non‐smoker, does not drink alcohol, completed higher education, works in a University setting, and is considered otherwise healthy. The subject was required to perform physical activity for a minimum of three times per week for 30 minutes per session. The subject fasted 16 hours from the time of his last meal of the evening to the start of his first meal the following day. Once the fast was broken, the subject was required to consume his daily caloric intake within an 8‐hour period, with no requirements on number of meals. Basal Metabolic Rate and Total Daily Energy Expenditure was utilized to calculate daily caloric amounts and macro nutrient goals. The Pittsburgh Sleep Quality Index and the Cohen Perceived Stress Scale were administered to determine sleep quality and self‐awareness on stress levels. Quality of Life was assessed through the 36‐Item Short Form Survey Instrument (SF‐36). Body composition was determined using the InBody 270 analyzer.

Results

At the conclusion of the 4 week intervention, there was an improvement in sleep quality (46.9%), reduction in stress (22.6%), and overall improvement in quality of life. In addition, there was a decrease in weight (4.9 kg), BMI (2.4 kg/m2), body fat (3.1 %), and waist circumference (5.1 cm), respectively.

Conclusion

We suggest the health benefits of Intermittent Fasting is a viable option to help decrease the risks of obesity related disease, decrease weight, and lower body fat. Results indicate that four weeks of Intermittent Fasting should be considered to improve sleep quality, decrease stress, improve quality of life, and enhance body composition. Our case study shows potential health related benefits from Intermittent Fasting that can be integrated into whole person wellness programs. Further research is needed to expand these positive findings.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Attenuation of High Glucose/High Insulin‐Induced Muscle Insulin Resistance by Rosemary Extract
Authors: Shamshoum Hesham , Tsiani Evangelia
Published: June 2021

Insulin is a key hormone affecting metabolism and plays a major role in the regulation of glucose homeostasis. Skeletal muscle accounts for ~80% of postprandial glucose disposal and is thus the major organ of glucose uptake. Insulin increases muscle glucose uptake by increasing the translocation of intracellularly stored GLUT4 glucose transporters to the plasma membrane through the phosphatidylinositol‐3 kinase (PI3K)/Akt signaling pathway. Impaired PI3K/Akt signaling is associated with insulin resistance (IR) in skeletal muscle, leading to chronically elevated blood glucose levels and followed by a compensatory rise in insulin levels. The elevated glucose and insulin levels, in turn, exacerbate IR. Polyphenols are phytochemicals derived from natural plant sources that were found to have antioxidant, anti‐inflammatory, anti‐cancer and anti‐diabetic effects.Rosemary extract (RE), rich in polyphenols, contains carnosic acid (CA), carnosol (COH) and rosmarinic acid (RA), which have been shown to counteract insulin resistance in both in vitro and in vivo animal models. However, the mechanisms involved are not known. Therefore, the aim of this study is to investigate the effects of RE on high glucose (HG)/high insulin (HI)‐induced skeletal muscle insulin resistance and the molecular mechanisms involved. Glucose uptake was assessed using [3 H]‐2‐deoxy‐d‐glucose. Total and phosphorylated levels of Akt and IRS‐1 were measured by immunoblotting. Exposure of L6 myotubes to 25mM glucose/100nM insulin for 24 h, to mimic hyperglycemia/hyperinsulinemia, abolished the acute insulin‐stimulated glucose uptake (I: 180±10.43, HG/HI + I: 108 % of basal). In addition, HG/HI attenuated the insulin‐stimulated Akt phosphorylation, while increased IRS‐1 Ser 636/639 phosphorylation indicating insulin resistance. Importantly, treatment with RE (5 μg/ml, 24 h) significantly restored the insulin‐stimulated glucose uptake (HG/HI + RE + I: 184%). Our data indicate a potential of RE to counteract muscle insulin resistance. The focus of our research is to investigate the mechanism involved in mediating this effect of RE.

