bluuebunny/crossref_metadata_2025 huggingface dataset:
Reductive Stress Promotes Heart Failure in Mouse and Human
Authors: Soorappan Rajasekaran Namakkal
Published: June 2021

Cardiomyopathy and heart failure (HF) is a growing cause of human morbidity and mortality across the globe. Antioxidant‐based treatments to detoxify ROS have been largely unsuccessful to protect humans from various cardiovascular diseases. Although transcriptional activation of antioxidants appear to be beneficial, their chronic effects remain elusive. Here, we tested a hypothesis that the sustained activation of Nrf2/antioxidant signaling will promote reductive stress (RS) and lead to hypertrophic cardiomyopathy. Using novel transgenic mouse models expressing Nrf2 in the heart (α‐MHC‐Nrf2‐TG) and their non‐transgenic (NTg) littermates, we studied the chronic effects of a hyper‐reductive condition on cardiac structure and function (echocardiography – Vevo2100 Imager). Furthermore, myocardial redox potential for glutathione (GSH/GSSG), transcript (qPCR), and protein (immunoblotting) levels for Nrf2‐related antioxidants in the myocardium of NTg and TG mice (n=6–12/gp.) were performed at different ages. Results from this novel study demonstrated ~40% mortality in TG mice compared to NTg by 60 weeks of age (Kaplan‐Meier survival plots). An abnormally higher GSH redox ratio was maintained along with a significantly diminished ROS in TG vs. NTg mice (p<0.05) indicated a hyper‐reductive state. This was strongly associated with a significant increase in Nrf2 function (promoter activity) with augmented levels of antioxidant genes and proteins (p<0.05) in TG vs. NTg mice. Subsequent analyses revealed that progressive cardiac remodeling and impaired cardiac output in TG relative to NTg mice. Thus, diminution of the obligatory oxidative signaling with chronic activation of Nrf2‐antioxidants could shift the redox equilibrium to the “reductive” side and cause cardiac disease. Recent clinical observations revealed a sub‐set of heart failure (HF) patients exhibiting a hyper‐reductive (i.e. RS) condition, suggesting a role for RS in the development of HF. Ongoing studies are focused on understanding the molecular mechanisms for RS‐pathogenesis in the myocardium.

Support or Funding Information

This work is supported by an R01 grant from NIH/NHLBI (HL118067).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isocitrate dehydrogenase‐1 deficiency exacerbates growth defects and metabolic alteration in Caenorhabditiselegans induced by glucose 6‐phosphate dehydrogenase deficiency
Authors: Chiu Daniel Tsun‐Yee , Yang Hung‐Chi , Yu Hsiang , Liu You‐Cheng , Arnold Stern
Published: June 2021

NADPH is a key reducing equivalent that maintains redox homeostasis and supports reductive biosynthesis. Lack of major NADPH producing enzymes predisposes cells to growth retardation and demise. Inactivation of the NADPH generating enzymes, glucose 6‐phosphate dehydrogenase (G6PD), the rate limiting enzyme of the pentose phosphate pathway, and isocitrate dehydrogenase (IDH) has been hypothesized to affect growth and development in the nematode, C. elegans. Toward this end, the G6PD/IDH1 double‐deficient C. elegans model was shown to display shrinkage of body size, growth retardation, slow locomotion and impaired molting. Global metabolomic analysis was employed to address whether or not metabolic pathways were altered by NADPH depletion by the G6PD/IDH1 double‐deficiency. Principal component analysis (PCA) points to a distinct metabolomic profile of G6PD/IDH1 doubledeficiency. Further metabolomic analysis revealed that NADPH‐dependent and glutamate‐dependent amino acid biosynthesis were significantly impaired. Consequently, the reduction in amino acids may affect protein synthesis, as indicated by the absence of NAS‐37 expression during the molting process. All in all, these findings support the notion that inactivation of G6PD and IDH1 causes growth retardation and molting defects, possibly mediated by impaired amino acid biosynthesis and metabolism in C. elegans.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining Impact of Learning Environment on Student Engagement, Bloom's Higher Order Skill Proficiency, and Exam Performance of First Year Medical Students
Authors: Hopper Mari K. , Kaiser Alexis K.
Published: June 2021

