bluuebunny/crossref_metadata_2025 huggingface dataset:
Mentorship In Converting Research Into A CURE: The Mentoring The Integration Of Research Into The Curriculum (MIRIC) Initiative
Authors: Wolyniak Michael , Barton Lance F. , Resendes Karen K.
Published: April 2020

The American Association for the Advancement of Science (AAAS) Vision and Change document has spurred several initiatives designed to improve the way in which undergraduates learn science. Often, these initiatives have been disseminated as one‐time workshops that generate awareness of and interest in developing authentic research experiences for undergraduate STEM classrooms. Conversely, whole “standardized” curricula have been developed and made available for adoption. However, successfully generating the sustainable change necessary to bring real reform to undergraduate science education should benefit both students and faculty scholarship. To create sustainable change, long‐term faculty development initiatives focused on mentorship are needed so that instructors can develop and implement sustainable curricula with local relevance. Experienced instructors seasoned in developing and implementing course‐based undergraduate research experiences (CUREs) based on their own scholarship and local resources can convey their experiences to mentees interested in using these pedagogical techniques as the centerpiece of their own teaching. The Council on Undergraduate Research (CUR) Biology Division has created the Mentorship for Integrating Research Into the Classroom (MIRIC) program to provide a means for members with an interest in developing improved and sustainable active learning techniques to gain experience in this style of teaching through close, long‐term interaction with a veteran teaching mentor. Developed from the former ASCB Mentorship in Active Learning and Teaching (MALT) program, MIRIC focuses on the development of instructors who wish to develop a dynamic CURE. Current and future life science instructors pair themselves up with seasoned veterans of CURE development and work with them and their students over the course of a semester or longer to develop a CURE that will allow the mentee to bring authentic research into his or her classes. In our pilot studies, we collected qualitative data based on participant interviews that suggest that MIRIC mentorships have made positive gains in promoting sustainable active learning techniques among participants. Since then, we have surveyed the first cohort on both the success of their CURE development process as well as the benefits of the mentoring relationship on both the mentors and mentees using validated questions derived from Mathur (2012) and Pamuk and Thompson (2009).

Support or Funding Information

‐The Council on Undergraduate Research (CUR)
Cux1 regulates cilia length in polycystic kidney disease
Authors: Holthaus Conner , Kumar Emmanuel , Rumschlag Matthew , Wee Zhi Nee , Pasillas Melissa , Heuvel Greg Vanden
Published: April 2020

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common inherited disorders affecting the kidney. Renal cyst development in ADPKD results from mutations in the PKD1 or PKD2 genes, which encode the proteins polycstin1 (PC1) and polycystin2 (PC2), respectively. PC1 and PC2 proteins are localized to primary cilia where they are proposed to form a receptor channel complex that detects flow transmitting a calcium‐mediated signal. Primary cilia are critical to the pathogenesis of ADPKD, which is one of many ciliopathies that exhibit renal cystic disease. Cux1, a murine homolog of the Drosophila gene Cut, is a cell cycle dependent transcriptional repressor that regulates expression of the cyclin kinase inhibitor p27. Cux1 is highly and ectopically expressed in mice carrying a collecting duct specific mutation of Pkd1 (Pkd1CD) and in human ADPKD cells where it promotes cell proliferation. Mice carrying mutations in both Cux1 and Pkd1 have reduced cystic disease and an increased life span. A role for Cux1 in regulating genes involved in cilia assembly and function has recently been identified in the Galapagos cormorant, however, the role of Cux1 in cilia in the mammalian kidney is not known. To begin to determine whether Cux1 regulates ciliogenesis we evaluated cilia morphology and the expression of the ciliary protein, OFD1 (oral‐facial‐digital‐1), identified as a target of Cux1 in the Galapagos cormorant. Cilia analysis was performed on kidneys isolated from wild type, Cux1 transgenic, Pkd1CD, and Pkd1CD/Cux1 double mutant mice. Cilia morphology was assessed by immunofluorescence labeling of alpha‐tubulin and the collecting duct marker dolichos biflorus agglutinin (DBA) to identify cells in which Pkd1 was deleted. Cilia in Pkd1/Cux1 double knockout kidneys were significantly shorter than cilia in the Pkd1CD kidneys, consistent with previous studies showing that decreased cilia length corresponds to decreased cystic disease. OFD1 is an inhibitor of ciliogenesis and OFD1 expression in the various mouse models demonstrate that OFD1 expression corresponds to Cux1 expression. OFD1 protein levels were the lowest in the kidneys of mice constitutively expressing Cux1 and were highest in mice with deletions of Cux1. Taken together, our results reveal a novel role for Cux1 in regulating ciliogenesis in the kidney and suggest that the reduced cystic disease in the Pkd1/Cux1 double mutant mice results from reduced cilia length.

