bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and Bioinformatic Analysis of Streptomyces Bacteriophage Cluster BF
Authors: McClendon‐Mosss Tianeaka , Hughes Lee
Published: June 2021

Streptomyces bacteria are at the forefront of the world's battle to control infectious bacteria. More than 70% of the antibiotics currently in use were derived from the secondary metabolites of various Streptomyces bacterial species. In the search to find new ways of combating antibiotic‐resistant infectious bacteria, bacteriophage‐derived genetic tools are quickly emerging with promising possibilities. Research to identify and characterize new Streptomyces bacteriophages is currently underway with the hopes of creating new genetic engineering tools that can be used to introduce variety into the genetic material of the host, thereby causing the production of new secondary metabolites. In this study, 8 Streptomyces bacteriophages in Cluster BF were analyzed genetically using bioinformatic tools such as DNA Master, Glimmer, GeneMark, and Starterator software. The bacteriophages in this study were isolated from soil samples gathered by student researchers at the University of North Texas using Streptomyces griseus 10137 as the host. Isolation techniques used were developed by the Science Education Alliance‐Phage Hunters Advancing Genomics and Evolutionary Science (SEA‐PHAGES) program. The average genome length of the bacteriophages in this cluster was approximately 46000 base pairs and each contains between 62 and 64 putative genes. The average G+C content for the phages in this cluster is 59.7%. Low G+C content, while common among the Cluster BF phages, is highly unusual given the fact that most phages tend to reflect the genomes of their hosts, and Streptomyces bacteria are high G+C organisms at approximately 70% or higher G+C content. Cluster BF members also contain an abnormally high number of tRNA genes, ranging in number from 20 to 22. Host range experiments on eight other Streptomyces species were also conducted. The results of this work show that the Cluster BF phages have interesting genomes that merit additional study. Future experiments will be designed to explore mechanisms that allow these low G+C phages to infect a high G+C host like Streptomyces, with particular emphasis on the role that the tRNA genes play.

Support or Funding Information

This research was funded by the University of North Texas Department of Microbiology. A portion of this project was funded by the Science Education Alliance‐Phage Hunters Advancing Genomics and Evolutionary Science (Sea‐Phages) Program and a grant from the Howard Hughes Medical Institute HHMI‐Transition Students Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing a Loss of Function Mutation in the NOD2 gene in Puerto Rico
Authors: Acevedo‐Sanchez Yamilex , Martínez‐Cruzado Juan Carlos
Published: June 2021

Loss‐of‐function (LoF) mutations are genetic variants predicted to truncate the structure of protein coding genes, nullifying their ability to fulfill their biological role. The distribution of these variants was outlined in Phase 1 of the 1000 Genomes Project, which included 55 individuals from the Puerto Rican population. Here, a frameshift mutation in the NOD2 gene, which has been previously associated with Crohn's Disease, was identified, having an allelic frequency of 0.029 in the Puerto Rico sample set of the 1000 Genomes Project, Phase 3. NOD2 encodes a protein that has a role in immune response via activation of the nuclear factor‐kappa‐B. The purpose of our study was to identify the distribution and frequency of this mutation in the Puerto Rican population. Genomic DNA obtained from saliva samples was genotyped using the polymerase chain reaction‐restriction fragment‐length polymorphism (PCR‐RFLP) technique. The presence of the mutant allele introduces an ApaI recognition site when a mismatched primer is used for PCR‐amplification, resulting in two fragments of 92bp and 18bp instead of one fragment 110 base pairs long. Fragment length is analyzed by agarose gel electrophoresis. Preliminary results show that from 42 samples analyzed, there is one heterozygous individual for the mutant allele in the northeast region of Puerto Rico. Further analysis is needed to provide an accurate estimation of the frequency and distribution of this polymorphism.

Support or Funding Information

Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number T34GM008419

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C‐terminal domain of Mad1 interacts with Mad2 to catalyze O‐C Mad2 conversion in coordination with mitotic checkpoints
Authors: Nguyen Nhan Do Minh , Ahmad Ejaz , Liu Song‐Tao
Published: June 2021

Aneuploidy occurs when chromosomes are unequally separated. It is important that all chromosomes' kinetochores attach to spindle microtubules before the cell starts to divide; if not, the chromosomes will be distributed unevenly between the two daughter cells. In order to prevent chromosome missegregation, the mitotic checkpoint complex (MCC) prevents chromosomes that are unattached to spindles from division. Human MCC is composed of multiple proteins: Bub3, BubR1, Cdc20, and Mad2. Kinetochores that are unattached to spindles give out the signal that triggers the MCC to assemble. Once formed, the MCC is able to inhibit anaphase‐promoting complex/cyclosome (APC/C). Without the activation of APC/C, the anaphase progression is temporarily stopped. In our study, we investigated Mad2, which is a critical part of the MCC. There are two functional conformations of Mad2, closed and opened, also known as C‐Mad2 and O‐Mad2, respectively. In the MCC, Mad2 exists in closed form (C‐Mad2). Therefore, to assemble the MCC, O‐Mad2 needs to be converted to C‐Mad2. Our research focuses on the pathway where Mad1 recruits O‐Mad2 and converts it to C‐Mad2. Previous research was concentrated on the middle part of Mad1 (Mad1‐MIM), and it was posited that Mad1 could not independently recruit O‐Mad2 without forming a complex with C‐Mad2. However, our research on the C‐terminal domain (CTD) of Mad1 indicates that Mad1‐CTD binds to both O‐ and C‐Mad2. This result suggests that CTD‐Mad1 could potentially recruit O‐Mad2, and convert it to C‐Mad2 which then assembles into the MCC.

