bluuebunny/crossref_metadata_2025 huggingface dataset:
High Fat Diet Impacts Gut Microbiome Differently in Male and Female Sprague Dawley Rats
Authors: Gonzalez Agnes P. , Rodriguez Christine D. , Brofen‐Quiñones Samuel , Sambolin‐Escobales Lubriel , Cruz Myrella , Appleyard Caroline B. , Chompre Gladys
Published: June 2021

Obesity has increased during the last couple of decades and is strongly related to cardiovascular disease, hyperlipidemia, diabetes, and neurological disorders, such as depression, which tend to be more prevalent in women. A high fat diet (HFD) is associated with negative effects on the gut microbiome, including increased Firmicutes/Bacteriodetes ratio (F/B ratio) which may affect the integrity of the intestinal barrier. Gut microbiome transplants from male to female rats have been shown to decrease diabetes type 1 in females, suggesting sex differences in the gut microbiome. Previous studies in our lab have shown that Sprague Dawley male rats fed with a HFD showed a depressive‐like behavior and also a decrease of tight junction proteins in the gut suggesting gut permeability. However, it is not clear whether the effects of a HFD on the microbiome are based on gender differences.

AIM

To ascertain whether the administration of a HFD will affect differently the gut microbiome in males and females.

METHODS

There were four groups of Sprague Dawley rats (n=12/group): Female Low‐Fat Diet (FLFD), Female High Fat Diet (FHFD), Male Low‐Fat Diet (MLFD), Male High Fat Diet (MHFD). Female and male postnatal rats were fed with a LFD (10% fat) for 6 weeks. Then, half of each group were transferred to a HFD (60% fat) for 8 weeks. We measured absolute weight daily, and rats were tested for depressive like syndrome in a porsolt swim test one day before sacrifice. Fat and feces were collected from all animals to assess differences in the gut microbiome.

RESULTS

Rats showed no differences in absolute weight nor in percent weight gain in either group, however, MHFD showed an increase of epidydimal fat tissue compared to MLFD. FHFD group showed a significant increase in immobility compared with the FLFD group suggesting depressive like syndrome (p<0.05). Measurement of depressive like syndrome in males was limited by their size and did not reach significance. In gut microbiome, MHFD showed a significant increase in the F/B ratio compared with the MLFD, however, in females there were no significant differences. Females on a high‐fat diet had a significant increase in Proteobacteria compared to FLFD (p<0.05). In contrast MHFD showed a significant increase in Verrucomicrobia compared with the MLFD (p<0.05).

CONCLUSIONS

A HFD affects the gut microbiome of males and females differently. The significance of this study is that sex differences may be a potential factor in how a HFD might contribute towards various pathophysiological conditions via differing effects on the gut microbiome.

Support or Funding Information

Supported by NIGMS/INBRE P20 GM103475‐14 and RCMI G12 MD007579

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic plasticity through pregnancy with normal pregestational BMI
Authors: Delgadillo‐Velazquez Jaime , Patiño‐Gonzalez Nayeli , Nambo‐Venegas Rafael , Millán‐Cortés Alexis , Vadillo‐Ortega Felipe , Palacios‐Gonzalez Berenice
Published: June 2021

During normal pregnancy, metabolic changes occur causing weight gain and insulin resistance to provide the fetus with substrates for growth and development. Hence, a metabolic plasticity must exist to allow the mother to adapt correctly upon the pregnancy metabolic overload, preventing adverse outcomes like gestational diabetes. The aim of the study was to analyze the metabolic plasticity during pregnancy in women with normal pregestational BMI. Glucose, triglycerides and total cholesterol were determined, and indirect calorimetry was performed during fasting and after the consumption of a high‐carbohydrate meal. Fasting triglycerides and total cholesterol were significantly higher during the third trimester. Postprandial glucose concentrations were significantly lower at second trimester and higher in third trimester compared to first trimester. There was a significant increase in fasting respiratory quotient (RQ) during the last trimester compared to the first and second trimester. Regarding ΔRQ, there was a significant decrease during the last trimester compared to the first trimester. In conclusion, at the end of pregnancy there is a lower metabolic flexibility, associated with the presence of physiological insulin resistance.

Support or Funding Information

This study was funded by CONACYT grant FOSISS 2016 # 273110

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Partial Sleep Deprivation on Lipid Profile
Authors: Covassin Naima , Bukartyk Jan , Cundrle Ivan , Singh Prachi , Calvin Andrew D. , St Louis Erik K. , Somers Virend K.
Published: June 2021

Background

Increasing evidence suggests that insufficient sleep is associated with increased morbidity and mortality. Studies linking short sleep with metabolic derangements such as obesity, insulin resistance and dyslipidemia, are especially persuasive. Nevertheless, the vast majority of these data have been obtained from observational studies. We sought to evaluate the effects of 9 days of partial sleep deprivation compared to normal sleep on lipid profile in healthy subjects undergoing an in‐laboratory, experimental protocol.