The primary aim of this study was to determine if levels of student engagement, higher order skill proficiency, and knowledge acquisition demonstrated by medical students would differ when completing the same course in three diverse learning environments. Following IRB approval, 54 first year medical students registered at the same medical school, but attending class at three different campus centers were enrolled in the study. All participants were completing a Medical Physiology course that utilized the same learning objectives but incorporated diverse methodologies (percent of class devoted to active learning strategies), course format (6 week block versus 17 week semester), and student attendance rates. Students completed a validated survey of student engagement (SSE), a proctored online problem‐based assessment of higher order skill proficiency (Collegiate Learning Assessment (CLA+) (http://cae.org), and the National Board of Medical Examiners (NBME) Physiology subject exam. Results indicated levels of engagement were lower than expected when compared to published values for graduate students. Higher order skill proficiency assessed by CLA+ was significantly higher than values reported for college seniors nationally. Importantly, there were no significant differences between campus sites for any of the variables assessed. Surprisingly, assessed higher order skill proficiency (CLA+) offered no prediction of performance on NBME as there were no significant correlations between the SSE and CLA+, or between CLA+ and NBME scores. These data indicate that although first year medical students may not perceive themselves as highly engaged, they are adept in using higher order skills and excel in meeting course objectives regardless of learning environment.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transparent Assessments: The Potential Benefits of Training Students in the How and Why of Evaluation
Authors: Hull Kerry , de Carvalho Oliveira Vanessa , Lawford Heather , Hood Suzanne , Malmquist Sarah , Jensen Murray
Published: June 2021

The ideal assessment accurately measures student knowledge and skills without causing debilitating anxiety. This study investigates the impact of two interventions on student anxiety, perceptions, and performance: increased transparency in evaluative techniques prior to the assessment, and working with peers during the assessment.

Evaluation anxiety can reflect discrepancies between self‐perceived assignment quality and the received mark, which may reflect instructors' use of tacit (i.e. knowing good work when I see it) as well as explicit knowledge (e.g. marking schemes). The impact of increased knowledge transfer from instructor to students was examined in an upper year Physiology seminar class using concept maps as summative evaluations. Students engaged in a training procedure involving rubric presentation (explicit knowledge transfer), small group evaluations of exemplar concept maps, and an instructor‐led discussion of the exemplars (tacit knowledge transfer). Students found the instructor‐led discussion the most helpful of the three steps. Students consistently ranked concept maps as better performance indicators than multiple choice exams, but results varied as to their anxiety‐provoking potential. Working in groups was only deemed useful if students already had a preliminary concept map to discuss. In comparison to students in previous classes who were not provided explicit training, students reported more positive perceptions towards concept mapping and generated higher quality maps.

The second study involved a large, inquiry‐based, upper year Physiology course using homework, group quizzes, and individual multiple choice and short answer exams. Since these evaluative techniques are relatively straightforward, we focused on differences between individual and group assessments. Group quizzes invoked less anxiety than individual exams in all students, but the difference was greater in self‐reported B students than in self‐reported A students. Yet, B students considered these assessments poorer indicators of their knowledge than A students. Comments indicated that A students worry about “free‐loaders”, and B students often feel that quiz results reflect the smartest student's knowledge, not their own. Explicit instructor explanations of the formative advantages of group quizzes, such as self‐elucidation for stronger students and peer teaching for weaker students, might increase student buy‐in. These results highlight the importance of explicit instructor explanations of how and why instructors evaluate student work.

Support or Funding Information

This work was supported in part by the Senate Research Committee of Bishop's University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Standardized Critical Thinking Scores Improve from Freshmen to Senior in an Exercise Science Program
Authors: Dicus Jeremy Raymond , Jensen Brock , Pierce Patty , Lynn Jeff
Published: June 2021

Institutional variability in undergraduate exercise science education may be greater than biology or chemistry programs due to the minimal requirement of national standardized examinations for program accreditation. Yet, programmatic discipline skills and outcomes, such as critical thinking, may be very similar across these disciplines. Additionally, evidence suggests that academic educators and employers agree that critical thinking is an important skill needed in both college and the workplace. Moreover, there is agreement that critical thinking is often more highly regarded by employers than mastery of discipline‐specific content. However, conclusive evidence that critical thinking outcomes are being sufficiently met in higher education remains unclear. The lack of conclusive evidence is unacceptable when critical thinking outcomes are commonplace at the university, college, department, and programmatic levels. Further, limited evidence exists regarding the acquisition of adequate skills related to critical thinking in exercise science education.

PURPOSE

The purpose of this investigation was to evaluate critical thinking in an exercise science program following an explicit critical thinking objective/outcome based intervention.

METHODS

Critical thinking scores were obtained using the Health Science Reasoning Test (HSRT), a valid and reliable standardized assessment, which is commonly used in undergraduate and graduate health science programs. Exercise science students (N= 121; 59% female) completed the HSRT as first semester freshmen and in their last didactic semester as seniors. Following pre‐testing, exercise science students completed a curriculum with a tiered critical thinking outcome. Descriptive statistics were calculated as MEAN±SD and a t‐test was performed to determine differences in critical thinking HSRT scores.

RESULTS

Freshmen exercise science HSRT percentile scores were 57.8±25 and improved to 74.2±18 by Senior year (P < 0.05). Similarly, the absolute HSRT critical thinking score was 2.5±3.4 higher following the completion of an exercise science program (P < 0.05). Categorical breakdowns of critical thinking skill scores revealed the greatest degree of observed improvement Freshmen to Senior was in inductive and deductive reasoning (mean difference = 0.93±1.5 and 0.92±2.0, respectively; P < 0.05).