Support or Funding Information

American Association of Anatomy FGAP, Western Michigan University Homer Stryker MD School of Medicine
INFLUENCE OF BODY POSITION ON PULMONARY VENTILATION DURING ARREST AND RELEASE OF LOWER LIMB CIRCULATION AFTER EXERCISE IN HEALTHY HUMANS
Authors: da Silva Cesar Tamires , Sayegh Ana Luiza , Oliveira Diogo , Rabelo Mariana , Nery Luiz Eduardo , Taylor Bryan , Silva Bruno
Published: April 2020

Pulmonary ventilation (V̇E) response to exercise is greater when exercise is performed seated versus supine. Maybe this phenomenon is partially attributed to interaction between neural mechanisms involved on the regulation of V̇E. For instance, venous return is reduced during seated exercise, which activates the cardiopulmonary baroreflex. Therefore we sought to investigate if the cardiopulmonary baroreflex interacts with the muscle metaboreflex and the carotid chemoreflex to the regulation of V̇E in healthy humans. Thirteen young healthy subjects participated in the study (7 men). Subjects performed isometric plantar flexion with the dominant limb for 1.5 min in the semi recumbent position. Close to the end of exercise, the circulation of the dominant leg was arrested at the thigh level via cuff inflation. Then, subjects stopped exercising and their position was shifted from semi‐recumbent to either: 1) seated with the non‐dominant limb lowered to inhibit the cardiopulmonary baroreflex; or 2) supine with the non‐dominant limb raised to activate the cardiopulmonary baroreflex. Circulatory arrest took 2 min and aimed to trap metabolites into the exercised muscles to sustain muscle metaboreflex activation. Next, the tight cuff was deflated, releasing metabolites from skeletal muscles to the systemic circulation. Another experiment, under free flow recovery, served as unconcluded control. A subset of the subjects (n = 3) have undergone hyperoxia breathing (100% O2) only during release of circulatory arrest at the seated position to inhibit the carotid chemoreflex. A normoxic experiment served as control. A rebreathing system was used in an attempt to maintain the end‐tidal partial pressure of CO2 constant throughout all experiments. We found that circulatory arrest did not change V̇E versus unconcluded control in the seated (P > 0.05) and supine (P > 0.05) positions throughout the 120‐s period of cuff inflation. V̇E increased above unconcluded control in the seated position after 60 s of circulatory release (mean ± SD: Δ = 20 ± 29%, P = 0.05) and it remained elevated until the end of data recording at 120 s (Δ = 21 ± 31%, P = 0.04). However, release of circulatory arrest did not change V̇E versus unconcluded control in the supine position (P > 0.05). Furthermore, hyperoxia did not change the V̇E increase after circulatory release at the seated position. Collectively, these results suggest that the cardiopulmonary baroreflex did not interact with either the muscle metaboreflex or the carotid chemoreflex. The underling mechanisms of the V̇E increase during release of circulatory arrest at the seated position deserve further investigation.