Methods

Eight healthy, nonobese, normolipidemic subjects (4 males, age 25.4±6.8 years, body mass index 25.2±3.9 kg/m2) completed a randomized, controlled, crossover, 16‐day in‐patient study. Each sequence consisted of a 4‐day acclimation period, followed by 9 days of experimental partial sleep deprivation (4 hours of sleep opportunity; 00:30 AM – 4:30 AM); or normal sleep (9 hours of sleep opportunity; 10:00 PM – 7:00 AM), and a subsequent 3‐day recovery segment. Subjects consumed a controlled, weight maintenance diet (15% protein, 55% carbohydrates, and 30% fat) during the study. Fasting blood samples for measures of total cholesterol, high‐density lipoprotein (HDL) cholesterol, low‐density lipoprotein (LDL) cholesterol, and triglycerides were obtained in both study sequences on Days 4, 7, 10, 13, and 16.

Results

Compared to normal sleep, experimental partial sleep deprivation did not cause significant changes in total cholesterol (partial sleep deprivation vs normal sleep: 160.1±21.8 mg/dL vs 164.9±23.7 mg/dL, P=0.724), HDL cholesterol (54.1±14.7 mg/dL vs 52±12.8 mg/dL, P=0.75), LDL cholesterol (86.5±18.9 mg/dL vs 89.5±19.7 mg/dL, P=0.329), or triglycerides (97.7±26.6 mg/dL vs 96.6±38.3 mg/dL, P=0.633). Analysis of time course of lipid values across study days did not reveal appreciable patterns (all P>0.295). Likewise, body weight was similar during both study sequences (partial sleep deprivation vs normal sleep: 69.6±14.2 kg vs 69.2±13.5 kg, P=0.459).

Conclusions

Experimental sleep restriction does not alter circulating lipids or weight in healthy subjects. Aberrant metabolic effects of insufficient sleep may be mediated by dietary intake and occur when access to food is unrestricted, or may emerge only after longer exposure to sleep curtailment.

Support or Funding Information

American Heart Association grant 16SDG27250156; National Institutes of Health R01 grants HL114676 and HL114024; European Union project FNUSA‐ICRC no. CZ.1.05/1.1.00/02.0123.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sagittal Craniosynostosis and Related Cranial Anomalies: Genetic Insights
Authors: Olatunde Josh , Frolov Andrey , Tersigni‐Tarrant MariaTeresa , Martin John
Published: June 2021

Background

Sagittal craniosynostosis (scaphocephaly) is a pathologic condition that results in premature fusion of the sagittal suture restricting the transverse growth of the skull leading to elevated intracranial pressure and consequent neurodevelopmental delay. It can occur most commonly as an isolated event resulting in nonsyndromic craniosynostosis, or it can occur less commonly in conjunction with other anomalies resulting in syndromic craniosynostosis. Although in past years close attention was paid by researchers regarding the mechanisms that govern development of craniosynostosis, much more remains to be learned about this cranial anomaly, in particular, the genetic mechanisms associated with scaphocephaly. The clearer understanding of the latter not only could shed more light on the respective mechanisms but could also help to improve and predict better outcomes of the respective corrective clinical procedures.

Results

The present study reports a unique case of a male cadaver 56 years of age with sagittal craniosynostosis with a 0.56 cranial vault index as measured from respective radiographic images. Upon closer examination of the individual's head it was concluded that he underwent, most likely early in his life, a sagittal strip synostectomy with replacement. The additional maxillofacial anomalies included edentulous, mandibular prognathism with class 3 malocclusion and 15 mm of negative overjet, and exostosis of the hard palate. The latter was preliminary characterized as torus palatinus. One of the most interesting features of the present case was the abnormal appearance of the replacement bone strip and its vertical displacement that were accompanied by a presence of abnormal dura and venous sinuses with no brain indentations on the dural surface of the strip. Histologic analyses of cranial soft and hard tissues should help to clarify a nature of the respective pathologies. In order to better understand molecular mechanism(s) responsible for the development of sagittal craniosynostosis with the particular maxillofacial anomalies, the gene coding region (exome) of DNA extracted from the cadaveric tissue was screened for putative mutations employing Illumina Next Generation Sequencing Platform. This will help to unveil new genetic variations associated with the aberrant skull development in humans and optimize clinical approaches for its correction.

Conclusion

The current case most likely represents syndromic sagittal craniosynostosis where surgical intervention was mainly unsuccessful resulting in abnormal development of the replacement bone strip and intracranial tissue.

Support or Funding Information

This study was supported by the Center for Anatomical Science and Education, Department of Surgery, Saint Louis University 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.
New in vivo microRNA biotechnology reveals specific roles for the miR‐200 family in craniofacial development
Authors: Sweat Mason E. , Yu Wenjie , Eliason Steven , Sweat Yan Yan , Cao Huojun , Hong Liu , Amendt Brad A.
Published: June 2021

The molecular regulators of stem cell differentiation are a hot topic and the proteins involved in this process have been well studied, but the function of microRNA (miR) remains elusive.

Objectives

To investigate the role of the miR‐200 family in the regulation of craniofacial development and dental epithelial stem cells (DESCs). Previously, we found that the miR‐200 family was highly expressed in the differentiated cell types of the lower incisor but was absent from the stem cell niche, suggesting it may contribute towards differentiation.