Support or Funding Information

The work was carried out during the summer of 2017 in the Department of Biological Sciences at the University of Toledo in Dr. Song‐Tao Liu's lab with support from the Department of Chemistry at Wabash College.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transient Receptor Potential Vanilloid 4 Channel Regulates TGFβ1‐Mediated Fibroblast Differentiation And Airway Remodeling Through RAC/NADPH Oxidase 4 Axis
Authors: Ghebreigziabher Samrawit , Al‐Azzam Nosayba , Snyderman Matthew , Thodeti Charles , Paruchuri Sailaja
Published: June 2021

Transient Receptor Potential Vanilloid 4 (TRPV4) is a stretch‐activated, mechano‐sensitive calcium channel. We have shown recently that TRPV4 regulates TGFβ1‐mediated fibroblast differentiation in vitro and D. farinae‐induced airway remodeling in vivo. Here, we present a unique finding that this regulation requires NADPH oxidase 4 (NOX4), a member of the NADPH oxidases. Our results reveal that TGFβ1 transcriptionally up‐regulated NOX4 gene expression and this expression was sensitive to TRPV4 inhibition. Further, TGFβ1‐induced fibrotic gene expression was inhibited by NOX4 antagonist (GKT137831), antioxidants and NOX4 siRNA. Interestingly, inhibition of Rac, a member of small family GTPases attenuated TGFβ1‐induced NOX‐4 upregulation and fibroblast differentiation, highlighting a novel TRPV4‐RAC‐NOX4 interaction. Importantly, TRPV4 KO mice which were protected from D. farinae‐induced asthma also exhibited lower levels of NOX4 gene expression, suggesting that TRPV4 promotes airway remodeling through NOX4. Our data unravels a unique role for TRPV4‐RAC‐NOX4 interaction in TGFβ1‐mediated fibroblast differentiation and lung remodeling.

Support or Funding Information

This work is supported by James Foght Assistant Professor support.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Disruption of a Short Open Reading Frame (sORF) In The mRNA 5′ Leader Sequence (5′LS) of the Type 1 Angiotensin Receptor (AT1R) Increases Angiotensin II (Ang II)‐ Induced AT1R Internalization and Signaling through the Extracellular Signal‐Regulated Kinases (ERK1/2) Pathway
Authors: Kadam Parnika , Ji Hong , Pai Amrita V. , Speth Robert , Mueller Susette , Sandberg Kathryn
Published: June 2021

Antagonists of the AT1R are widely prescribed clinically to treat hypertension and associated cardiovascular and renal diseases. We investigated the effects of a sORF in exon (E) 2 in the AT1aR mRNA 5′ leader sequence on Ang II‐induced AT1aR internalization and signaling through the ERK1/2 signal transduction pathway. The start codon of this sORF was disrupted [E1,2(‐108T),3‐AT1aR] and along with the unmutated sORF [E1,2,3‐AT1aR] were cloned into an expression plasmid containing enhanced green fluorescent protein [EGFP‐N2]. Human embryonic kidney‐293 (HEK) cells were transfected with intact or disrupted sORF (AT1aR‐EGFP‐N2) plasmids. Radioligand binding studies with 125I‐Ang II (300 pM) in transfected HEK cells demonstrated disruption of the sORF increased AT1aR binding by 1.4‐fold after 30 min of Ang II stimulation [AT1aR (cpm): E1,2,3‐AT1aR, 5865±1040 vs. E1,2(‐108T),3‐AT1aR, 8201±878; p<0.05; n=2;] and 1.3‐fold more AT1aR was internalized after 15 min of Ang II treatment [AT1aR (cpm): E1,2,3‐AT1aR, 9576±399 vs. E1,2(‐108T),3‐AT1aR, 12,440±292; p<0.05, n=2]; however, the rate of Ang II‐induced AT1aR internalization did not differ between the two constructs. Ang II (10 nM) stimulation rapidly increased ERK1/2 activation with peak activity occurring between 5–10 min in HEK cells expressing both the disrupted and intact sORF. ERK1/2 activity rapidly declined between 10–40 min in cells expressing the intact sORF. In contrast, disruption of the sORF led to sustained ERK1/2 activation up to at least 40 min. In addition, a dose response curve indicates that disruption of this sORF increases ERK1/2 signaling induced by Sar1, Ile4, Ile8‐Ang II even when normalized to AT1aR Bmax determined by radioligand binding. This AT1aR biased agonist selectively activates the β‐arrestin‐ERK1/2 signaling cascade. Thus, this study supports previous findings that a seven amino acid peptide (PEP7) encoded by this sORF is a selective inhibitor of AT1R‐mediated ERK1/2 activation. PEP7, therefore, may serve as a valuable tool in dissecting the physiological and pathophysiological role of β‐arrestin‐dependent and ‐independent AT1R signaling cascades.