Support or Funding Information

CAPES and CNPq
CUREing Ocean Plastic Microbes: Positive Experience of Engaging Non‐traditional Undergraduate Students in a Hispanic‐Serving Institution
Authors: Barral Ana Maria , Simmons Rachel E. , Boman-Davis Marie , Bowman Jeff S.
Published: April 2020

Early participation in research experiences is considered a high‐impact educational practice shown to enhance student engagement in STEM. This, in turn, can result in increased enrollment and retention of majors, as well as increased appreciation of science by nonmajors. However, traditional research opportunities such as summer internships are difficult to expand and are often limited to students already invested in STEM. Course‐based undergraduate research experiences (CUREs) broaden the impact of student exposure to research by making it a required part of a course.

We report the result of the integration of a CURE addressing microbial colonization of plastic in coastal Pacific waters in both non‐majors and major’s biology courses during a one‐year period. This CURE, a collaboration between a large private non‐profit university serving nontraditional students and a world‐renowned research‐oriented institution, received NSF funding in 2018 (NSF‐HSI #1832545).

The CURE incorporates an existing research project, which explores the nature of microbial populations colonizing different types of plastic using metagenomic sequencing and traditional microbiology techniques. Students participate in field and laboratory research, and interact with research scientists and graduate students. Course level assessment involve surveys and focus groups to evaluate student engagement. Institutional assessment is planned to monitor biology degree retention.

As of November 2019, students from four non‐majors and 2 majors’ courses (one lower and one upper division) have participated in the CURE. Students were asked fifteen questions related to knowledge of different aspects of the course, laboratory skills, plastic research, and appreciation of scientific research results. The questions were framed in an after/before format which allowed for a two‐tailed t‐test (alpha = 0.05) in the one‐group posttest only design. Results showed statistically significantly differences in all questions indicating improvements across all assessed areas (n=49). Student open‐ended comments about the experience were overwhelmingly positive. Future plans include assessing BS Biology majors’ retention using a longitudinal cohort model, and expanding nonmajors’ participation in the CURE to promote enrollment in the major.

Due to the modular nature of the CURE, its framework can easily be adopted and modified to other courses and curricula. We anticipate that this CURE could be a widely applicable model for biology and ocean science education and disseminated to institutions serving nontraditional adults.

Support or Funding Information

This work has been supported by NU internal grants, as well as the NSF HSI’s new to NSF award #1832545
Biventricular Shape Modes Associated with Clinical History and Pulmonary Regurgitation in Repaired Tetralogy of Fallot
Authors: Govil Sachin , Mauger Charlene , Hegde Sanjeet , Perry James , Suinesiaputra Avan , Omens Jeff , McCulloch Andrew , Young Alistair
Published: April 2020

Surgical repair of tetralogy of Fallot (ToF) often involves damage to the right ventricular (RV) outflow tract typically resulting in pulmonary valve impairment and subsequent pulmonary regurgitation. Residual pulmonary regurgitation leads to progressive RV remodeling in the form of RV dilation and apical bulging, and plays an important role in long‐term outcomes. Patients with severe pulmonary regurgitation and RV decompensation undergo pulmonary valve replacement (PVR) to reduce the risk of arrhythmia, heart failure, and sudden death. However, the clinical decision on whether and when to perform PVR remains unclear. We sought to quantify biventricular shape modes associated with clinical history and pulmonary regurgitation prior to valve replacement using a biventricular geometric atlas constructed using principle component analysis. In this cross‐sectional retrospective study, biventricular shape models were customized using cardiovascular magnetic resonance images from 97 repaired ToF patients. Patients were divided into two groups: those that subsequently had PVR and those that did not. Regression and clustering techniques were used to quantify shape variations associated with type of surgical repair, time following surgical repair, time leading up to valve replacement, and pulmonary regurgitant volumes. Principal components of biventricular shape variation were associated with RV dilation and apical bulging, overall size and sphericity, and valve plane orientation. Biventricular shape modes were quantified using statistical shape modeling techniques and are correlated with clinical history and pulmonary regurgitation.