Methods

We constructed a plasmid‐based miR inhibitor system (PMIS) and used it to inhibit the miR‐200 family in mice. One inhibitor line specifically targets the seed sequence specific for miR‐200a, and ‐141, while the other is specific for the seed sequence found in miR‐200b,‐200c and ‐429, and crossing the lines allows the functional inhibition of the entire miR‐200 family.

Results

Double inhibitor mice are neonatal lethal and have poorly differentiated lower incisors. The teeth are smaller and they lack cells expressing cell differentiation markers (DSP and amelogenin) of mature odontoblasts and ameloblasts. Several transcription factor regulators of stemness which are direct targets of the miR‐200 family, including Sox2, are ectopically expressed in normally differentiated cells. A proliferation cell labeling BrdU assay demonstrated that the majority of dental epithelial cells are proliferating in double inhibitor mouse incisors.

Conclusions

This study is among the first to use targeted inhibition to study specific miRs and we found that the miR‐200 family is required for the appropriate differentiation of DESCs and the family functions by reducing the expression of transcription factors promoting stemness outside of the stem cell niche compartment of the lower incisor. Overall, craniofacial development is affected due to a lack of cell differentiation.

Support or Funding Information

University of Iowa College of Dentistry

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Patient‐derived MAFB Missense Mutation Is Associated with Cleft Palate in Mice
Authors: Paul Brian J. , Palmer Kristina , Padilla Crystal , Sharp Jocelyn C. , Pratt C Herbert , Murray Stephen A. , Dunnwald Martine
Published: June 2021

Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with complex etiology. Amongst the numerous genes involved, mutations in MAFB (MAF BZIP Transcription Factor B) were recently associated with increased risk for NSCL/P. MAFB, a single‐exon gene of 3.3kb, encodes the protein MAFB, which is a transcription factor expressed in the craniofacial region at time points critical for palatal development. Previous sequencing of MAFB in cases revealed a novel missense mutation which was significantly associated with an increased risk of NSCL/P. This patient‐derived MAFB mutation, leading to the amino acid change H131Q, was knocked‐into the mouse Mafb, resulting in the allele MafbH131Q. In contrast to Mafb nulls, which die shortly after birth, MafbH131Q mice survived into adulthood at Mendelian ratios. MafbH131Q embryos were harvested for serial sectioning and histological characterization at time points critical for craniofacial development. One MafbH131Q/H131Q animal at embryonic day (e) 18.5 (N = 12) exhibited highly abnormal craniofacial morphology that included a cleft palate and shortened snout. At e13.5, our preliminary data indicate the presence of mild oral adhesions in MafbH131Q/+ and MafbH131Q/H131Q mice that were absent in wild type animals. However, immunofluorescence for periderm markers did not indicate altered expression of keratin 6 and keratin 17. These data demonstrate functional significance of the patient‐derived H131Q mutation and its role in the etiology of NSCL/P.

Support or Funding Information

MD ‐ NIH/NIAMS AR067739. Additional partial financial support was provided by a grant from the NIH R37DE08559 and by the FaceBase consortium (grants DE020052 and DE020057)

CP ‐ Iowa Biosciences Academy, funded by NIH/NIGMS R25GM058939, the University of Iowa (UI) Office of the Vice President for Research, and the UI Chief Diversity Office.

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 Relationship Between Joint Size and Trabecular Bone Density in Human and Nonhuman Primates
Authors: Chirchir Habiba , Ruff Christopher
Published: June 2021

Biomechanically, a relationship is expected between loading and joint size, i.e., an organism with greater mechanical loading may have a large joint surface in order to mitigate greater joint stresses. However, among primates, hominoids have large long bone joint surfaces relative to diaphyseal strength, and also demonstrate generally greater joint mobility. Consequently, it has been proposed that relative joint size may also be related to greater joint excursion. Subchondral trabecular bone mass may be more directly influenced by mechanical loading of a joint. Thus, since subchondral trabecular bone density is a function of both joint size (volume) and trabecular bone mass, both mechanical loading and joint excursion could influence it. Although there has been some research investigating the relationship between trabecular bone mass and joint volume, the potential impact of joint size on trabecular bone has not been fully tested across many species in multiple limb elements. This study investigates trabecular bone density (trabecular bone fraction, TBF) in multiple proximal epiphyses in primates with varying modes of locomotion. We used a peripheral Quantitative Computed Tomography (pQCT) scanner to estimate TBF in the femur, humerus, 3rd metatarsal and 3rd metacarpal. This was done in modern humans, chimpanzees, orangutans, vervet monkeys, olive baboons, gelada baboons and patas monkeys. Pearson's correlations were used to test relationships between joint size and trabecular density in each species. The results show that TBF has broadly similar values across a large range of joint sizes. There is no strong correlation between the variables; modern humans, despite having larger joint sizes than the other primates, display low trabecular density, while orangutans and chimpanzees with intermediate joint sizes have relatively low trabecular density compared to the cercopithecines. Olive baboons with larger joints among the cercopithecines have trabecular densities that are most similar to that of vervets and patas monkeys, whose joints are relatively smaller. These results support the proposal that increased joint size is likely correlated with greater joint mobility to increase joint excursion, and is not necessarily a function of increased mechanical loading. Therefore, relationships between subchondral bone density and mechanical loading are not straightforward when compared across taxa with differences in joint excursion.