Support or Funding Information

Funding agency & grant #: NIH/NHLBI 1R01‐HL121456 to KS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beta‐Adrenergic Receptor Signaling in Distal Convoluted Tubules
Authors: Cheng Lei , Wu Qi , Fenton Robert A.
Published: June 2021

The sympathetic nervous system (SNS) plays a central role in blood pressure regulation. Recent studies have shown that cells of the distal convoluted tubule (DCT) can be stimulated directly via the beta‐adrenergic receptor resulting in activation of the NaCl cotransporter NCC. Whether these effects are mediated by the beta1‐ or beta2‐adrenergic receptor is unclear, and the signaling cascades rapidly activated by beta‐adrenergic receptor stimulation in the DCT are unknown. RT‐PCR identified alpha1B‐, alpha1D‐ and beta2‐adrenergic receptors, but no beta1 receptors, in mpkDCT cells (a model of the DCT). Treatment of mpkDCT cells with the short‐acting beta2‐adrenergic receptor agonist salbutamol (1μM) for 15 min resulted in significantly increased cAMP levels, and phosphorylation of VASP at a PKA‐dependent site. Global changes in signaling networks following 15 min of salbutamol treatment of mpkDCT cells were assessed using stable isotope labeling by amino acids in cell culture (SILAC) based quantitative phosphoproteomics. A total of 43773 unique phosphopeptides containing 13599 confidently assigned phosphorylation sites were identified, 9209 of which were suitable for additional statistical analysis. Salbutamol increased phosphorylation at 81 sites; with kinases including PAKB, CLK, p70S6K and PKA predicted to be responsible. 71 sites, including several targets for CDK and GSK3 kinase groups, were decreased in phosphorylation. The predicted kinases fit well with motif analysis, where sites increased in phosphorylation were basophilic (RXXS), whereas the downregulated motif (SXXXS) is predicted to be a target for GSK3. Pathway analysis predicted that CREB and AKT are also important pathway nodes altered by salbutamol. Phosphorylation of the NCC‐modulating kinase OSR1 was increased at S339, a site previously shown to be regulated by PI3K‐mediated signaling. In summary, beta2‐adrenergic receptor signaling in mpkDCT cells involves PI3K and cAMP‐mediated pathways. Incorporation of our data into known signaling networks may provide novel clues linking the sympathetic nervous system, DCT regulation and NCC activity.

Support or Funding Information

Funding is provided by the Novo Nordisk Foundation, the Lundbeck Foundation, Marie Skłodowska‐Curie Individual Fellowship and the Danish Medical Research Council.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐fat diet augments muscle atrophy by changes of muscle atrophy‐related genes in mice
Authors: Goto Ayumi , Tsuzuki Takamasa , Yoshihara Toshinori , Furuichi Shinya , Tsukioka Kei , Kakigi Ryo , Naito Hisashi
Published: June 2021

Introduction

Recently, it has been reported that obesity accelerates muscle atrophy and protein degradation induced by 14‐days denervation in mice. Muscle atrophy primarily results from accelerated protein degradation and is associated with increased expression of two muscle‐specific ubiquitin ligases: MuRF1 and atrogin‐1. PGC1α is the master transcription regulator that stimulates mitochondrial biogenesis, and expression level of PGC1α was reduced in obesity and muscle atrophy. However, the expression level of these genes in response to muscle atrophy on obesity is still unclear. The purpose of this study was to investigate the expression level of MuRF1, atrogin‐1 and PGC1α during muscle atrophy in mice fed a high‐fat diet.

Methods

This study was approved by the Juntendo University Animal Care Committee (H28‐14). Fifty‐two male C57BL/6J mice (6 weeks old) were randomly divided into two groups: 1) normal diet (ND, n=26), 2) high‐fat diet (HFD, n=26). HFD were fed a high‐fat diet (60% calories from fat) for 16 weeks, while ND received a chow diet (10% calories from fat). Following 16 weeks of each experimental diet intake, all mice were lightly anaesthetized with the inhalant isoflurane and one hindlimb was immobilized in the plantar flexion position with casting tape. After immobilization for 1, 3, and 7 days, the soleus and plantaris muscles were removed. Statistical significance was analyzed by using two‐way (diet and time) analysis of variance (ANOVA). Statistical significant level was set at P < 0.05.