Support or Funding Information

Supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to ET

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histological Study of Bovine Hoof Development
Authors: Hung Jun , Grossman Ari , Rodriquez‐Sosa Jose R. , Ruiz Saul , Hall Margaret I. , Plochocki Jeffrey H.
Published: June 2021

The ungulate hoof is a complex integumentary organ formed by epidermal and dermal layers that primarily serve to protect the enclosed digits. Each layer serves specific functions and their histological structure may vary throughout prenatal and postnatal development. Importantly, epidermal regions of the hoof interface with the underlying connective tissue layers of the dermis. This tissue interaction includes a system of dermal laminae that interdigitate with the epidermally‐derived horn to increase contact area between the tissues, thereby anchoring the outer hoof to the distal phalanx. Functionality of this system is necessary at birth to reduce risk of lameness, which is associated with bovine mortality. However, the development of this tissue interaction has not been thoroughly investigated in the cow. Here, we study hoof development in bovine fetuses (n=21) aged 60, 80, 110, and 165 days of gestation, which lasts approximately 283 days. Hindlimb hooves were dissected, sectioned at 5 microns, and stained with H&E. Micrographs of the sections show the major layers are present by day 60 of development. Distances between laminae vary slightly with age, but the morphological complexity of the laminar system is similar among the age groups, suggesting growth and maturation of these structures occurs earlier in development, closer to the time of organogenesis. Thus, the tissue morphology necessary to provide mechanical stability to the bovine hoof are present early in prenatal development.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Links between Anatomy, Systems biology, Epigenetics, etho‐eco‐morphological mismatches, evolutionary trends, and organism behavior: a unifying view of anatomical evolution
Authors: Diogo Rui
Published: June 2021

It is a puzzling paradox that eco‐morphological mismatches occur so frequently in an evolutionary process that often leads to macroevolutionary trends and in which organisms are said to be ‘designed’ for the habitats they inhabit. Here I present a new framework ‐ Organic Nonoptimal Constrained Evolution (ONCE) ‐ to address this paradox, and to explain why organisms look as they look: organisms themselves, and in particular their behavior, are the major active players of evolution. That is, within this framework, internal factors can both decrease and increase plasticity/hidden variation and therefore, together with epigenetic factors influenced by the external environment, can allows organisms to shift their behavior, for instance as a response to environmental changes. Importantly, due to behavioral persistence related to behavioral/ecological inheritance, organisms as diverse as bacteria, plants and animals help to construct their own niches, thus being crucial to direct evolution. Darwinian natural (external) selection then comes into play as a secondary ‐ but still crucial ‐ player. That is, due to organismal behavioral persistence, the random mutations/epigenetic factors that happen to be advantageous within the niches constructed by the organisms will be selected, further directing evolution and increasing the match between behavior, phenotype, and environment. This process can extend for long periods of time, leading to macroevolutionary morphological trends and further increasing this match, potentially resulting in successful phenotypic overspecialization. However, behavioral persistence, loss of plasticity due to natural selection, genetic drift, overspecialization, and internal constraints can often make it difficult for the organisms to respond behaviorally and/or anatomically to new environmental changes, resulting in potential mismatches between behavior, ecology and form, and eventually in extinction. This new framework about anatomical evolution therefore bridges the gap between ideas that have been long considered to be opposing views of biology since Aristotle, including internalism and externalism, Cuvier's vs. Geoffroy's take on form and function, Darwinism and Lamarkism, the ideas of Baldwin, Waddington and Goldschmidt, and current Evo‐Devo ideas such as a physicalist framework, niche construction and epigenetic plasticity. This talk therefore covers ‐ and aims to link ‐ areas as diverse as evolutionary developmental biology, paleontology, evolutionary biology, comparative anatomy, ecomorphology, functional anatomy, ecology, ethology, biological anthropology, evolutionary medicine and history and philosophy of biology.