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 relationship of vascular anatomy to thermal physiology: linking anatomical and imaging studies to in vivo non‐invasive infrared thermography of free‐ranging birds
Authors: Porter Wm Ruger , Tattersall Glenn J. , Caggiano Emily G. , Witmer Lawrence M.
Published: June 2021

Vertebrates use the blood vascular system to moderate the temperature of specific anatomical regions. However, in many groups, such as birds, fundamental thermoregulatory mechanisms—the actual link between physiological function (heating and cooling) and the underlying vascular “plumbing”—have largely remained obscure, challenging our understanding of issues ranging from organismal responses to climate change to the human health consequences of vascular disease. In vivo physiological testing typically involves highly invasive procedures that may impose confounding artifacts due to stress responses mediated by the sympathetic nervous system. Fortunately, high‐resolution infrared (IR) thermal imaging provides a non‐invasive means of in vivo physiological measurement of surface temperatures in free‐ranging, normally behaving animals. In our study, thermal imaging was used to document heat maps of freely ranging birds (e.g., vultures, pelicans) in South Carolina and Florida. Quantitative thermographic data were analyzed to assess sites of heat exchange. Thermal data were compared to existing and new anatomical data based on microCT scanning of radio‐opaque (barium‐latex) vascular injections on legally obtained cadaveric bird specimens. Specific regions of the head where vascular devices in birds are suspected to be important for shedding heat as well as for cooling venous blood destined for the brain and eye were analyzed for anatomical and physiological correlations. This study seeks to establish the mechanistic links that ultimately allow birds to manage thermal stressors. Thermal images of investigated sites of thermal exchange indicated heat flow was found to correspond to the location of known blood vessels. These blood vessels were discernable in times of both heating and cooling. For example, in pelicans, the location of the ophthalmic rete corresponds to the location of a cool spot located behind the eye. Blood vessels in the pelican's pouch correspond to a streak of high temperature in the pouch. These results indicate that blood vessels play an active role in the thermophysiology of free‐ranging and unstressed birds and that the details of thermoregulatory mechanisms are discernable with non‐invasive methods.

Support or Funding Information

United States National Science Foundation (IOB‐0517257, IOS‐1050154), Ohio University Heritage College of Osteopathic Medicine, Ohio University Research Committee, Natural Sciences and Engineering Research Council of Canada, and the National Geographic Society.

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 Cadaver Specimens to Better Understand Ulnar Nerve to Median Nerve Transfers
Authors: Dorius Geoffrey T. , Morton David A. , Mahan Mark A.
Published: June 2021

The recurrent branch of the median nerve innervates the thenar muscles. Therefore, injury to the median nerve as it courses from the axilla to the hand has profound negative effects on the functional use of the thumb. The surgical procedure of peripheral nerve transplants in order to regain function in denervated muscle is well documented. Using the lumbrical branch of the ulnar nerve to the 4th digit as a nerve transplant with the recurrent branch of the median nerve in order to regain function is not well documented. As such, the capacity of grafting these ulnar nerve branches from their anatomical position including neuron count of each nerve and their ability to be spliced together was investigated. Cadaveric dissections were performed to document the distribution of the deep branch of the ulnar nerve as it enters the lumbrical musculature and the recurrent branch of the median nerve as it courses through the carpal tunnel. After the dissection was completed, the ulnar branch to the lumbrical muscle was drawn towards the recurrent branch of the median nerve. Using a digital camera, and electronic calipers the nerves were photographed for visual confirmation of the ability to transfer and measured to compare the size of the nerves. Sections of each branch were then studied for a comparison of neuron counts. Data suggests the nerve transfer to be a viable option, and could assist neurosurgeons in performing surgeries to help patients regain functionality in their thenar muscles. A continuation of data collection will allow greater insight into the number of neurons that can potentially re‐innervate the thenar muscles as well as assist in desired surgical outcomes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Non‐Invasive Brain Stimulation for Negative Symptoms in Schizophrenia: An Updated Systematic Review and Meta‐Analysis
Authors: Barros Mirna Duarte , Osoegawa Caio duarte , Cordeiro Quirino duarte , Gomes July Silveira , Brietzke Elisa duarte , Trevizol Alisson Paulino
Published: June 2021

Background

Schizophrenia is a mental disorder with significant social and economic burden. Although current pharmacological treatments are effective for controlling the positive symptoms, medications have small‐to‐no effect for the treatment of negative symptoms.

Objective

To assess the efficacy of non‐invasive brain stimulation (NIBS) for negative symptoms in schizophrenia in randomized clinical trials (RCTs).

Methods

A systematic review in the Medline and Cochrane Library databases was performed up to May 31, 2017. The primary outcome was the Hedges' g for continuous scores in a random‐effects model. Heterogeneity was evaluated with the I2 and the χ2 test. Publication bias was assessed using the Begg's funnel plot. Meta‐regression was performed using the random‐effects model modified by Knapp and Hartung.