Support or Funding Information

Funding was provided by the National Institutes of Health 1R01HL121754. SG acknowledges National Institutes of Health NHLBI 1T32HL105373. CM acknowledges New Zealand Heart Foundation Grant 1695. AY acknowledges Health Research Council Grant 17/234.
Structural Determination of the CCT:PhLP1:Gβ5 Folding Complex
Authors: Ludlam William Grant , Stewart Michael , Zocca Sean , Shen Peter , Willardson Barry
Published: April 2020

Before the Gβ subunit of the G‐protein heterotrimer can be assembled into signaling complexes, it must first be folded by the cytosolic chaperonin CCT and by the co‐chaperone Phosducin‐like Protein 1 (PhLP1). PhLP1 has been observed to release the Gβ1 isoform from CCT after folding. In contrast, PhLP1 increases the affinity of the Gβ5 isoform for CCT. Here we present a structure of CCT in complex with PhLP1 and Gβ5 at less than 4 angstrom resolution. Using a combination of cryo‐electron microscopy and crosslinking coupled with mass spectrometry, we found this structure differs markedly from the previously published complex of CCT with PhLP1 and Gβ1. In the case of Gβ5, the substrate sits deep in the CCT folding chamber between the CCT rings, whereas Gβ1 sits high in the folding chamber associated with the apical domain of the CCT3 subunit. The position of PhLP1 is also distinct, presumably to accommodate the differences in substrate position. These structural observations provide insight into the different roles PhLP1 plays in folding Gβ1 and Gβ5 and their subsequent dimerization with Gγ subunits or RGS proteins, respectively.

Support or Funding Information

R01‐EY012287
Microbiome: Its role in health and disease
Authors: Raynes Edilberto Alcantara , Harris Matthew Justin , Bossuah Kwaghdoo Atsor , Bradley William Chris
Published: April 2020

The resident microorganism of the human body, termed human microbiome, encompasses whole gamut of microbial genes, gene products, and genomes. These microbial organisms, also known as microbiota, regard the human body as home. Normally colonizing the airway passages, skin, oral cavity, gastrointestinal, and vagina to prime immunity and to maintain host health. Specifically, the human gastrointestinal tract is populated with millions of microorganisms termed gut microbiota. These gut microbiota control the host physiology including immune system maturation and function as well as the nervous system thereby orchestrating the microbiota‐gut‐brain communication. Disruptions of the microbial interactions called dysbiosis alter gut homeostasis due to dietary changes, stress, illnesses, intake of drug particularly antibiotic, or other disturbances within the human host. The purpose of the study was to conduct a descriptive research on the role of microbiome in health and disease thereby increasing public awareness on the significance of alterations of the microbiome. Using different databases including PUBMed, Google Scholar, and CINAHL, articles were gathered and reviewed. Results of the study revealed that DNA sequencing, RT‐qPCR method, and fecal samples were used to identify genes and microorganisms that disrupt the human microbiome. As a result of the alterations, some of the microorganisms were identified include Prevotella, Clostridia, Atopobium, and Lactobacillus. These organisms altered the fermentation, produce extracellular enzymes and growth factors in the intestinal tract. Several diseases such as rheumatoid arthritis, diabetes, autism spectrum disorder, inflammatory bowel diseases, kidney diseases, to name a few, were associated with the dysbiosis of the microbiome. The findings suggest that gut dysbiosis in patients with the above mentioned disorders are associated with host metabolism and inflammation.
Characterization of Pancreatic Cancer‐Targeting Peptides MCA1 and MCA2
Authors: Asar Mallika , Soendergaard Mette
Published: April 2020

Pancreatic cancer is the fourth leading cause of cancer deaths in the U.S., characterized by a 5‐year survival rate of 3%. The dire prognosis is due to inadequate detection methods. However, peptides offer advantages regarding in vivo molecular targeting due to their small size and ample binding affinity. Thus, peptides may be used in imaging of pancreatic cancer for the detection of the disease.