Support or Funding Information

NA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Smell Loss (Hyposmia) is a Lifelong, Chronic Sensory Disease
Authors: Henkin Robert
Published: June 2021

Smell loss (hyposmia) occurs in about 7% of the U.S. population. It is comprised of a heterogeneous group of diseases with three major groupings: direct (brain or nerve injury), congenital (genetic factors) or acquired (biochemical causes). Smell loss most commonly occurs with acquired factors with specific biochemical pathology initiating the disorder associated with a variety of clinical conditions, e.g., post‐viral infection, chronic allergic rhinitis, head injury, inhalational anesthesia and other conditions. The biochemical mechanisms underlying both acquired and congenital hyposmia relate to inhibition of olfactory epithelial receptor cell function due to altered secretion of specific growth or transcription factors in nasal mucus which are required to initiate olfactory receptor stem cell generation and perpetuation. Some growth/transcription factor secretions are decreased to cause these dysfunctions. They include hormones, vitamins, trace metals and a group of biochemical growth/transcription factors – cAMP, cGMP and sonic hedgehog. Some growth/transcription factor secretions are increased to cause these dysfunctions ‐ these are “death factors” which act to inhibit olfactory receptor function. These include cytokines such as TNFalpha. Treatment with oral or intranasal phosphodiesterase (PDE) inhibitors increase secretion of decreased growth/transcription factors and decrease secretion of “death factors” such that stem cells in the olfactory epithelium are stimulated to initiate receptor growth and development. Treatment with PDE inhibitors are required to provide continual stimulation of these stem cells since they initiate the cellular activity by which these receptors (which do not contain blood vessels, lymphatics or exhibit mitosis but turn over rapidly, commonly on a daily basis) are stimulated. In this sense, hyposmia is a chronic disease process similar to diabetes or arthritis. While the disease can be treated it is a lifelong, chronic disorder.

Support or Funding Information

NA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of cocaine cue re‐exposure after prolonged cocaine self‐administration and forced abstinence on DNA methylation and mRNA expression in the rat dmPFC
Authors: Ploense Kyle L. , Vieira Philip , Carr Amanda , Purpura Mari , Kippin Tod
Published: June 2021

Cocaine addiction is a chronic relapsing disorder whereby cocaine craving in response to drug related cues persists, and even intensifies through protracted drug‐free abstinence periods. Studies employing animal models observe cocaine‐seeking patterns that are similar to human addicts. Additionally, there are long term changes in neuron structure, receptor function, and neurotransmission associated with abstinence from cocaine in humans and animals. DNA methylation is an epigenetic modification to the DNA structure that mediates mRNA expression to confer different cell types, but has recently been implicated in learning and memory mechanisms. The long‐term control that DNA methylation has over gene expression in animals makes it a prime candidate for controlling gene expression over the course of abstinence in animals with previous drug experience. To test this, I trained rats to self‐administer either saline (1 h per day) or cocaine (6 h per day) for 15 days, then exposed them to 60 days of forced abstinence. After abstinence half of the rats were put back in self‐administration chambers and re‐exposed to cues for 2 h, while the other half were left in their home cages. After the cue manipulation, all rats were sacrificed, the dmPFC was dissected out, and DNA and mRNA were extracted. Methylated DNA was purified using methylated DNA immuno‐precipitation, and mRNA was reverse‐transcribed into cDNA; levels of each were then quantified via digital PCR procedures. Interestingly, I observed cue‐induced changes in DNA methylation within Homer2, Dlg4, Npas4, and Grin1 promoter regions. These context‐specific changes indicate that DNA methylation is much more dynamic than previously shown. Additionally, I saw changes in mRNA expression for Homer2, Dlg4, and Npas4 that appear to be highly reflective of the changes in DNA methylation. These results indicate re‐exposure to cocaine cues induce gene‐specific mRNA expression via rapid changes in DNA methylation.

Support or Funding Information

This research was supported by NIH grants DA‐027115 and DA‐027525 (TEK) and DA024038 (KKS), as well as funding from and the W.M. Keck Foundation (TEK).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of systematic stimulation mapping and functional/structural imaging to improve localization of seizure onset in patients with drug‐resistant epilepsy
Authors: Prado Rossana Cristina Blanco , Drees Cornelia , Thompson John A.
Published: June 2021