Results

We included 31 RCTs (n=1272); most with small‐to‐modest sample sizes. Active stimulation was significantly superior over sham for negative symptoms (Hedges' g = 0.23; 95% CI 0.11 – 0.34). The funnel plot and the Eggers test showed that heterogeneity and the risk of publication bias were low (I2=2.3%, p=0.429 for the χ2 test; p=0.179 for the Egger's test). Meta‐regression showed no influence of any variable on the results found. Both transcranial magnetic stimulation and transcranial direct current stimulation were superior to sham. In a subgroup analysis, no trial was alone responsible for the positive results observed.

Conclusion

NIBS active was superior to sham stimulation for the amelioration of negative symptoms in schizophrenia. We found no considerable heterogeneity or publication bias in our analysis, corroborating to the strength of our findings. Further RCTs with larger sample sizes are needed to clarify the precise impact of NIBS in negative symptoms in schizophrenia.
Towards A Structural Characterization of The Leopard Gecko Cerebellum: Purkinje Cells And Cells of The External Granular Layer
Authors: Bradley Stefanie , Bailey Craig D.C. , Vickaryous Matthew
Published: June 2021

The cerebellum is a major component of the hindbrain, and plays important roles in motor coordination, posture, and cognitive functions. Although the gross anatomy and histological organization of the cerebellum has been documented across many species, less is known about the contributing cell types, especially among non‐mammals. Here we focus on Purkinje cells (PC) and cells of the External Granular Layer (EGL) in a representative lizard, the leopard gecko (Eublepharis macularius). PC serve as the major motor output of the cerebellum. Similar to other vertebrates, gecko PC are large, calbindin‐immunopositive neurons located at the junction between the molecular layer and the granular layer. Golgi‐cox impregnation reveals a dendritic arborization broadly comparable with that of alligators (and distinct from that of mammals). There is typically a single, lengthy primary dendrite that passes towards the molecular layer. This primary dendrite divides into multiple short dendritic branches that conclude in thin spiny branchlets with abundant, closely packed spines. Adult geckos also retain an EGL, a progenitor cell population that characteristically disappears prior to adulthood in mammals. Cells of the gecko EGL are immunopositive for the transcription factor SOX2, a hallmark protein of neural stem/progenitor cells, and the intermediate filament (and radial glia marker) glial fibrillary acid protein, but are immunonegative for NeuN, a marker of mature neurons. Using a bromodeoxyuridine pulse‐chase strategy, we determined that a subset of gecko EGL cells were label retaining after a 140 day chase period. Combined, these data provide important new information about the evolutionary variability of the cerebellum, with implications for functional adaptations in a species capable of tail loss.

Support or Funding Information

Natural Sciences and Engineering Research Council Discovery Grant #400358 (MKV), #436190 (CDCB) and The Canada Foundation for Innovation #25371 (MKV) and #30381 (CDCB)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Catalytic functions of RECQ1 helicase are important in defying genomic stress and maintaining cell proliferation
Authors: Parvathaneni Swetha , Lu Xing , Sharma Sudha
Published: June 2021

RecQ helicases are an important class of DNA repair proteins with critical roles in genome maintenance. RECQ1, a structure‐specific DNA helicase and the most abundant RecQ homolog in humans, is frequently upregulated in rapidly proliferating cells and has dual roles in genome maintenance and transcription regulation. RECQ1's function as well as expression levels modulates patient survival and response to anti‐cancer therapy. More recently germline mutations in RECQ1 have been linked to breast cancer susceptibility. However, the mechanism by which altered RECQ1 expression or mutations in functional domains of RECQ1 modulate response to chemotherapy and increase cancer risk is unclear. Our study aimed at generating a RECQ1 knockout cell line using CRISPR‐Cas9 technology to investigate phenotypes of cells that lack RECQ1 expression completely or express breast cancer risk associated RECQ1 mutants in an isogenic background. Our results demonstrate that we have successfully generated a human RECQ1 knockout MDA‐MB‐231 breast cancer cell line using the CRIPSR‐Cas9 technique which recapitulates previously established phenotypes of RECQ1 knockdown resulting in reduced cell proliferation, growth and migration as well as increased sensitivity to camptothecin. We further demonstrate that the missense mutations in the evolutionarily conserved residues within the catalytic core of RECQ1 that predispose individuals to breast cancer, also lead to impaired cell proliferation, increased sensitivity to replication stress and accumulation of spontaneous DNA damage indicating that defective helicase functions of RECQ1 may eventually contribute to genomic instability and drive cancer progression.