Bacteriophage (phage) display technology using a 15‐mer fUSE5 peptide library (1013 virions) was previously performed by an initial negative selection round against 106 normal human pancreatic (hTERT‐HPNE) cells. Unbound phage were amplified in E. coli K91BK and isolated using PEG/NaCl precipitation. Phage (1013 virions) were then subjected to four subsequent rounds of positive selection against 106 human pancreatic cancer (Mia Paca‐2) cells. Following the last round, amplicons were produced by PCR and submitted to next‐generation sequencing. The results identified two clones, pMCA1 and pMCA2, which were comparatively prevalent in the fourth positive round.

In order to evaluate the pancreatic cancer binding properties of phage‐displayed peptides (MCA1 and MCA2), the ligands were synthesized with an N‐terminal GSG spacer and a biotin group. A modified ELISA and fluorescent microscopy were performed, in which 10 μM MCA1, MCA2, J18 (negative control), or DMSO (vehicle) was incubated with Mia Paca‐2, hTERT‐HPNE, ovarian adenocarcinoma SKOV‐3, prostate cancer LNCaP, or immortalized human embryonic kidney HEK 293 cells for 1 h. For the modified ELISA, bound peptides were probed by HRP‐conjugated streptavidin, and detected spectrophotometrically at 405 nm after the addition of ABTS. For fluorescent microscopy, bound peptides were detected by FITC‐conjugated streptavidin. Results showed that MCA1 and MCA2 exhibited significantly (p<0.01) higher binding to Mia Paca‐2 cells, compared to J18 and DMSO, while no binding was detected to hTERT‐HPNE, SKOV‐3, LNCaP, or HEK 293 cells. Fluorescent microscopy validated these results by showing significantly (p<0.0001) increased fluorescent intensity of MCA1 and MCA2 to Mia Paca‐2. However, only minimal fluorescent intensity was observed to hTERT‐HPNE, SKOV‐3, LNCaP, or HEK 293 cells. This study indicates that peptides MCA1 and MCA2 specifically target pancreatic cancer cells. Hence, these ligands may be utilized in further studies to improve the detection of pancreatic cancer.
Tcf19 Knockout Mouse Islets Have Increased Stress‐related Gene Expression and Reduced Proliferative Capacity
Authors: Blumer Joseph T. , Han Jee Young , Yang Grace H. , Lodh Sukanya , Fontaine Danielle A. , Davis Dawn B.
Published: April 2020

Transcription factor 19 (Tcf19) is a putative transcription factor associated with both Type 1 and Type 2 diabetes. Tcf19 is expressed in human and rodent pancreatic β‐cells and is upregulated in proliferating islets and obesity. We generated a whole body knockout (wbKO) of Tcf19 and the resulting lean, 15‐week‐old mice have normal fasting glucose, insulin secretion, and glucose tolerance compared to control. RNASeq led to the identification of 733 upregulated and 763 downregulated genes in wbKO islets compared to control. Overrepresented GO terms include upregulated apoptotic process, and negative regulation of cell proliferation, and downregulated vesicle‐mediated transport. We verified markers of proliferation, β‐cell identity, cell stress, and pro‐apoptosis using RTqPCR. Ki67, Pdx1, Nkx6.1, and Nkx2.2 were significantly decreased while Chop, Bak, Gadd45α, and Dtx3l were significantly increased in islets from wbKO mice. Whole pancreas was harvested to measure β‐cell area and although total area is not different, wbKO mice have altered islet size distribution with an increased number of small islets. Next, β‐cell proliferation and apoptosis were measured in frozen pancreatic sections using Ki67 and TUNEL staining, which revealed significantly less proliferation with no change in apoptosis rates in wbKO mice. In adulthood, β‐cell mass expansion occurs due to proliferation as an early compensation for insulin resistance in response to obesity. To determine the role of Tcf19 in this adaptive response wbKO and control mice were put on a one‐week high fat diet (HFD). After one week on HFD, islets from wbKO do not appropriately upregulate Ki67 and cyclin D2 as measured by RTqPCR. In summary, Tcf19 is involved in proliferation and stress related processes both of which are involved in regulating β‐cell mass, which declines in Type 1 and Type 2 diabetes.