CONCLUSIONS

Students in an exercise science program at a 4‐year liberal arts university demonstrate a significant increase in critical thinking scores. Using a standardized critical thinking assessment tool may be valuable for standardization of program or university 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.
Collaborative Testing and Reflection Papers as Supplementary Assessment Tools in a Clinical Physiology Course
Authors: Anderson Lisa Carney
Published: June 2021

Collaborative testing and reflection papers area are a novel way to supplement traditional assessments such as multiple‐choice exams. The course setting for implementing and studying these two supplementary assessments was Clinical Physiology I and II, a two‐course series in the Department of Integrative Biology and Physiology. Of the 30 students registered for the fall 2016 session of the course, 13 were undergraduates in physiology/biological sciences, 5 were graduate students in the biological sciences and 12 were doctor of Nursing practice (DNP) students in nursing anesthesia. Of the 30 students registered for the spring 2017 semester, 11 were undergraduates in physiology/biological sciences, 15 were graduate students in the biological sciences, 2 were post baccalaureate students, and 12 were DNP students. Students were assigned by the instructor to 1 of 5 groups in which students of different. disciplines were evenly divided among the groups.

On the scheduled assessment day, students first completed a closed‐book, 40‐ question, multiple‐choice exam. After a short break, students gathered into their groups and re‐took a clean version of the same exam. Students discussed each question until they reached a consensus as to the correct answer. When all groups were finished, students were given an exam key and a reflective writing assignment. Students were instructed to pick three question that inspired a lot of discussion and to write a paragraph about the student thought process during the individual exam and the group exam. Reflection essays were due prior to the start of the next weekly class session.

Group exam scores averaged about 7 points higher than individual exam scores and it was not unusual for the group exam score to be higher than any individual exam score within the group. Students reported the experience of the group exam was enjoyable and a helpful learning tool.

Though writing about their thought process was difficult for the students, reflection papers were enlightening to both the students and the instructor. Student identified knowledge gaps, misconceptions, and logic errors. They also gained confidence in their critical thinking skills. The instructor learned which concepts continued to confound students and gained insight as to why certain multiple‐choice questions were poorly received. A major finding of this study is that assessments are powerful tools for teaching critical thinking.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Head Circumference (HC) as a Predictor of Maximum Cranial Width (MCW), Maximum Cranial Length (MCL) and Cranial Index (CI) in Nigerian Children (Less than 3 years old)
Authors: Musa Muhammad A. , Zagga Abdullahi D.
Published: June 2021

Head circumference is one of the commonest and very important anthropometric parameters investigated in children during physical examinations in hospitals within our locality. The aims of this study were to determine the relationship between head circumference and cranial parameters (maximum cranial width [MCW], maximum cranial length [MCL], cranial index [CI]) of study participants as well as to use head circumference values to predict cranial parameters of study participants. A total of 153 children (80 Males and 73 Females) aged between 0 and 3 years were recruited for this study with mean ages of 20.85±9.05 months in males and 19.37±9.07 in females. The head circumference (cm), maximum cranial length (cm), maximum cranial width (cm) of the study participants were measured and cranial index (%) values calculated. Pearson's product‐moment correlation coefficients between variables were computed using Microsoft Excel Spreadsheet. The mean values for head circumference were 46.31±3.48cm in males and 46.52±2.91cm in females. The mean value for maximum cranial length was 14.67±1.13cm in males and 14.80±1.27cm in females while the maximum cranial width mean values were 11.46±0.91cm and 11.89±1.16cm in males and females respectively. The mean value for the cranial index in males was 78.14±3.26% and 80.29±3.02% in females and this difference was not statistically significant (p > 0.05). A moderate positive correlation was observed between HC and MCL (r = 0.521; p < 0.0001) in males, between HC and MCL (r = 0.591; p < 0.0001) in females respectively. A weak positive correlation was found between HC and MCW (r = 0.390; p < 0.0001) in males while a moderate positive correlation existed between HC and MCW (r = 0.546; p <0.0001) in females. Results of linear regression analysis indicated that HC significantly predicted MCW and MCL in both sexes (p < 0.05) while CI was not significantly predicted by HC (b = −0.20, p = 0.0523) in males and (b = 0.06, p = 0.6019) in females respectively. However, the slope of the regression lines (b) between head circumference (cm) and all the cranial parameters was positive in females and same in males except for cranial index (b = −0.20, p = 0.0523) and this was not statistically significant (p < 0.05). From this present study, there is a moderate positive linear relationship between HC and cranial parameters (MCW; MCL) with no statistically significant relationship between HC and CI in both sexes. HC significantly predicted the values for MCW and MCL in both males and females. Routine cranial evaluation such as ours as demonstrated in this study can be of clinical importance in assessing and monitoring cranial growth in children