Despite the advantages of using Stereotactic Electrode Encephalography (SEEG) to demarcate epileptogenic onset zones, there are limitations to utilizing SEEG as a method to accurately map the epileptogenic network in patients with drug‐resistant epilepsy. The objective of this study is to use patient‐specific cortical and subcortical segmentations from pre‐electrode implantation structural MRI and post‐electrode implantation CT scans to assess the likelihood that SEEG electrodes are implanted in common eloquent cortex foci across subjects. We assessed the association between seizure foci, semiology, and cortical localization of electrode implantation in 10 patients to provide a three‐dimensional neuroanatomical representation of a patient's epileptogenic network. We hypothesized that there is a common set of eloquent cortical areas targeted among analyzed patients and that functional features of patient semiology will predict cortical localization of seizures and correlate with neurologist‐identified, SEEG‐defined, epileptogenic sites. We used a novel analysis pipeline based on a recently published method (MATLAB iElvis toolbox) and FreeSurfer to prepare pre‐electrode implantation patient MRI and post‐electrode implantation CT scans to build a patient‐specific three‐dimensional model, corrected for brain shift. Electrodes were mapped in 3D‐space in BioImage Suite software. Cortical brain areas were later mapped and merged with 3D‐model of patient‐specific electrodes. Data were analyzed for correlation of electrode, cortical location, and patient semiology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An fMRI Study Examining Inhibition and Threat Vigilance Among Adolescents with Severe Worry
Authors: Feiler Jacob B.
Published: June 2021

Severe worry is a common psychiatric phenomenon that increases in prevalence during adolescence and affects multiple aspects of emotion and cognition, including executive function. Inhibition, one domain of executive function, is the ability to suppress particular thoughts or actions in the pursuit of goals and is thought to be mediated by coordinated activity across a network of prefrontal cortical regions. Individuals with severe worry are often hypervigilant for threat, a process which may compete for cognitive resources and contribute to deficits in inhibitory control; these mediating brain processes, however, are poorly understood. In this study, we have thus far examined a sample of 50 adolescents, ages 13 to 18, with varying degrees of worry, as measured by the Penn State Worry Questionnaire (PSWQ‐C) (Chorpita et al., 1997). We collect fMRI data from each participant, utilizing a mixed block/event‐related design and go/no‐go paradigm to examine behavioral inhibition under conditions of threat (i.e., unpredictable aversive sound) and non‐threat (i.e., no chance of aversive stimuli). Under conditions of threat, we hypothesize that individuals with severe worry will show less effective inhibitory control during no‐go trials and, therefore, demonstrate lower task performance when compared to low worriers. Task performance, or sensitivity index, is measured by the variable d’, which accounts for a participant's normalized task accuracy (hit rate) and false alarm rate. Additionally, we hypothesize that individuals with severe worry will demonstrate greater activation in brain regions related to inhibition and threat vigilance, including, the anterior cingulate, ventrolateral and dorsolateral prefrontal cortices, and limbic regions like the amygdala and insula. Preliminary results of these findings will be presented, as well as the role of these variables in the origination and maintenance of severe adolescent worry.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of the Activating Transcription Factor 5a (atf5a) gene in Hair Cell Development and Regeneration
Authors: Morales Roberto Rodriguez , Colon‐Cruz Luis , Burgess Shawn , Sharney Gaurav , Behra Martine
Published: June 2021