Support or Funding Information

VA Merit Award 1I01BX001880

VA Merit Award 1I01BX004715

TL1 Award TR000429

ICTR Clinical and Translational Science Award UL1TR000427

NIDDK 5R01DK110324
Timing and Specificity of Cotranslational Nascent Protein Modification in Bacteria
Authors: Yang Chien-I , Hsieh Hao-Hsuan , Shan Shu-ou
Published: April 2020

Selection of newly‐synthesized proteins into correct protein biogenesis pathways is crucial for cellular homeostasis. The ubiquitous N‐terminal methionine excision (NME) process is mediated by peptide deformylase (PDF) and methionine aminopeptidase (MAP), two essential enzymes in bacteria. This reaction takes place near the nascent peptide exit site of the ribosome, where multiple ribosome‐associating protein biogenesis factors (RPBs) also compete for the access to the nascent chain. How NME achieves its efficiency and specificity at this crowded environment is unknown. Here, using kinetic measurements on purified ribosome‐nascent chain complexes, we show that the ribosome accelerates the MAP reaction for optimal substrates by 102–104 folds. Kinetic competition with translation elongation and selective regulation from other RPBs enhance the specificity of NME by narrowing the processing time window for reactions on suboptimal substrates. With the kinetic data, we constructed a mathematical model and accurately predicted the cotranslational NME efficiency in cytosol. Our data demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity. Moreover, the remodeled MAP activity prompted us to develop a cotranslational assay to screen for MAP inhibitors in the physiological context of translating ribosome. The results explain the discrepancy between traditional peptide‐based assays and cellular data, providing a powerful tool for the development of antibacterial agents targeting the NME machineries.

Support or Funding Information

This work was supported by National Institutes of Health grant GM078024 and a grant from the Weston Havens Foundation to S.‐o. Shan, and a Think Global Education Trust Fellowship from Taiwan to C.‐I. Yang.
Tough Slug Glue: How Transition Metal Binding Proteins Cross‐link an Unusual Bioadhesive
Authors: Christoforo Courtney , Haws Haley , Smith Andrew
Published: April 2020

In this study, we characterize the role of transition metals in the structure and function of the adhesive secretion of the slug Arion subfuscus. A deeper understanding of this glue’s cross‐linking mechanisms is useful because of its potential to guide the design of novel medical adhesives. We hypothesized that transition metal ions, in particular iron, play an important role in cross‐linking and thus strengthening the glue. We used immobilized metal ion affinity chromatography to identify iron and zinc binding proteins in the glue, and to determine the pH at which these proteins bind and dissociate. Atomic absorption spectroscopy was also used to measure how tightly zinc was bound to the glue at different pH values. The glue was then subjected to tensile tests at pH values where the proteins were bound to metals, and at pH values where they dissociated, to determine if the loss of metal‐binding made the glue weaker. An iron‐binding protein and several zinc‐binding proteins were identified. The zinc was readily removed from the glue as the pH dropped from 6.5 to 4.5, but this had no effect on the glue’s strength. In contrast, the iron‐binding protein stayed bound across a pH range from 3 to 8, but dissociated from iron at a pH of 9. This dissociation appears to impact the glue’s mechanical properties. The precise nature of this effect is still under investigation. These data suggest that iron may be more important to the glue’s strength than zinc. This information should be valuable in guiding the design of synthetic glues that can adhere to wet surfaces, and deform with tissues but maintain toughness.