Support or Funding Information

This work was funded by the NIGMS/NIH grant SC1GM093999 to Sudha Sharma

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generating and characterizing resistance to ionizing radiation in experimentally evolved populations of Escherichia coli.
Authors: Trimarco Joseph D. , Bruckbauer Steven T. , Cox Michael M.
Published: June 2021

Efficient and accurate DNA repair processes are essential to maintaining genomic integrity. While most DNA repair mechanisms are remarkably effective, extreme sources of DNA damage can overwhelm these processes. One such source is ionizing radiation (IR). Our lab has developed a novel approach to determining the molecular basis of bacterial IR resistance. Using directed evolution, we have generated four populations of IR‐resistant E. coli with high‐energy electron beam IR delivered via a clinical linear accelerator (Linac). The Linac administers controllable and consistent doses of ionizing radiation, allowing for accurate and repeatable conditions of selective pressure. Now at round 90 of selection, we have generated the most IR‐resistant organisms ever produced within a laboratory. Using whole‐population genomic sequencing, we have identified mutations in proteins conducting and regulating DNA repair, transcription, anaerobic metabolism, cell wall biogenesis and amelioration of reactive oxygen species. We have shown mutations in DNA double strand break repair machinery (RecA, RecBCD, RecN) and RNA polymerase (RpoB, RpoC) to confer IR resistance. Using these mutations and others, we describe genetic modifications necessary for nascent mechanisms of IR resistance.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Phototherapy on Gene Expression in the Treatment of Psoriasis
Authors: Abdo Salam N. , Tulp Orien L. , Einstein George P.
Published: June 2021

Proliferative skin disorders such as psoriasis have been an enigma in medicine for many years with cutaneous and systemic manifestations that vary in severity. Moreover, therapeutic approaches range from over‐the‐counter topical ointment to systemic biologics which may place patients at risk of death due to immune suppression. While phototherapy has been utilized in the treatment of psoriasis, previous research studies have focused on utilizing ultraviolet radiation and other wavelengths without investigating a specific emission/absorption pattern of psoriasis patient DNA. As psoriasis affects approximately 2% of population, the results of this study will affect the treatment and improve the quality of life of numerous psoriasis patients. The purpose of this study is to analyze the effects of phototherapy on the DNA of psoriasis patients. There have been numerous studies that have conducted the genetic analysis of the psoriatic patient DNA to identify gene consequence that may be associated with a disease while others attempted ultraviolet spectrophotometric analysis of the DNA. We are seeking access to the National Psoriasis Foundation samples for analysis of pre‐and post‐ultraviolet (UVB/UVA) exposure. We will record any change in absorbance due to the exposure to UVB/UVA in a qualitative and quantitative assay using DNA Quantitation Biotechnology Spectrophotometer.

Support or Funding Information

Supported by Institutional Resources of USAT‐Montserrat and the Einstein Medical Institute, North Palm Beach, FL

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 regulation of curcumin on cacinogenesis and modulation through Wnt/β‐catenin signaling in ovarian cancer cell line
Authors: Liu Chinyu , Yen Hsingyu , Tsao Chihwei , Kuo ChihChi , Lin Yawen
Published: June 2021

Ovarian cancer (OC) is the most lethal gynecologic malignancy and the top ten cancer of females in Taiwan and all over the world. Oncogenic activation of the Wnt signaling pathway is common in cancers, but mutation of beta‐catenin in ovarian cancer is rare. In addition to genetic events, epigenetic modification of secreted frizzled‐related protein (SFRP) family has been shown to be important in regulating Wnt signaling. The SFRPs act as antagonists of the Wnt ligands and inhibit the Wnt/β‐catenin signaling pathway. In the past research indicated the curcumin rich in curcuma longa has the capacity for anti‐ inflammatory, anti‐ oxidant and anti‐cancer effects as well as involve in the epigenetic regulation. It is helpful for inhibiting the tumor suppressor gene methylated and re‐expression. Therefore, the aim of this study is going to investigate the effects of curcumin on sFRP5 DNA methylation modification in ovarian cancer cell (SKOV3). Cell was treated with DMSO, 10 μM 5‐aza‐2′‐deoxycytidine (DAC), 5 μM DAC, 20 μM curcumin and respectively combined with 5 μM DAC in 96 hours, extracting the RNA and proteins for further analyze. The results of present study show that curcumin combined with 5 μM DAC may inhibit cancer cell colony formation and migration, the total DNMT activity, DNMT3a protein expression, also regulate tumor suppressor gene SFRP5 expression through Wnt/β‐catenin signaling pathway. As a result, it can attenuate or inhibit the ovarian cancer 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.
A Role of Iron in the Pathogenesis of Idiopathic Pulmonary Fibrosis
Authors: Huang Chaoqun , Xu Dao , Senavirathna Lakmini Kumari , Liu Lin
Published: June 2021