Hair cells (HC) are sensory mechanoreceptors that are localized in sensory patches of the vertebrate inner ear. HC loss is the single leading cause of hearing and balance deficiencies. Mammals are unable to regenerate damaged HCs unlike lower vertebrates like birds, amphibians and fish. The two latter, also regenerate HCs in a superficial mechano‐sensory structure called the lateral line (LL) that allows them to sense water movements. Significant progress has been made in recent years by exploiting the experimental accessibility of the LL in a genetic tractable animal model: the zebrafish. The LL is composed of stereotypically distributed small sensory patches called neuromasts (NM). Each NM is a bulb‐shaped structure made of ~60 cells. It is externally delimited by ~10 mantle cells (MCs) which are forming an outer apical constricted ring through which protrude cilia from 12–15 centrally located HCs that are engulfed by 30–40 support cells (SC). Synchronized HC destruction will trigger a well described 3‐day regeneration program. New HCs are sequentially added from dividing and differentiating SCs. In parallel, other SCs proliferate replenishing their own stock, thus fitting the stem cell definition. Our lab has previously identified several genes with SC enriched expression. One of them is the activating transcription factor 5a (atf5a). It is a cAMP‐response element‐binding (CREB) transcription factor which was previously shown to have an important role in olfactory sensory neuron (OSN) differentiation in rodents, but was never described in the LL. To assess the role of this gene during OSN and HC development and regeneration, we first confirmed by in situ hybridization using an antisense probe directed against atf5a that it was expressed in both the olfactory pits and the LL. Next, we generated loss‐of‐function atf5a (KOs) using CRISPR‐Cas9 genome editing technology. So far, we have identified a 7 bp deletion (atf5a7D) that should represent a null allele, as reading frame shift is predicted to introduce an early stop codon in amino acid 93. We will present our preliminary results of the phenotypic analysis of homozygote (atf5a7D−/−) animals which we are focusing on the development and the regeneration of HCs in the LL and OSN in the nasal pits. Understanding the role of atf5a in the development and regeneration of these sensory tissues will help advance therapeutic regenerative approaches.

Support or Funding Information

Funding: R25GM061838 MBRS‐RISE and NSF CREST Grant #HRD‐1137725

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bacteriophage DNA thymidine hypermodification biosynthesis is identified via an amino acid‐modified nucleobase intermediate
Authors: Lee Yan‐Jiun , Guan Chudi , Dai Nan , Correa Ivan , Weigele Peter
Published: June 2021

Double stranded DNA viruses of bacteria (i.e. bacteriophages) contain the greatest diversity of modified nucleotides in their DNA. These non‐canonical nucleotides are appear to completely replace one of the canonical bases or substitute for a subset of one of them. Base modifications expand the properties of DNA, conferring additional essential functions such as DNA protection and epigenetic gene regulation.

Recently, we have discovered and chemically characterized two new naturally occurring thymidine modifications: 5‐aminoethyl‐2′‐deoxyuridine from Pseudomonas phage M6 and 5‐(2‐aminoethoxy)thymidine from Salmonella phage ViI. These phage DNA thymidine modifications begin from 5‐hydroxymethyl‐2′‐deoxyuridine (5hmdU), where the C‐5 of the heterocycle is covalently modified with substituents. We have identified the subsequent steps in the biosynthesis of these novel hypermodified bases and in turn have been able to partially reconstitute their formation in vitro. Briefly, bioinformatic comparisons of hypemodifying and canonical phage genomes were used to determine candidate hypermodifiying function‐encoding genes. We developed an assay utilizing bead immobilized DNA substrates and in combinations of lysates containing recombinantly expressed candidate hypermodifying genes which allowed us to examine base modification activities in a systematic and reproducible fashion. By this approach, we have discovered that bacteriophage M6 and ViI thymidine DNA modification occurs through three steps. First, a bacteriophage encoded DNA kinase transfers the γ‐phosphate from adenosine triphosphate (ATP) to the 5hmdU on the DNA forming 5PmdU and activating the adjacent hydroxymethyl group at the C5. Second, a novel enzymatic activity ‐‐ amino acid transferase ‐‐ displaces the phosphate with the amino acid; glycine in the case of M6 and serine in the case of ViI. Lastly, a final processing step catalyzed by a pyridoxal phosphate dependent enzymes (PLPDE) or radical s‐adenosylmethionine (rSAM) enzymes leads to the decarboxylation of the amino acid decoration, resulting in the final bacteriophage DNA thymidine modifications. We are currently investigating the individual enzyme mechanisms (particularly on the novel amino acid transferase) to understand the thorough biochemical basis of the phage DNA thymidine hypermodification processes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fluoxetine and citalopram significantly alter gene expression in the midbrain of neonate mice uncovering possible antidepressant‐mediated epigenetic programming changes during development.
Authors: Henry L. Keith , Rodriquez Meghan A. , Allen Michael D. , Shetty Madhur , Perley Danielle
Published: June 2021