Support or Funding Information

This work was partially supported by an Ithaca College summer research grant.
Examination of Protein Folding and Metabolic Activity in a Saccharomyces cerevisiae Model for TPI Deficiency
Authors: Hrizo Stacy , Skolnick Kathryn , Schaffold Hailey , Schlosser Lily Jean , Barnes Carol Jean , Koziara Kortnee , Fisher Isaac
Published: April 2020

Triose Phosphate Isomerase (TPI) is a glycolytic enzyme that catalyzes the inter‐conversion of DHAP to G3P. Mutations in TPI have been identified that are associated with a neurodegenerative disease in humans known as TPI deficiency. To study the cause of TPI deficiency pathogenesis, we constructed Saccharomyces cerevisiae yeast strains expressing six different TPI alleles that are associated with the development of TPI deficiency in humans and Drosophila melanogaster. Interestingly, not all of the TPI alleles exhibit similar characteristics in terms of stability, temperature sensitivity and activity. Yeast with the M80T allele exhibits significant changes in growth and metabolic activity at all temperatures. Yeast with the E104D allele exhibit an acute temperature sensitive phenotype for growth and metabolic activity at 37C. All of the alleles except C41Y demonstrate protein instability at 37C. This study further examines the role of protein folding in TPI deficiency disease pathogenesis. Yeast expressing the mutant TPI alleles were examined for activation of HSF, a cytoplasmic protein folding stress response transcription factor. Understanding the activation of cell stress pathways in TPI deficient yeast may lead to understanding of disease pathogenesis in TPI deficiency patients and provide potential pharmacological targets.

Support or Funding Information

Faculty Research Development Grant from Slippery Rock University
Role of AIG1 and ADTRP in Endogenous FAHFA Regulation
Authors: Ertunc Meric Erikci , Kok Bernard P. , Parsons William H. , Wang Justin G. , Tan Dan , Donaldson Cynthia J. , Pinto Antonio M. , Vaughan Joan M. , Ngo Nhi , Lum Kenneth M. , Henry Cassandra L. , Coppola Aundrea R. , Niphakis Micah J. , Cravatt Benjamin F. , Saez Enrique , Saghatelian Alan
Published: April 2020

Androgen‐induced gene 1 (AIG1) and Androgen‐dependent TFPI‐regulating protein (ADTRP) are atypical transmembrane hydrolases that rely on a catalytic threonine for their enzymatic activity. In vitro characterization of AIG1 and ADTRP in lysates and cells identified fatty acid ester of hydroxy fatty acids (FAHFAs) as their putative substrates. Here, we generate ADTRP knockout (Adtrp‐KO), AIG1 knockout (Aig1‐KO), and ADTRP/AIG1 double deficient (DKO) mice using CRISPR‐Cas9 technology to test whether these enzymes regulate FAHFAs in vivo. AIG1, ADTRP, or a deficiency in both enzymes leads to decreased FAHFA hydrolytic activity in tissue lysates. Quantitative measurement of FAHFA levels in several tissues revealed increased FAHFA levels in brown adipose tissue (BAT), subcutaneous adipose tissue (SQWAT), and perigonadal WAT (PGWAT) of Adtrp‐KO mice consistent with the loss of FAHFA degrading activity. Furthermore, contribution by AIG1 was modest and only observed in the kidney and BAT of DKO mice. Lipidomics of tissues from knockout and wild type control mice detected no significant changes in other lipid classes to indicate that these enzymes are specific for FAHFA substrates. Furthermore, we developed a potent and selective, dual AIG1/ADTRP inhibitor to enable pharmacological interrogation of these enzymes in vivo. Chemical inhibition of AIG1 and ADTRP raised FAHFA levels demonstrating acute regulation of FAHFAs. In aggregate, the results establish AIG1 and ADTRP as the only endogenous FAHFA hydrolases known, and describe resources (mice, inhibitors) needed to elucidate the biochemical and physiological role of these exciting enzymes.

Support or Funding Information

This research was supported by the NIH (DK106210, DK114785, DA033760), The Leona M. and Harry B. Helmsley Charitable Trust (grant #2012‐PG‐MED002 to A.S.), NCI Cancer Center Support Grant P30 (CA014195 MASS core, A.S.), Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.), a NIH F32 postdoctoral fellowship, DK111159 (M.EE.), a Hewitt Foundation for Medical Research Fellowship (W.H.P.), Mass Spectrometry Core of the Salk Institute with funding from NIH‐NCI CCSG: P30 014195, NIH 1S10OD021815‐01 and the Helmsley Center for Genomic Medicine, and Transgenic Core Facility of the Salk Institute with funding from NIH‐NCI CCSG: P30 014195.
Ketone Ester Supplementation Does Not Impair Cognitive or Motor Performance and Delays CNS Oxygen Toxicity in Male Sprague‐Dawley Rats
Authors: Stavtizski Nicole M. , Landon Carol S. , Hinojo Christopher M. , Poff Angela M. , Rogers Christopher Q. , D’Agostino Dominic P. , Dean Jay B.
Published: April 2020