Support or Funding Information

Nil

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ethyl Pyruvate Reduces Swine Barn Dust Exposure Induced Brain Microglial Cell Inflammatory Responses via Targeting HMGB1‐RAGE Signaling
Authors: Massey Nyzil , Puttachary Sreekanth , Mahadev‐Bhat Sanjana , Shrestha Denusha , Kanthasamy Anumantha G. , Charavaryamath Chandrashekhar
Published: June 2021

Swine production in the US is a major agricultural economy with 550,000 jobs and $39 billion added to GDP. Large swine farms employ full time workers who are exposed to occupational contaminants including organic dust, lipopolysaccharide (LPS), peptidoglycan (PGN) and gases. Exposed workers mainly report bronchitis, asthma and loss of lung function. Majority of the studies have focused on the mechanisms of inflammation underlying the respiratory symptoms. But effect of barn exposure on other vital organs such as brain remain largely elusive. Brain microglial cells are like macrophages of other organs in their innate response and their activation is known to cause neuroinflammation. Therefore, we tested a hypothesis that swine barn dust extract (SBDE) activates microglial cells of the brain using an in vitro model.

Settled dust from commercial swine operations was collected, buffer extract prepared and filter sterilized. Mouse (C57BL6) microglial cell line were treated with SBDE or LPS or PGN or medium and stained for the expression of Iba1 (microglial activation), gp91 phox (reactive oxygen species (ROS)), HMGB1 (high‐mobility group box‐1), RAGE (receptor for advanced glycation end products) and DAPI at 0, 6, 24 and 48 h post treatment. Microscopy and ELISA were used to measure morphological activation and cytokine quantification respectively. Along with SBDE or LPS or PGN, microglia were co‐treated with Ethyl Pyruvate (EP) or Mito‐Apocynin (Mito‐Apo) and HMGB1 translocation or 3‐NT (reactive nitrogen species (RNS) marker) expression were measured respectively.

Microglia morphologically appeared reactive with an upregulation of Iba1 and gp91 phox by 24 hours post LPS or PGN or SBDE treatment. At 48 hours, SBDE treated microglia demonstrated nuclear to cytoplasmic translocation of HMGB1 and co‐localization with RAGE in the cytoplasm. EP treatment reduced nucleocytoplasmic translocation of HMGB1 and RAGE expression while Mito‐Apo treatment reduced the expression of 3‐NT. EP treatment reduced SBDE‐induced microglial cell activation (morphological), ROS, TNF‐α and IL‐6 concentrations.

Our data indicate that, EP reduces SBDE induced microglial activation and inflammation via targeting HMGB1‐RAGE signaling.

Support or Funding Information

Iowa State University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fractones and basement membranes: matrix for patterning, growth and development
Authors: Lee Kiana D. , Douet Vanessa , Arikawa‐Hirasawa Eri , Davis Harry B. , Alarcon Vernadeth B. , Fronville Alexandra , Mercier Frederic
Published: June 2021

More and more evidence supports the view that extracellular matrix (ECM) is essential for growth and patterning during development. However, the mechanisms underlying patterning and morphogenesis are unknown. In both adult and developing mammalians, we have characterized a new type of extracellular matrix structure that we have named fractones. Fractones are chemically similar to basement membranes but differ from the latter by their location. Fractones reside in the parenchyma of tissues and organs. They are are visualized as particles by light microscopy but often display a fractal ultrastructure. In contrast, basement membranes are located as sheaths at the connective tissue/parenchyma interface. We have shown that fractone‐associated heparan sulfates bind and activate growth factors to regulate stem cell proliferation in the neurogenic zone of the adult brain. Here, we used multiple fluorescence immunohistochemistry to visualize fractones, basement membranes and cell proliferation throughout mouse development, with a focus on brain structuration. Fractones preceded basement membranes and were associated with proliferating pluripotent stem cells in the inner cell mass of the blastocyst. During gastrulation, fractones were found in the three germinal tissues. During neurulation, fractones were visualized at the vicinity of proliferating neuroepithelial cells in the notoplate and in the ventricular zone. During corticalization, fractones gradually displaced through the neural tissue according to an in‐out gradient towards the basement membrane of superficial meninges. At this time, fractone distribution negatively correlated with neural cell proliferation. For white matter tract formation, axonal growth was accompanied by fractones along meningeal elongations that progressively patterned the forming brain. Then, brain fractones disappeared only to reappear soon after birth throughout the sub ventricular zone, a location that persisted into adulthood. Together, our results strongly suggest that fractones and basement membranes are essential for patterning and morphogenesis.