Selective serotonin (5‐HT) reuptake inhibitors (SSRIs) are the primary medication prescribed for depression during pregnancy and act by increasing serotonergic tone via binding and inhibiting 5‐HT reuptake by the serotonin transporter (SERT). In utero exposure to SSRIs has been linked to developmental delays and malformations and could be related to the trophic actions of 5‐HT. In longitudinal studies, in utero exposure to SSRIs in humans has been associated with increased mood disorder diagnoses in adults. Similar findings have been observed in rodent models where neonate treatment with SSRIs results in depressive and anxiety‐like behaviors in adult animals. These models exploit specific developmental windows when the brain has increased susceptibility to changes in serotonergic tone. Early‐life challenge by SSRIs and long‐term behavioral consequences implicates the involvement of epigenetic mechanisms. To investigate this possibility, RNA‐Seq analysis was performed on midbrain, cortex and hippocampal tissue from neonate mice challenged with the SSRIs fluoxetine (FLX), citalopram (CIT) or paroxetine (PXT) from postnatal days (PND) 4–21 and 4–28. In midbrain samples, FLX‐treated PND 4–21 mice had 43 differentially expressed genes (DEGs), whereas CIT‐ treated PND 4–28 mice had 193 DEGs. These results were encouraging, as the midbrain is enriched with serotonergic cell bodies in the raphe nuclei. In contrast, gene expression changes were not identified in cortex or hippocampal tissues which contain serotonergic termini and an increased heterogeneous cell population in comparison to the midbrain. This heterogeneity presents significant challenges for detecting SSRI‐mediated changes in gene expression. Pathway analysis revealed an enrichment of transcripts associated with serotonergic and dopaminergic signaling, and with multiple cellular functions involved in neuronal communication and nervous system development in the midbrain of CIT and FLX‐treated mice. Importantly, several key genes involved in epigenetic modifications, such as histone acetylation and methylation of RNA, mRNA and histones were differentially expressed in CIT‐treated mice, implicating a link to epigenetic mechanisms. Additionally, neonate treatment with CIT affects ribosome function and mRNA splicing and processing. Collectively, these data provide reasonable candidate genes which can be evaluated for a role in SSRI‐mediated developmental changes in the brain and how these alterations can lead to persistent behavioral changes in adult animals.

Support or Funding Information

NIH‐NIGMS P20 GM104360

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Presence of N6‐methyladenine and 5‐methylcytosine in Trypanosoma brucei RNA
Authors: Keplinger Amanda J. , Coffey Jordan M. , Militello Kevin T.
Published: June 2021

Trypanosoma brucei is the parasite responsible for Human African Trypanosomiasis, commonly referred to as African Sleeping Sickness. In T. brucei there is a lack of promoters and other regulatory sequences normally present in DNA. Thus, posttranscriptional RNA alterations such as methylation may be important in this organism. To investigate the occurrence of RNA base methylation, a series of ELISAs for N6‐methyladenine (N6‐MeA) and 5‐methylcytosine (5‐MeC) were performed. The presence of N6‐MeA in total RNA and small RNA was detected with minimal differences between procyclic and bloodstream forms. ELISAs identified 5‐MeC in both procyclic and bloodstream form total RNA samples, and low 5‐MeC levels in small RNA. Using a robust bisulfite sequencing technique, total RNA and small RNA 5‐MeC residues were mapped with base resolution. There was an elevated frequency of 5‐MeC in tRNA with numerous residues having a level of methylation greater than 50%. To investigate the source of 5‐MeC in RNA, BLAST identified seven putative T. brucei cytosine RNA methyltransferases (TbCRMTs). Of the seven TbCRMT RNAi lines generated, 2 of the 7 resulted in growth defects when the RNAi system was activated. To study the TbCRMTs in vitro, recombinant pET28‐TbCRMT plasmids were introduced into E. coli. The TbCRMTs were expressed as N‐terminal 6x‐his tagged proteins and characterized by SDS‐PAGE and western blot analysis. The TbCRMTs are soluble and, in the future, successful protein purification will enable in vitro RNA methylation assays. In summary, the data indicate that T. brucei transcriptome contains modified RNA bases, and we have identified the 5‐MeC positions and putative enzymes responsible.