Results

Body weight and epididymal fat pad mass were significantly higher in the HFD group compared with the ND group. In the 7 days, the HFD significantly augmented loss of mass rate as compared with ND. The expression level of MuRF1 mRNA 3d in soleus and plantaris muscles was significantly higher in the HFD group compared with the ND group. The expression level of atrogin‐1 mRNA was significantly increased in response to immobilization, but no changes in ND and HFD. The expression level of PGC1α mRNA was significantly reduced in response to immobilization and was lower in HFD compared with ND in soleus and plantaris muscle.

Conclusion

HFD augments muscle atrophy by immobilization, at least in part by increasing the expression level of MuRF1 mRNA.

Support or Funding Information

This study was supported in part by Japan Society for the Promotion of Science KAKENHI Grant (16H07182), President's Grant for Interfaculty Collaboration, Juntendo University (Goto A: K1613, K1708) and MEXT‐Supported Program for the Strategic Research Foundation at Private Universities.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing an ELISA for Avian Corticosterone Binding Globulin
Authors: Johnson Elizabeth , Antunes Ivy , Malisch Jessica L. , Mertz Pamela S.
Published: June 2021

The chemical response to environmental stressors has been highly conserved throughout evolution and is largely consistent amongst vertebrate animals; this allows avian endocrinologists to use birds as a model for the human stress response. Corticosterone binding globulin (CBG) is a carrier protein for the avian equivalent to cortisol: corticosterone (CORT). Research indicates that CBG's role as a CORT carrier is far more complex than just transport. It is widely accepted that free CORT is the biologically active form, but it is unclear if being bound to CBG (CBG‐CORT complex) inactivates CORT. There is evidence that CBG bound CORT can increase cyclic adenosine monophosphate and evidence that receptors exist for the CBG‐CORT complex in humans. However, it is not known if this is true for birds as well. With so much uncertainty surrounding CBG and its interactions with CORT, it is important to quantify CBG in avian stress studies; including CBG measurements may provide a more complete picture of the avian stress response. CBG is often not quantified in avian CORT research because radioactive assaying is the most common technique for CBG measurement in birds. This project developed an avian CBG enzyme linked immunosorbent assay (ELISA), using a novel CBG antibody developed for the zebra finch (Taeniopygia guttata), and a recombinant form of zebra finch CBG. This study also examined the reactivity of the CBG antibody against zebra finch on different species of songbirds (Junco hyemalis, Zonotrichia albicollis, and Zonotrichia leuocophrys) via western blotting, as their CBG sequences vary slightly.

Support or Funding Information

2017 ASBMB Undergraduate Research Award to Elizabeth Johnson

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Single Nucleotide Polymorphisms on HPA Axis Functionality in Depression
Authors: Kelly Claire E. , Cohen Brian D.
Published: June 2021

The hypothalamic‐pituitary‐adrenal (HPA) axis plays a primary role in stress response through the regulated secretion of the glucocorticoid hormone cortisol. Diseases of cortisol dysregulation such as Cushing's syndrome (hypercortisolemia) and Addison's Disease (hypocortisolemia) are both associated with depression. Based on this we, and others, have hypothesized that mutations in the genes for the glucocorticoid receptor, the closely related mineralcorticoid receptor (MR), and regulatory proteins associated with cortisol or GR function may contribute to depression in the absence of hyper‐ or hypo‐cortisolemia.

Our study investigated the genotypic frequency in a clinical population of several single nucleotide polymorphisms (SNPs) that affect GR and MR sensitivity to cortisol binding or in genes for proteins that regulate these receptors. Buccal swab DNA samples were acquired from patients clinically diagnosed with depression and from a random population. Extracted DNA was analyzed utilizing multiple allele‐specific polymerase chain reactions to determine genotypic frequency of SNPs associated with hypersensitivity or resistance to cortisol. In addition, patients suffering from depression completed the multiple measures of depression and anxiety. Preliminary results have shown that a specific allele of the GR BclI polymorphism (rs41423247) is more frequently seen in depressed patients compared to a random population and is also associated with elevated measures of depression. Understanding the role of geneotypic variation in cortisol function could lead to more specific and targeted therapies for depression with the goal of improving patient 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.
Defining the expression domains of the Arabidopsis glutaredoxin genes AtGRXS5, AtGRXS6, and AtGRXS8
Authors: Davalos Oscar , Ehrary Ahmad , Rosas Miguel , Fernandez Francisco , Escobar Matthew A.
Published: June 2021