Support or Funding Information

Supported by JSPS S09109

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of BRCA1 by SIRT2
Authors: Minten Elizabeth , Zhang Hui , Li Chunyang , Head PamelaSara , Yu David
Published: June 2021

In our everyday lives, we are constantly being exposed to DNA damaging agents, both from internal and external sources. The most deleterious type of DNA damage a cell can suffer is the DNA double‐stranded break (DSB). Breast Cancer 1 (BRCA1) is a protein necessary for the proper repair of resultant DNA DSBs through the homologous recombination (HR) pathway. Defects in BRCA1 have been linked to different types of cancer in both men and women, including breast, ovarian, and pancreatic cancer. However, the regulation of BRCA1 is not yet well understood, and in many cases, how defects in this pathway lead to an increased risk of developing cancer is also unknown, making preventative care and treatment of resulting cancers more difficult. Our lab has discovered that SIRT2, a histone deacetylase and putative human tumor suppressor, plays a crucial role in the DNA damage response (DDR) and repair of DNA DSBs. We have shown that depletion of SIRT2 impairs HR and increases cell sensitivity to IR in a deacetylase‐dependent manner. A mass spectrometry analysis showed SIRT2 interacts with a number of proteins involved in DDR, including BRCA1. We validated the interaction between SIRT2 and BRCA1 and also found SIRT2 deacetylates BRCA1 both in vitro and in cells. Depletion of SIRT2 and subsequent deacetylation of BRCA1 decreases BRCA1 protein levels in cells and thus impairs HR. Our results show SIRT2 is a novel regulator of BRCA1 and is critical for the repair of DNA DSBs through HR. These findings provide insight into how dysregulation of SIRT2 or BRCA1 can result in genomic instability and lead to the development of cancer.

Support or Funding Information

Dr. David Yu, NIH R01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Link Between Mutations in MutS DNA Repair Protein and Lynch Cancer Syndrome
Authors: Kessler Emily , Liu Juan , Awad Helena , Hingorani Manju
Published: June 2021

The Mismatch Repair (MMR) pathway is responsible for correcting errors in DNA and is thus essential for maintaining genome stability in all living organisms. Several studies have shown that mutations causing defects in MMR protein structure and function lead to hypermutation. The increased level of mutations results in serious consequences for human health, highlighted by Lynch Syndrome (LS), a hereditary predisposition to colorectal and other cancers (1). The first protein in the MMR pathway, MutS, recognizes mispaired bases or short insertion‐deletion loops (IDLs) and utilizes its ATPase activity to initiate DNA repair (2). A long‐term study in the Hingorani laboratory examines cancer‐linked single amino acid mutants of the human MutS protein (using Thermus aquaticus MutS as a model system) in order to understand how the changes affect protein structure and function, and alter MMR. This work involves kinetic analysis of MutS activities, and related dynamic conformational changes in the protein as it utilizes ATP to work on DNA. The results will enable construction of a complete mechanism of action for each mutant protein, and help us determine whether the mutants are compromised in their ability to target mismatches, and/or bind and hydrolyze ATP, and/or undergo necessary conformational changes, and therefore understand why they disrupt DNA mismatch repair and give rise to Lynch Syndrome. My project focuses on fluorescence‐based stopped flow kinetic experiments to monitor the conformational changes of wild type MutS and several LS‐linked mutants as they bind +T insertion‐containing DNA and catalyze ATP hydrolysis. In addition to generating new information on each mutant, the findings will advance the proof‐of‐principle concept that detailed analysis of the structure, dynamics, and catalytic activities of individual MutS mutants is both feasible and can reveal critical information for understanding the molecular basis of Lynch Syndrome.

Support or Funding Information

National Institutes of Health R15 GM114743; The Beckman Scholars Program, Arnold and Mabel Beckman Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Connections between the DNA damage response and mRNA processing
Authors: NAZEER FATHIMA I. , Skidders Morgan , Dean Emily , Hofler Alex , Moore Claire
Published: June 2021

Eukaryotic cells have evolved multiple mechanisms to deal with DNA damage in order to prevent disease. In the presence of helix distorting DNA damage, RNA polymerase II (RNAPII) blockage at damage sites can lead to RNAPII degradation and activation of the DNA damage response (DDR), if the damage is not repaired promptly. Our hypothesis is that proteins involved in RNAPII degradation, Elc1 and Def1 are also involved in regulating the DDR and other related cellular processes like mRNA maturation, in response to DNA damage. Our preliminary data suggest that Def1 may be involved in activating the DNA damage response while Elc1 is involved in blocking mRNA maturation after UV‐type DNA damage. We are currently working on purifying the Yeast ElcI complex using affinity chromatography to assess if our purified Elc1 complex is able to degrade factors involved in mRNA maturation, in‐vitro. In addition, we are also constructing deletion strains to test if other protein factors involved in DNA repair play a role in blocking mRNA maturation. Taken together, our data point to an unexpected interconnection between the DNA damage response and mRNA processing pathways.