Iron is a trace element indispensable for nearly all living organisms and participates in a variety of biological processes, including electron transport, oxygen transport, and DNA synthesis. On the other hand, excessive iron can result in tissue damage due to the formation of free radicals. Abnormal iron homeostasis causes a broad spectrum of human diseases. A number of studies suggest that iron is associated with pulmonary fibrosis. However, it is still unclear about molecular mechanisms of iron action in the pathogenesis of pulmonary fibrosis. In the present study, we aimed to investigate the effects of iron on fibroblast activation and global gene expression in pulmonary fibroblasts. LL29 lung fibroblasts were treated with desferrioxamine (DFX) to deplete iron in the cells. Real‐time PCR showed that iron depletion by DFX suppressed TGFβ‐induced mRNA expression of collagens (COL1A1, COL3A1, COL4A1), α‐smooth muscle actin, and fibronectin mRNA expression. To explore the molecular mechanisms of iron action on fibroblast activation, RNA sequencing was employed to study the effects of iron on gene expression profile in LL29 fibroblasts. The expression of 1,203 mRNAs and 198 lncRNAs were found to be significantly changed by DFX. Gene ontology and functional annotation analyses indicated that iron‐downregulated mRNAs were involved in TGFβ and HIF signaling pathways and iron‐upregulated mRNAs were involved in the regulation of cell cycle. Moreover, functional annotation analyses showed that iron‐dysregulated lncRNAs were involved in the regulation of cell proliferation. Iron‐controlled SMURF2, DBF4, and MCM6 genes which contain potential iron response elements were further verified by real‐time PCR. These results suggest that iron may control fibroblast activation by regulating TGFβ signaling pathway and fibroblast proliferation by regulating cell cycle genes.

Support or Funding Information

NIH R01HL116876, 1R01HL135152, and P20GM103648

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 1,4‐dimethylnaphthalene on the Expression of Pathogenesis‐Related Protein 4 in Solanum tuberosum.
Authors: Aljahani Abrar , Campbell Michael
Published: June 2021

It has been shown in recent studies that 1,4‐dimethylnaphthalene (DMN) can halt cell division in tuber meristems of Solanum tuberosum. By preventing cell division, DMN can prevent sprouting and prolong storage. RNA sequence data of potatoes treated with DMN revealed that the gene encoding for pathogenesis‐related protein 4 (PR‐4) is significantly induced in response to DMN. The level of PR‐4 gene expression increased by three‐fold in the fall and winter and by six fold in the spring, which is during late storage and the tissues are no longer dormant. This demonstrates that dormant potato tubers are recalcitrant in response to DMN induction of PR‐4. The PR‐4 protein belongs to a large family of plant host proteins triggered in response to pathological situations, such as bacteria or virus invasion. Members of PR protein family have different functions. PR‐4 is specifically involved in wound response, and it has both RNase and DNase activity. Further experiments are being conducted to determine the level of gene product for PR‐4 in response to DMN. Currently, it is unclear how the expression of PR‐4 relates to the suppression of cell division in potato meristems.

Support or Funding Information

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

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of RNA Binding Protein RBM15 in m6A RNA Methylation During Megakaryocytic Differentiation
Authors: Ayala‐Lopez Nadia , Ross Robert , Halene Stephanie , Limbach Patrick , Krause Diane S.
Published: June 2021

N6‐methyladenosine (m6A) modification of RNA regulates RNA splicing, localization, export and degradation. Loss of m6A RNA is associated with defects in stem cell differentiation and gain of m6A RNA is associated with leukemogenesis. The RNA binding protein RBM15 was recently discovered to be involved in the m6A methylation of RNA. We have shown previously that RBM15 inhibits differentiation of myeloid cell lines, and its expression is decreased in megakaryocytes compared to bone marrow hematopoietic stem cells. We hypothesize that RBM15 m6A methylates RNAs that are involved in megakaryocyte fate specification and maturation. We tested whether RBM15 overexpression affects global m6A RNA levels during megakaryocytic maturation using two human erythroleukemia (HEL) cell lines: parental control (HEL‐pcDNA) cells carrying only the Tet‐repressor gene and HEL‐RBM15 cells, which encode the RBM15 transgene tagged with hemagglutinin (HA), under the control of a tetracycline‐inducible promoter. Megakaryocytic differentiation was induced by addition of 12‐Otetradecanoylphorbol‐13‐acetate (TPA) with and without doxycycline induction of the transgene for 24 hours. Immunoprecipitation using an anti‐HA antibody to pull down the HA‐tagged RBM15 followed by a Western blot revealed that WTAP (Wilms' tumor associated protein), which is part of the N6‐methyltransferase complex, binds to HA‐tagged RBM15. Global m6A RNA methylation levels were analyzed by ELISA and confirmed by LC‐MS/MS. HEL‐pcDNA (control) m6A RNA levels did not change with any of the treatments using the ELISA or LC‐MS/MS. In contrast, overexpression of RBM15 during megakaryocytic differentiation increased m6A RNA significantly when measured by ELISA (HEL‐RBM15= 307.5±83.0% increase in m6A RNA vs. untreated (no doxycycline, no TPA); N=3]. Confirmation by LC‐MS/MS however, showed no significant changes in m6A RNA levels (HEL‐RBM15= 7.14±7.26% increase in m6A RNA compared to untreated; N=3). These data show that RBM15 associates with WTAP, one of the methyltransferase complex proteins, in a megakaryocyte cell line, and that antibody assays to detect m6A RNA should be validated by other methods such as mass spectrometry. We are currently performing rigorous identification of the factors responsible for discordance of ELISA and LC‐MS/MS measures. Additionally, we will test the hypothesis that RBM15‐induced RNA methylation is a mechanism by which RBM15 regulates megakaryocytic maturation with the ultimate goal of identifying the mechanisms of translational control at the RNA m6A methylation level that can be targeted in megakaryocytic leukemia.