Glutaredoxins are oxidoreductase enzymes that can regulate the activities of target proteins through the reversible breakage of disulfide bonds. We recently identified a group of seven glutaredoxin genes that are upregulated by nitrate and control primary root length in the model plant Arabidopsis thaliana. The objective of this study was to characterize the gene expression patterns and subcellular localization of three nitrate‐regulated glutaredoxins: AtGRXS5, AtGRXS6, and AtGRXS8. Reporter lines consisting of the glutaredoxin promoter sequences fused to the β‐glucuronidase (GUS) gene were used to study glutaredoxin gene expression. Colorimetric GUS assays and histological sectioning showed that AtGRXS5, AtGRXS6, and AtGRXS8 are expressed exclusively in the phloem of A. thaliana roots and leaves. GUS activity assays demonstrated that AtGRXS6 and AtGRXS8 are strongly and specifically upregulated by nitrate. In contrast, AtGRXS5 gene expression was activated by both ammonium and nitrate. Nitrate concentrations as low as 50 μM activated glutaredoxin gene expression, with no further increase in expression at higher concentrations of nitrate. To characterize the subcellular localization of AtGRXS5, AtGRXS6, and AtGRXS8, translational fusions between the glutaredoxin gene and yellow fluorescent protein (YFP) were expressed in transgenic Arabidopsis and tobacco (Nicotiana benthamiana) plants. Preliminary results demonstrated that all three glutaredoxins are localized to cell nuclei as well as small, yet unidentified structures in the cytoplasm. Overall, these studies will further explain how plants control the growth of their root system to maximize the uptake of nitrogen from the soil, a behavioral response that is highly significant in agriculture.

Support or Funding Information

NSF IOS grant 1651584NIH RISE GM‐64783

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of Glycosomal and Cytosolic Nudix Hydrolases with Polyphosphate Hydrolyzing Activity in Trypanosoma brucei
Authors: Cordeiro Ciro D. , Negreiros Raquel , Ahmed Kyle , Docampo Roberto
Published: June 2021

Trypanosoma brucei is a protozoan parasite that causes the neglected tropical disease African trypanosomiasis, also known as sleeping sickness. T. brucei infection in humans leads to death if untreated and in cattle causes a disease called Nagana, which has considerable socioeconomic impact in developing nations of sub‐Saharan Africa. T. brucei is well adapted to fight the host immune response as well as various stress conditions it endures in its various hosts. The parasite relies only on glycolysis for ATP production when inside the mammalian host. It activates oxidative phosphorylation once in the fly vector. We are interested in understanding the metabolism and functions of the inorganic phosphate polymer polyphosphate (polyP) in trypanosomes. Previous work from our laboratory reported the importance of polyP for parasite homeostasis and survival. In this work, we identified and characterized the activity of two Nudix hydrolases, NH2 and NH4 that can degrade polyP. We found that NH2 is an exopolyphosphatase with higher activity on short chain polyP, while NH4 is an endopolyphosphatase that has similar activity on polyP of various chain sizes. Both enzymes have higher activity at around pH 8.0. NH2 has a glycosomal targeting signal and it was found previously in the proteome of glycosomes, peroxisome‐related organelles where glycolysis occurs. We confirmed NH2 localization using specific antibodies while endogenously tagged NH4 was localized to the cytosol. We also found that only NH2 can dephosphorylate ATP and ADP. However, NH2 has a higher affinity for polyP, which suggests that polyP could be relevant for glycosomal function. Additionally, NH2 could also participate in regulation of ATP/ADP levels. Furthermore, using the polyP‐binding domain (PPDB) of yeast exopolyphosphatase we localized long chain polyP to the glycosomes of trypanosomes. Our results suggest that Nudix hydrolases can participate in polyP homeostasis and therefore may help control polyP levels in glycosomes and cytosol.

Support or Funding Information

NIH (5R01AI077538‐09)

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 CRBN in molecular and cellular mechanism of AMPK inhibition.
Authors: Lee Kwang Min , Yang Seung‐Joo , Park Chul‐Seung
Published: June 2021

Cereblon (CRBN), initially identified as a target protein of mild mental retardation in humans, is a substrate receptor of cullin‐ring E3 ubiquitin ligase (CRL) and a primary target of thalidomide‐induced teratogenicity. An earlier study showed that CRBN negatively regulate the functional activity of AMP‐activated protein kinase (AMPK) by binding to the alpha subunit of the AMPK complex, and the ectopic expression of CRBN reduced the content of gamma subunit in AMPK complex. However, the molecular mechanism for the CRBN‐dependent reduction of gamma subunit from AMPK complex was not demonstrated. Thus, we investigated the molecular mechanism of CRBN‐dependent inhibition of AMPK. We noticed that the amount of gamma subunit in AMPK complex was increased in Crbn‐knockout (KO) MEFs and the gamma subunit was degraded more rapidly in wild‐type MEFs than in Crbn‐KO MEFs. After treating MG132, a proteasome inhibitor, the gamma subunit is accumulated more rapidly in wild‐type MEFs compared to Crbn‐KO MEFs. Moreover, exogenous expression of CRBN promoted the degradation of gamma subunit in Crbn‐knockout MEFs only in the presence of alpha subunit. These results strongly suggest that CRBN negatively regulates the AMPK activity by recruiting AMPK complex to CRL and degrading its gamma subunit.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxidative Stress and Obesity: Detection of Free Radicals in Leptin Resistant and Catalase Transgenic Mice
Authors: McCormick Garrett Lee
Published: June 2021