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‐Native Structure Present in the Unfolded Ensemble May Initiate Aggregation of ALS variants of Superoxide Dismutase (SOD1)
Authors: Cohen Noah , Bilsel Osman , Matthews C. Robert
Published: June 2021

Aggregates of Cu, Zn Superoxide Dismutase (SOD1) in the spinal cord are a hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Mutations throughout the SOD1 sequence have been found to cause ALS, suggesting a general gain‐of‐function mechanism for the toxicity of this disease. We have previously argued that the aggregation of the unfolded chain, following synthesis on the ribosome and prior to maturation, provides a parsimonious explanation for the common phenotype. To determine whether segments of unfolded SOD1 are capable of adopting structure prior to the exceedingly slow (>10 sec) productive folding reaction, we dissected SOD1 into peptides that were labeled with a donor/acceptor FRET pair at or near their N/C‐termini. Time‐resolved FRET (tr‐FRET) analysis revealed that the peptide corresponding the C‐terminal region of SOD1 forms urea sensitive structure. Utilizing 2‐dimensional Maximum Entropy Modeling (MEM), we found that this peptide undergoes a discrete transition between a compact state and a random coil‐like structure. Mutations show this collapse results from a non‐native intramolecular interaction involving a hydrophobic sequence at the C‐terminus as well as a polar/charged sequence at the N‐terminus. Ongoing studies are examining the behavior of these segments in the full‐length protein with tr‐FRET. These results are consistent with the results of proteolysis and crystallographic studies from other labs and together suggest that this region of SOD1 may present a potential target for novel therapeutics to treat ALS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilizing homology of Wound Inducible Transcript 3.0 (WIT3) as a stepping stone to investigate the function of Suppressor of IKKepsilon (SIKE)
Authors: Dawood Mariam , Bell J. Ellis , Bell Jessica K.
Published: June 2021

Wound inducible transcript 3.0 (WIT3.0) is a novel cytoskeleton protein that regulates fibroblast migration and initiates rapid wound closure. WIT3 shares ~50% sequence identity with an innate immune protein of unknown function, Suppressor of IKKepsilon (SIKE), that also associates with cytoskeletal proteins. We hypothesize that SIKE and WIT3 will have similar biochemical and biophysical characteristics that contribute to a shared function. To assess these similarities, we undertook characterization of WIT3 structure utilizing circular dichroism, fluorescence‐based thermal shift assays, ligand binding studies using an ANS reporter, and crosslinking with BS3. Using PHYRE2, a WIT3 model was predicted that consisted of an alpha helical coil with 30% disordered regions. The secondary structure of WIT3 was assessed via circular dichroism. Wavelength spectra is consistent with an alpha helical protein and thermal melt data showed a linear unfolding pattern with Tm of 37°C, suggesting a minimally stable protein. As part of the PHYRE2 model, 3DLigandSite proposed a zinc binding site. To assess an interaction between WIT3 and divalent cations, WIT3‐ANS titration and fluorescence‐based thermal shift assays were completed +/‐ Mg, Mn, Ca, Ni and Zn. An interaction between WIT3 and Zn was confirmed where, in the presence of zinc, WIT3's affinity for ANS was 2 fold higher than in the absence of zinc. The initial comparison suggests that WIT3 and SIKE share similar helical structure and similar FTS melt curve topography indicating exposed hydrophobic residues prior to denaturation, but different structural stability and a potential divalent cation binding site on WIT3. Together, these data suggest that WIT3 and SIKE share several biochemical characteristics that may allow these two proteins to have complimentary function.

Support or Funding Information

Studies were funded in part by USD SURE.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tuberculosis serine hydrolase variable expression, isolation, and characterization under hypoxia conditions
Authors: Severinac Luke Edward , Johnson R. Jeremy
Published: June 2021

Tuberculosis (TB), caused by infection by Mycobacterium tuberculosis, is the deadliest infectious disease worldwide. Complicating the treatment of TB is its ability to transition between an intransient dormant state and an active infectious state. One class of enzymes required for the maintenance of this dormant state and the transition into the active state is serine hydrolases. Our research goal was to globally characterize serine hydrolase activity in actively growing M. smegmatis and under hypoxia conditions that mimic dormant growth. To measure hydrolase activity, lysates were prepared under different growth conditions and then separated by native‐PAGE. Native‐PAGE gels were incubated with a library of fluorogenic ester substrates and hydrolase activity quantitated based on fluorogenic substrate activation. By following the shift in fluorogenic activation, we were able to measure changes in hydrolase protein expression versus fluorogenic substrate and in relation to induction of dormant growth conditions. Reactive hydrolases were excised from the gel and identified by mass spectrometry. Our results provided a global map of mycobacterial serine hydrolase activity and identified starting points for finding novel drug targets for dormant TB.