Support or Funding Information

NIH 1R15AI13342801

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigenetic Modifications in the Rat Neonate Olfactory Bulb and Anterior Piriform Cortex Associated with Olfactory and Tactile Experiences
Authors: McAnulty Jessica , Webb Lauren , Doherty Tiffany , Roth Tania
Published: June 2021

Rodents are macrosmatic; they depend heavily on the olfactory system, which includes the olfactory bulb and anterior piriform cortex, to navigate their environment. The olfactory system is key to the survival of neonatal pups in order to learn the scents of the dam, which aid in nipple attachment and overall attachment to the caregiver. This attachment‐based learning was mimicked in rodents using a laboratory‐based experimental design in which neonatal pups were classically conditioned to associate the novel scent of peppermint with stroking, an action that mimics sensory cues associated with caregiving. The effects of attachment‐based learning on mRNA levels of epigenetic regulators, as well as epigenetic modifications of Bdnf and Reln, genes involved in neuroplasticity, are unknown.

We investigated changes in gene expression of Bdnf, Reln, and epigenetic regulators (DNMT1, DNMT3a, DNMT3b, HDAC1, HDAC2, HDAC5, TET1, Gadd45B, and MeCP2) following infant olfactory learning. Using an odor‐stroke paradigm, Long‐Evans rat pups (postnatal days 6 or 7) (n=46) were assigned to either a paired (simultaneous peppermint odor and stroking), unpaired (non‐simultaneous peppermint odor and stroking), or odor‐/stroke‐only condition. Brains were harvested 30 minutes after conditioning. Using Real‐time PCR, we found experience‐induced changes in gene expression profiles in both brain regions. For example, Bdnf exon IV gene expression in the olfactory bulb significantly increased in rats that received contiguous presentation of both stimuli; however, gene expression of HDAC2, HDAC5, TET1, and Gadd45B significantly decreased in the same subjects. In the anterior piriform cortex, there were significant increases in gene expression of Bdnf exons I and IV, DNMT3b, and Gadd45B in both paired and unpaired subjects. Understanding the unknown epigenetic modifications involved in attachment‐based learning may elucidate the mechanisms of the emotional connection an infant learns for the caregiver and its profound influence on neurological development.

Support or Funding Information

Special thanks to Dr. Roth and Tiffany Doherty for their support and guidance along with Lauren Webb's and Tiffany's completion of odor‐stroke conditioning. This research is funded through grants P20GM103653, R01HD087509, and private donation funds to Dr. Roth.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying polyphosphorylation, a novel PTM, in mammalian cell lines
Authors: Holinier Charlotte , Bentley‐DeSousa Amanda , Bondy‐Chorney Emma , Davey Norman , Downey Michael
Published: June 2021

Polyphosphates are long chains of phosphates attached to one another via high‐energy phosphoanhydride bonds. The literature reports their implication in a variety of medically related functions, such as apoptosis, blood coagulation, and inflammation. The mechanisms involved with polyphosphate and its functions have been studied in bacteria and in yeast, but the synthesis pathway of polyphosphate in mammals is still unknown. Moreover, the concentrations of polyphosphate in mammalian cells (<1mM) are much lower than the ones found in yeast (100–200mM). This makes it challenging to study polyphosphate biology and polyphosphorylation in mammalian cell lines. Recently, Azevedo, et al. (2015) identified a new PTM in yeast called polyphosphorylation and two targets of this PTM: Nsr1 and Top1. It consists of the non‐enzymatic covalent attachment of polyphosphate chains to lysine residues found in a poly‐acidic serine and lysine‐rich (PASK) motif. Our lab has since identified 24 novel yeast targets being polyphosphorylated. Our next aim was to test if human proteins can also be polyphosphorylated. In order to modulate the concentrations of polyphosphate in the cell, we transfected mammalian cells with PPK1, an E. coli enzyme that synthesizes polyphosphate. This ectopic expression allowed us to perform a small “screen” and identify 6 human proteins that can be polyphosphorylated: nucleolin, hNop56, Mesd chaperone, DEK, eIF5B and UPF3B. We will present our latest work focused on determining the molecular function of polyphosphorylation in mammalian cells.