There is a homeostatic balance between the production of natural antioxidants and free radicals in the body. Catalase is an enzyme which behaves as an antioxidant by decomposing peroxide into oxygen and water. Oxidative stress occurs when free radicals are in excess in relation to antioxidants. Deficiencies in antioxidant mechanisms have been linked to obesity, type II diabetes, and cardiovascular disease. More than 33% of adults and 16% of children are obese, and to combat this crisis a better understanding of these mechanisms is needed. We hypothesized that an increase in catalase expression and activity should lead to a decrease in obesogenic pathways. Free radical concentrations were measured using electron paramagnetic resonance spectrometry in four different genotypes of mice: wild‐type, cat‐tg (catalase transgenic), ob/ob, and a new model bob‐cat (leptin resistant and catalase transgenic). Liver and adipose tissue were bathed in an EPR spin probe CMH, and incubated for 30 minutes. Tissues were then measured using the Bruker X‐Band 9.8 GHz EPR Spectrometer at Marshall University. It was observed that the wild‐type sample contained the highest concentration of free radicals, followed by cat‐tg. Bob‐cat contained the lowest concentration, slightly less than ob/ob. It was expected that cat‐tg would have a lower concentration than wild‐type. However, obtaining even lower measurements from ob/ob and bob‐cat shows that more work must be done to fully elucidate these processes.

Support or Funding Information

Summer Undergraduate Research Fellowship 2017 (SURF)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changes in membrane lipids in soybean leaves in response to soybean aphid infestation
Authors: MacIntosh Gustavo C. , Nguyen Khoi , Hohenstein Jessica
Published: June 2021

Aphids are specialized insects that feed on phloem sap. They alter plant metabolism, affect plant growth and development, and are also vectors for plant viruses. Their feeding habits result in little mechanical damage and avoid triggering many plant defenses commonly elicited by other herbivores. It has been proposed that aphids can suppress effective defenses through the induction of decoy responses. Several lines of evidence suggest that lipid‐derived signals and phytoalexins have an effective role as defenses against these insects. Previously, we showed that soybean aphids, Aphis glycines Matsumura, affect fatty acid accumulation in soybean plants. Here, we performed lipidomics and transcriptome analyses of soybean leaves that were infested with soybean aphids for 7 days to identify further changes in lipid composition triggered by aphid feeding. We found significant changes in membrane lipid species including digalactosyldiacylglycerol (DGDG), monogalactosyldiacylglycerol (MGDG), phosphatidylglycerol (PG), lysophosphatidylethanolamine (lysoPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidic acid (PA). Transcriptome data identified a large number of differentially expressed genes associated with lipid metabolism, including changes in different fatty acid desaturases, and a strong induction of phospholipase A, phospholipase C, and phospholipase D. We also observed a strong induction of the oxylipins biosynthesis pathway, and negative regulation of sphingolipids biosynthesis. In addition, a number of genes corresponding to PI‐binding proteins are upregulated in leaves of aphid‐infested plants. Our results suggest that membrane phospholipids play an important role in the soybean response to aphid feeding, and that fatty acid‐ and PI‐derived signals likely mediate this interaction.

Support or Funding Information

This work was supported by a grant from the Iowa Soybean Association

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Assessment on Biochemistry Threshold and Core Concepts in Students with Varying Chemistry Backgrounds
Authors: Keenan Kelly
Published: June 2021

Stockton University is one of a few number of universities that do not follow the traditional introductory Chemistry sequence. Instead of the usual year of General Chemistry followed by one year of Organic Chemistry, Stockton follows a 1:2:1 one model of one semester of General, two semesters of Organic Chemistry followed by one of General. The prerequisite for students to take Biochemistry at Stockton is the first two semesters of Chemistry or one semester of General and one of Organic. About half of the students take Biochemistry with these prerequisites while the other half have various combinations of Chemistry courses. Previous assessments have shown there is a stronger correlation between the students grade in Biochemistry compared to previous Chemistry courses rather than the number of Chemistry courses. An assessment was developed to test the level of knowledge on three threshold concepts for incoming Biochemistry students: biochemical pathways, physical basis of interactions and free energy. A fourth component was added based on functional groups and organic chemical reactions. Students were given the assessment on the first day of class as well as at the end of the term. To insure that the students took the assessment seriously, extra credit (2% of final grade) was offered for a high enough score on each test. For the analysis of results, the exam questions were divided into categories based on the threshold concept and average scores were calculated for students based on the chemistry background. Grades less than 50% were described as lacking an understanding of the topic, grades between 50 and 70% were described as having a basic understanding of the topic and grades greater than 70% were having a deep understanding of the topic. While there were differences in other threshold concepts between the groups, all students were lacking in the free energy question at the start and end of the semester and showed the smallest improvements.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stress & Burnout in Graduate Students: The Role of Work‐Life Balance and Mentoring Relationships
Authors: Evans Teresa M. , Bira Lindsay , Gastelum Jazmin Beltran , Weiss L. Todd , Vanderford Nathan L.
Published: June 2021

Many factors contribute to the success of graduate students such as mental health, work‐life balance and mentorship. Stress, a key component of one's mental health, and its management are correlated with training success among graduate students. The 2015 University of Arizona Graduate Student Mental Health National Association of Graduate Professional Students Report found that a majority of doctoral students experienced “more than average” stress or “tremendous” stress and endorsed school and education‐related issues as the most significant contributors. Additionally, multiple studies have shown that there is a correlation between occupational stress and the development of burnout. Burnout involves emotional exhaustion, depersonalization, and diminished sense of personal accomplishment.