Support or Funding Information

This work was supported by a grant from the National Institutes of Health (NIH 1 R15 GM110641‐01A1)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and functional characterization of active nicotinic acid dehydrogenase from Pseudomonas fluorescens Pf5
Authors: Brownstein Nathan Mark , Snider Mark J.
Published: June 2021

Many compounds created as a byproduct of industrial processes or synthetic products are having a significant, negative affect on the environment. N‐Heteroaromatic compounds (N‐HACs), make up many of these environmental pollutants. A proposed solution to environmental damage by N‐HACs is bioremediation: using engineered microorganisms to clean areas affected by pollution. Nicotinic acid (NA) is an N‐HAC which is present in a wide range of synthetic products and is an environmental pollutant. Many microorganisms have evolved the ability to catabolize N‐HACs, including several capable of using NA as their sole source of carbon. One organism that degrades NA is Bacillus niacini. B. niacini is of interest because it degrades NA by a pathway unlike other organisms. Characterizing B. niacini’s NA degradation pathway is potentially important for accomplishing the long‐range goal of developing effective bioremediation strategies. The first step in B. niacini's putative NA catabolism pathway is through the nicotinic acid dehydrogenase NicAB, which is encoded by several ORFs in the gene cluster hypothesized to be responsible for NA degradation. Previous attempts to characterize the activity of NicAB expressed in E. coli have been unsuccessful and have demonstrated that the B subunits of NicAB are deficient in molybdenum, an essential metal for its MCD cofactor. The activity of NicAB will be characterized upon expression in Pseudomonas fluorescens Pf5, a closely related organism to the NA degrading bacteria Pseudomonas putida. P. fluorescens Pf5 may have MCD maturation proteins more compatible with NicAB. We show that P. fluorescens Pf5 has no endogenous NA dehydrogenase activity via HPLC. Future work will test whether knock‐in of the genes encoding NicAB into P. fluorescens Pf5 will show whether NA dehydrogenase hydroxylates NA and 6HNA. The activity and substrate binding of functional NA dehydrogenase will be characterized by kinetic analysis.

Support or Funding Information

Henry J. Copeland Fund for Independent Study

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Insights into Peptide Recognition and Modification by the Radical SAM Enzyme SuiB
Authors: Davis Katherine M.
Published: June 2021

Posttranslational modification of ribosomally synthesized peptides provides an elegant means for the production of biologically active molecules. Unlike nonribosomal peptides, which are assembled by large multimodular enzymes, ribosomally synthesized and posttranslationally modified peptides, or RiPPs, have comparatively simple biosynthetic pathways. Modifications to the core sequence of the precursor peptide are installed by tailoring enzymes, however, the structural basis for substrate recognition in these reactions remains unknown. Using X‐ray crystallography and computational modeling, we examine the role of the N‐terminal leader peptide in the biosynthesis of a streptide homolog, a recently identified peptide natural product with an intramolecular lysine–tryptophan crosslink installed by the radical S‐adenosylmethionine (SAM) enzyme, SuiB. We evaluate a series of structures that not only help to elucidate the formation of the C–C crosslink but also provide insights into the interplay between RiPP precursor peptides and tailoring enzymes more generally. Surprisingly, although the N‐terminal domain of SuiB adopts a typical RRE (RiPP recognition element) motif, which has been implicated in precursor peptide recognition, we observe binding of the leader peptide in the catalytic barrel. Computational simulations support a mechanism in which the leader peptide guides posttranslational modification by positioning the cross‐linking residues of the precursor peptide within the active site. Together, these findings reveal unanticipated roles for the RRE and other domains during RiPP biosynthesis by radical SAM enzymes.

Support or Funding Information

This work was supported by the Arnold O. Beckman Postdoctoral Fellowship 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.
Synthesis of Mono‐substituted Anthraquinone Derivatives to be Used in the Purification of Lactate Dehydrogenase
Authors: Retzlaff Brandon David , Mueller Jaime
Published: June 2021

Cibacron Blue 3G‐A has been previously used in biochemical laboratories for purification of enzymes containing the dinucleotide fold. Lactate dehydrogenase is one such enzyme that can be purified using Cibacron Blue 3G‐A in affinity chromatography. Rising costs and inaccessibility to Cibacron Blue 3G‐A have created a demand to find a more cost effective replacement. Electron withdrawing and donating properties of the anthraquinone core substituent groups are believed to affect the affinity of the core for various enzymes. A range of anthraquinone dyes were synthesized to determine the importance of the substituents' electronic properties with the coupling of ortho substituted aniline nucleophiles to the anthraquinone core via a diazonium salt link. The aniline substituents include: methyl, methoxy, chloro, and sulfonyl groups. For each anthraquinone dye synthesized, the λmax value was determined to effectively monitor the kinetics of the dyes. Spectrometric assays measured the oxidation of NADH to NAD+ determining the dye's affinity for enzymes containing the dinucleotide fold.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of Hybrid Inhibitors for Metalloproteinase Targeting
Authors: Quinto Laura B. , Berumen Gregory I. , Kehoe Haixing P. , Van Deventer James A.
Published: June 2021

Although cancer‐promoting activities of extracellular proteases and peptidases in the tumor microenvironment are well known, the disruption of individual enzymes in this environment remains a fundamental challenge. Individual matrix metalloproteinases (MMPs), in particular, have proven difficult to selectively inhibit due to their structural similarities. To better understand the roles of these enzymes in cancer progression, and to potentially begin to target them in vivo, selective inhibitors of these enzymes are needed. We are developing a protein‐small molecule “hybrid” approach to meet this need by conjugating inhibitory small molecules to single‐chain variable antibody fragments (scFvs) that selectively bind a unique target.