Support or Funding Information

NIH 2T32HL007974‐16

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A screen for novel targets casts polyphosphorylation of lysine as a common post‐translational modification
Authors: Bentley‐DeSousa Amanda , Moteshareie Houman , Holinier Charlotte , Tseng Yi‐Chieh , Bondy‐Chorney Emma , Davey Norman , Golshani Ashkan , Downey Michael
Published: June 2021

Polyphosphates (polyP) are long chains of linked inorganic phosphates ranging from 3–1000s of residues in length. They are found in all organisms and play critical roles in a range of functions including blood clotting and bacterial virulence. Given its diverse roles, polyP is an attractive target for novel therapies. Recently, it was shown that polyP can be non‐enzymatically added to protein targets within poly‐acidic, serine, and lysine rich (PASK) motifs as a lysine post‐translational modification termed polyphosphorylation. This modification was described for two yeast proteins, Nsr1 and Top1. In yeast, polyP is initially synthesized by a polyP synthetase (VTC4) and subsequently added as a PTM non‐enzymatic. Our lab has developed a screen to assess polyphosphorylation of yeast substrates wherein we uncovered 25 novel substrates including a conserved network of proteins functioning in ribosome biogenesis. Disruption of this polyP synthesis through vtc4D deletion results in translation defects as measured by polysome profiling. Finally, we found that expression of E. coli polyP synthetase (EcPPK1) in vtc4D mutant yeast results in extreme sensitivity to rapamycin and cycloheximide, despite its ability to restore polyphosphorylation of multiple yeast targets. We propose a model wherein polyphosphorylation of unique targets regulates multiple aspects of cell growth. Moreover, regulation of polyphosphorylation in space and time is critical to preserve essential cell functions, such as ribosome function.

Support or Funding Information

The funding agencies that support this research are: University of Ottawa and 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.
The Regulation of Cellular Proliferation by VACM‐1/CUL5 is Dependent on its Post‐translational Modifications by NEDD8
Authors: Lee Si Eun , Sundquist Skylar , Burnatowska‐Hledin Maria
Published: June 2021

VACM‐1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase complex formation and subsequent protein degradation by ubiquitin‐dependent pathways. In vivo, VACM‐1/CUL5 is expressed in endothelial cells, and when expressed in vitro it attenuates cellular growth. While the specific mechanism by which VACM‐1/CUL5 suppresses cell proliferation is unknown, its antiproliferative effect is dependent on its post‐translational modification by an ubiquitin‐like protein, NEDD8. To investigate the effect of NEDD8 on the structure and function of VACM‐1/CUL5, we have mutated its cDNA sequence at the putative neddylation sites and expressed the mutated construct in Rat Adrenal Medulla Endothelial Cells (RAMEC) and COS‐1 cells in vitro. Our results demonstrate that the antiproliferative effect of wt VACM/CUL5 was attenuated in cells transfected with the mutated VACM‐1/CUL5 cDNA. Furthermore, the mutated VACM‐1/CUL5 affected the anti‐angiogenic phenotype of wt VACM‐1 when expressed in RAMEC cells and grown on Matrigel® support. Finally, the immunoblotting results show that the mutant is modified at multiple sites by NEDD8 in vitro. These results suggest that antiproliferative effect of VACM‐1/CUL5 is regulated by its neddylation status.

Support or Funding Information

This work is supported by the Paul A. Schaap Fellowship and Hope College Department of Biology.

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 and Characterization of a Model Post‐Translationsally Modified Protein Library
Authors: Major Emily K. , Magnani Roberta , Houtz Robert L. , Bradley Luke H.
Published: June 2021

From plants to humans, calmodulin lysine methyltransferase (CaM KMT) is a highly conserved protein responsible for the trimethylation of Lys‐115 in calmodulin (CaM). Recent findings suggest that CaM trimethylation is important in a wide range of metabolic, cellular, and developmental functions. Furthermore, we used CaM KMT as a protein engineering tool to post‐translationally modify a high‐quality protein library of the CaM central linker, using a coexpression system in E. coli. We showed that trimethylation differentially altered the conformational changes of CaM associated with the binding of calcium, CaM's thermal stability, and binding specificity towards CaM‐peptide target sequences. However, the substrate specificity of CaM KMT is unknown. A total of 40 conservative and non‐conservative amino acid substitutions were designed and introduced 3 residue positions (positions P(−3) to P(+3)) flanking the target Lys‐115 trimethylation site. In vitro trimethylation assays, using bacterially expressed and purified CaM KMT with purified mutants, identified positions and putative target sequences for CaM KMT. These results suggest alternate in vivo substrates and cellular roles for CaM KMT. Furthermore, these results provide further design information towards producing an unbiased and targeted post‐translationally modified library of novel sequences, thereby providing an additional tool for designing and generating proteins with stringent protein‐target specificities.

Support or Funding Information

NSF KY ESPCoR 0IA‐1355438 Bucks for Brains‐ University of Kentucky Office of Undergraduate Research

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