Despite knowing that graduate students have high rates of stress, key questions about the impact of situational factors such as work‐life balance and mentorship on stress and burnout has not been defined in this population. As such, the current study explores the interaction between stress, burnout, work‐life balance and mentoring relationships in a diverse population of graduate students.

We deployed a comprehensive survey, via social media and direct email, that included validated scales for stress and burnout as well as questions related to work‐life balance and mentorship. We surveyed a total of 2,231 respondents (223 Master's students and 2,008 PhD students) who represented the following fields of study: science (39%), humanities and social sciences (55%), engineering (2%), and other (4%).

Master's and PhD students had significantly high rates of moderate to severe stress, 81% and 82% respectively. Seventy‐three percent 73% of Master's students and 70% of PhD students indicated moderate to severe burnout. Area of study, quality of mentoring relationships and work‐life balance were significantly correlated with stress and burnout.

The results of this study support the need to bolster ongoing initiatives that are focused on improving the work‐life balance and mentor/mentee relationships of graduate students. We also suggest integrating measures of mental health into the evaluation of ongoing initiatives in order to fully measure the impact of a particular program. Additionally, we suggest that there is a need for developing new interventions that could perhaps have a greater impact on graduate student well being.

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 Frankenstein to Teach Ethics and Code of Conduct in an Undergraduate Science Course
Authors: Tansey John T.
Published: June 2021

Numerous agencies (including ASBMB, ACS, IUBMB, NSF, and NIH) have called for formal ethics education in the undergraduate curriculum, yet there is widespread confusion over what this means, and therefore how to accomplish the task. Furthermore, while bioethics, animal rights, or medical ethics are rich fodder for discussion, the discussions are often polarizing, difficult to manage, and fail to achieve the desired outcomes (broadening the students understanding of ethics and ability to conduct science in line with a code of conduct). In this exercise, students read the novel Frankenstein by Mary Shelley and used the ethical situations in the novel as a launching off point for discussion of modern codes of conduct and ethical dilemmas. These include broader themes such as the responsibility of a creator to their creation, but also topics such as working safely in a laboratory, use of human subjects, and following an institutional code of conduct. Student discussions occurred in class in small groups (less than three students), class wide discussions (approximately 24 students) and in online forums through a closed Facebook group. Feedback from students was largely positive. Preliminary results indicate that this method is a viable way to broach sometimes difficult topics in scientific ethics from a safe distance and introduce professional codes of conduct and ethics to undergraduates.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Math‐Up Skills Test (MUST): As a Predictor of Success in General Chemistry
Authors: Chen Ashley , Mamiya Blain , Mason Diana
Published: June 2021

General Chemistry is known as a gateway course to many STEM careers. Some of these careers include nursing, technology, and engineering. However, General Chemistry is also known as a high‐risk course where thirty percent of students taking it will be unsuccessful. Unsuccessful is defined as a grade of D, F, or W (withdrawal). Recent educational trends have shown that number‐sense decline is causing students to become reliant on calculators. Students are unable to do basic math without the use of an electronic calculator. As such, thirty to sixty percent of college students require remedial coursework in both math and science.

The MUST test, or the Math‐Up Skills Test, has been developed to test math skills in topics that are taught in algebra courses and below. Some of the math skills include multiplying numbers in a scientific notation form, and conversions from a fraction to a decimal. The MUST Test has been distributed to participating General Chemistry I and II faculty members throughout different universities in Texas. For data collection, students must fill out an IRB form stating that they will give their consent for using their scores and demographics for research purposes. On different days in the semester, the MUST test is done twice: Once without the calculator, and once with a calculator. The presence of a calculator does not make a statistical difference with the scores of the student taking the MUST. Using multiple statistical tests, the MUST test scores are correlated with student success in General Chemistry I and II. The MUST correlations were divided into following different categories: Gender, classification, and institution.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CRISPR/Cas9 in Yeast as a Tool for Teaching and Reinforcing Molecular Biology Concepts to Undergraduates
Authors: Ulbricht Randi J.
Published: June 2021