To this end, we have established a platform to introduce a versatile range of chemical functionalities into scFvs on the yeast surface. Using amber suppression, we can introduce noncanonical amino acids (ncAAs) at specific sites in displayed scFvs that are predicted to be close to the antigen binding site. In our proof‐of‐concept model, we have been investigating the possibility of adding enzyme active site‐targeting chemical groups to scFvs that bind to MMP‐9. Flow cytometry results show that constructs containing ncAAs with an azide side chain retain MMP‐9 binding. Importantly, introducing the azide functionality enables us to conjugate MMP‐targeting small molecules to displayed scFvs using bioorthogonal copper‐catalyzed azide‐alkyne cycloadditions (“click chemistry”). We have investigated installation of MMP‐targeting small molecules in different antibodies and at various scFv positions. In some cases, we see almost complete reaction, but the extent of reaction across antibodies and sites is dependent on the small molecule used and the site of ncAA incorporation. Systematic exploration of reaction conditions including time and click chemistry ligand has led to conditions that enhance the extent of reaction of small molecules at different sites. Work to evaluate enhancements to binding and inhibition of MMP‐9 with the resulting antibody‐small molecule “hybrid” conjugates is ongoing. We anticipate that adding more chemical functionality into proteins will facilitate selective targeting of broad classes of enzymes active in the tumor microenvironment and in other complex diseases.

Support or Funding Information

Tufts Summer Scholars, Tufts Faculty Research Fund

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 hybrid PLGA‐based nanoparticles as a smart nanoplatform for imaging‐guided and near‐Infrared light‐triggered combination cancer therapy
Authors: Shen Xue , Xie Xiaoxue , Yang Hong , Wu Chunhui , Liu Yiyao
Published: June 2021

Cancer theranostics, the combination of imaging and therapeutic agents in a smart nanocarriers, has been a promising approach towards cancer [1]. And the chemotherapy combining with photothermal therapy to treat tumors can efficiently improve therapeutic efficacies [2].

Herein, we report a theranostic nanoplatform based on PLGA nanoparticles, which are constructed by simultaneous encapsulating indocyanine green (ICG), a widely used near‐infrared (NIR) dye in photothermal therapy, and doxorubicin (Dox), a chemotherapy drug, into biodegradable poly(D, L‐lactic‐co‐glycolic acid) (PLGA) polymeric nanoparticles and decorated with bovine serum albumin (BSA) using the double emulsion solvent evaporation method (W/O/W). The as fabricated IDP NPs with the mean diameter of 220 nm exhibited good dispersity, excellent size stability, especially enhanced photostability (fluorescence stability) compared with free ICG, and is able to efficiently load chemotherapeutic drug doxorubicin (Dox) into the PLGA core. When the weight ratio of added ICG to Dox was 0.075 (μg ICG/μg Dox), the encapsulation efficiency of the Dox and ICG can be reached approximately 42.0% and 55.3%, respectively. It was confirmed that IDP NPs showed preferable photothermal effect, favorable photoacoustic (PA) imaging properties and faster release of Dox when exposed to near‐infrared (NIR) laser irradiation or acidic conditions. Moreover, the NIR light‐induced photothermal heat can enhance the permeability of the lysosomal membrane resulting in efficient Dox translocation from lysosomes to cytoplasm indicating by intracellular distribution assay. In comparison with the chemo‐ or photothermal treatment alone, the combined treatment of IDP NPs with laser irradiation achieving a marked synergistic therapeutic effect. The tumor cell viability was ~5% when treated with IDP NPs ([ICG] =15 μg/mL, [Dox] =3.9 μg/mL) under NIR laser irradiation (808 nm, 1.5 W/cm2). Our work validates that the developed co‐loaded BSA‐decorated IDP NPs could serve as a promising platform for PA imaging‐guided combined chemo‐ and photothermal therapy with reducing side effects in cancer theranostics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating interactions between zebrafish innate immune system and cancer progression
Authors: Lou Meng , Powell Davalyn , Huttenlocher Anna
Published: June 2021

In recent years, the innate immune system has been increasingly implicated in cancer prognosis. Neutrophils have been shown to migrate to the tumor microenvironment, release pro‐invasion factors, and even enhance angiogenesis. Here, we provide a glioblastoma tumor‐initiation model by transforming astrocytes with a constitutively activated form of kRasV12 in hopes of elucidating the contribution of neutrophil presence to tumorigenesis and progression. Constitutive expression of this oncogene has been demonstrated to induce malignant transformation in several models including the larval zebrafish. Additionally, the role of tumor suppressor protein p53 in relevance to this model remains largely unconfirmed. By expressing the oncogene in different p53 backgrounds, we demonstrate an increase in abnormal growth and mass formation in the absence of p53. Further experimental design perturbing neutrophil recruitment has the potential to identify novel interactions with the innate immune system and aggression of glioblastomas.

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