Support or Funding Information

This project is funded by the Canadian Institutes of Health Research (CIHR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding How Two Similar RNA Binding Domains of Paralogous Proteins Mediate Different Protein‐Protein Interactions
Authors: Marshall Collin McCarty , Pina Jeffrey , Keppetipola Niroshika Monerawila
Published: June 2021

Polypyrimidine Tract Binding Protein (PTBP) is an RNA binding protein that has a high affinity for pyrimidine rich regions of RNA sequences. Studies have shown that it acts primarily as a negative regulator of alternative pre‐mRNA splicing events (ASEs); however, it has been shown in some cases to enhance ASEs. Improper regulation of ASEs is linked to neurodegenerative diseases and cancer. Two well studied paralogs of PTBP are PTBP1 and PTBP2. The two proteins demonstrate different levels of splicing repression activity despite sharing high amino acid sequence identity (74%) and similar domain arrangement consisting of four RNA recognition motifs (RRMs) connected by three linker sequences and an N‐terminal region. Recent studies generating chimeras of the two proteins demonstrated that multiple regions of PTBP1 can confer splicing repression activity when integrated in the PTBP2 backbone. These studies also highlighted that despite the high amino acid sequence identity, the RRM 2 region of the two proteins demonstrate different affinities for a partner protein, Raver 1. This protein is required for complete PTB‐mediated splicing repression of the α‐tropomyosin exon 3 sequence. The PTBP‐Raver1 interaction motif has been identified, and PTBP1 and PTBP2 share 87% amino acid sequence identity within their respective Raver1 interaction motifs. These results suggest that additional factors such as post‐translational modifications (PTMs) might influence the PTBP‐Raver1 interaction and in turn, splicing repression activity. To test this, we assayed PTBP1 RRM2 for PTMs via immunoprecipitation followed by mass spectrometry and have identified several ubiquitin modifications. Next, we plan to examine whether the ubiquitin modifications in PTBP1 RRM2 contributes to the Raver1 interaction specifically, as well as to other protein‐protein interactions required for splicing repression activity.

Support or Funding Information

This work was supported by a Maximizing Access to Research Careers grant to CSUF from the National Institutes of Health [5T34GM008612‐21]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Broad‐Based Labeling Reagents for Peptide and Protein Footprinting
Authors: Nieves Wilson , Zhang Bojie , Cheng Ming , Gross Michael
Published: June 2021

Interactions between macromolecules in living organisms are governed by fundamental biophysical principles. Different methods to study these biophysical properties exist; including footprinting. Footprinting refers to the structural probing of interaction between macromolecules using chemical reactions ranging from HDX to reactive species such as free radicals and carbenes. Modern footprinting methods allows for a fast, specific, and precise determination of proteins' structure, interaction sites, conformational changes, folding, misfolding, and binding affinity. The complete understanding of these, in‐vitro and in the cell, is crucial for drug development and antibody synthesis for therapeutics and diagnostics. These advances contribute to the understanding of the human proteome. Methods for determining a protein footprint include Mass Spectrometry (MS), which is advantageous given its sensitivity. Covalent modification of amino acid residues coupled with MS has been useful in characterizing non‐membrane and membrane proteins; a field where current biophysical techniques are limited. Covalent modification can be achieved via Fast Photochemical Oxidation of Proteins (FPOP) using radical chemistry. Residue labeling using current radical species lacks generality or broad reactivity among amino acids. Thus, the development of novel chemical labeling methods that increases amino acid labeling are needed. To implement this, we propose the use of carbene species (double radicals), trifluoro‐methyl radicals, and chloride cations. Carbene compounds will be generated via FPOP from diazirine precursors, which were synthesized from ketone precursors. The Togni's Reagent will be used as a precursor for covalent modification through trifluoromethyl radicals and Palau's reagent will be used for modification through chloride cations. If a robust footprinting method is developed to further understand protein biophysics it would lead to further advances in the study of protein folding, protein‐protein interactions, and drug development.

Support or Funding Information

Amgen Foundation. Michael Gross's Laboratory. NIH/NIGMS Biomedical Mass Spectrometry Resource.

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