Undergraduate students in an upper‐level molecular biology laboratory class used their training in biochemistry, cell biology and molecular biology to design and perform CRISPR/Cas9 genome editing on yeast. After an introduction to DNA repair and CRISPR/Cas9 in the lecture, the students took on the task to design an experiment using Cas9 to generate auxotrophic yeast mutants. They used readily available bioinformatics databases to retrieve yeast genome sequence and design CRISPR‐associated sgRNA to target the gene of interest. They cloned genes for the sgRNAs into a commercially available CRISPR/Cas9 vector, and then transformed it with a template for homology‐directed repair into yeast. Next, the students phenotyped and genotyped the potential auxotrophic mutants generated by CRISPR/Cas9. As the penultimate laboratory course in the program, this lab introduced new skills, provided opportunity to apply concepts discussed in lecture, and reinforced skills and knowledge from previous courses. A survey, exam and formal lab report assessed positive outcomes in learning and affect. The reagents in this laboratory course design are readily available and financially feasible for undergraduate laboratory courses, and the protocols/emphasis are adaptable to diverse program goals. Overall, incorporating the tractable, yet modern gene editing technology into undergraduate laboratory courses is both realistic and favorable to learning.

Support or Funding Information

This work is supported by Missouri State University, Department of Biomedical Sciences

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FRESH – Freshman Research Engagement in Science: Early Results
Authors: Watters Michael K. , Bouyer Patrice , Clark Robert
Published: June 2021

We describe here some early results of the FRESH (Freshman Research Engagement in the Sciences) program. A program with the goal to expose freshman to an ongoing research project during the academic year to promote student growth and improve retention in the STEM disciplines. Freshmen worked with a faculty mentor and were also chaperoned by a more senior student researcher in order that they learn lab techniques and the capacity to work independently. Participants were fully engaged in a research project (performing experiments, analyzing and discussing results), not a classic classroom projects, but discovery based projects. By bringing students into the research lab at this early stage, our aim was to improve retention by allowing science students to actually act as scientists, providing an enhanced experience over the usual freshman survey course content. Of the 13 students in two cohorts who joined the program as freshmen, 12 are still in their major and have co‐authored over 20 different papers and conference presentations to date. Based on these initial successes, we have modified our approach, tracking qualified applicants who we were unable to fund to serve as our control group in order to study the impact of the FRESH approach on student success.

Support or Funding Information

This work was supported by grants from the Indiana Space Grant Consortium (INSGC).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion Of Muscle Acsl1 Caused Myopathy And Fiber Switch
Authors: Zhao Liyang , Bacudio Lawrence , Suchanek Amanda L. , Young Pamela A. , Pascual Florencia , Coleman Rosalind A.
Published: June 2021

Skeletal muscle exhibits plasticity and complexity in the choice of fuel substrate. During starvation, muscle initiates protein degradation to provide amino acids for liver gluconeogenesis, during acute exercise, muscle uses glucose as the primary fuel, while during rest or prolonged exercise, muscle switches to use primarily fatty acids. Defective fatty acid metabolism is one of the leading causes of metabolic myopathy. Long chain acyl‐CoA synthetase isoform‐1 (ACSL1) converts long chain fatty acids to long chain acyl‐CoAs, before acyl‐CoAs enter mitochondria for β‐oxidation or are esterified to form complex lipids. Although 5 Acsl isoforms exist, deletion of Acsl1 reduces 90% of ACS activity and fatty acid oxidation (FAO) in gastrocnemius, indicating that ACSL1 is the predominant ACSL isoform in the muscle. The objective of this study was to examine how reduced FAO through deletion of Acsl1 affects muscle physiology. We hypothesized that ACSL1‐directed fatty acid metabolism is essential for energy balance in muscle, and that lack of ACSL1 causes myopathy. To test this hypothesis, we used a mouse model in which ACSL1 is specifically deleted in skeletal muscle (Acsl1M−/−). Compared to control mice (Acsl1flox/flox), Acsl1M−/− mice demonstrated 3.4‐ and 1.5‐fold increases in plasma creatine kinase and aspartate aminotransferase activities, indicating that deletion of Acsl1 led to muscle damage. Gastrocnemius from Acsl1M−/− mice also showed the presence of central nuclei, a marker of muscle regeneration. In addition, we detected expression of caspase 3 protein only in Acsl1M−/− muscle, suggesting that the apoptosis pathway was involved. Different muscle fiber types prefer to metabolize different substrates. To investigate the consequence of Acsl1 deletion on muscle fiber type, succinate dehydrogenase staining was performed and it showed a significantly increased percentage of oxidative fibers in Acsl1M−/− gastrocnemius compared to controls. In addition, gene expression of myosin heavy chain I, an oxidative muscle fiber marker, was 6 fold higher in Acsl1M−/− glycolytic fibers than in controls, suggesting a switch from glycolytic to oxidative fibers. We further hypothesized that the fiber switch in Acsl1M−/− gastrocnemius muscle was caused by increased damage to glycolytic fibers compared to oxidative fibers. In summary, deletion of Acsl1 caused muscle damage and regeneration and promoted a glycolytic to oxidative fiber switch. Ongoing studies are examining protein turnover to determine its contribution to muscle damage.

Support or Funding Information

Supported by DK56598.

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