Oxygen acts as a drug at increased atmospheric pressures, such as occurs during exposure to hyperbaric oxygen (HBO) during hyperbaric and undersea medicine. Use of HBO is limited by the risk of developing CNS oxygen toxicity (CNS‐OT) which manifests as seizures. The ketogenic diet (KD) is used clinically as an anticonvulsant therapy for refractory epilepsy but requires significant dietary restrictions to maintain ketosis. Studies suggest that exogenous ketone ester (KE) induces ketosis and delays the onset of CNS‐OT without the need for dietary restrictions; however, an optimal dose of KE that provides neuroprotection and delays the onset of CNS‐OT without hindering cognitive or motor performance has yet to be elucidated. Sprague Dawley rats received either no gavage, vehicle gavage, or 7.5 g/kg KE gavage and were subjected to a battery of behavioral tests: DigiGait (DG), Light/Dark (LD), open field (OF), and novel object recognition (NOR). No significant differences were observed in motor performance on the DG tests in all treatment groups, suggesting that there is no effect of KE on motor performance. No differences in the time spent in the light or dark zones in the LD test were observed between groups, nor in the latency time to the dark zone. KE‐treated rats exhibited reduced distance traveled in the dark zone, suggesting the KE may be anxiolytic, a property that has been reported previously. No differences in time spent in the perimeter or center zones between the groups in the OF test were noted; however, KE‐treated rats exhibited decreased distance moved and speed, similar to what was seen in the DigiGait test which again may reflect an anxiolytic effect. To further explore this result, we evaluated overall activity level of the rats using the OF test. KE‐treated rats exhibited increased overall activity, indicating that the animals were exploring the areas and were less anxious than vehicle controls. Although there were no significant differences between the groups in the NOR test, KE‐treated rats exhibited a trend in both exploratory preference and discrimination index suggesting that the animals learned and were able to recall that a novel object was present. Additional experiments are currently being conducted in which vehicle‐ or KE‐treated rats are implanted with radio‐telemetry modules, allowing for real‐time monitoring of EEG, ECG, and respiratory EMG during an HBO dive protocol. These experiments are underway but have thus far shown that the KE increases latency time to seizure over vehicle (24.9±6.7; 15.5±6.5, n=8). Together, these findings suggest that a single administration of KE will delay onset of CNS‐OT without hindering performance. This work is funded by the Office of Naval Research.

Support or Funding Information

Office of Naval Research
Reconsidering the Role of the RecF Protein in Repair of Post‐replication Gaps
Authors: Beyer Hope A. , Wood Elizabeth A. , Chitteni-Pattu Sindhu , Henry Camille , Cox Michael M.
Published: April 2020

In Escherichia coli, the RecA protein is an important factor in homologous recombination and DNA repair. When a post‐replicative gap is formed, RecA is loaded onto the single strand (ss) DNA coated by SSB, a process facilitated by recombination mediator proteins (RMP), RecF, RecO, and RecR. Contrary to current models, RecF, RecO and, RecR do not form a complex. In vitro, RecR can interact with either RecO or RecF but they compete for RecR binding. In vivo, RecF and RecO have distinct locations within the cell. Only RecF colocalizes with the replisome after UV stress, suggesting that RecF and RecO have distinct functions. Moreover, in some conditions only RecF or RecOR appear to be required. The RecOR complex is believed to load RecA onto ssDNA. Nevertheless, the RecF’s role and the function of its ATPase activity remains enigmatic.

Building evidence suggests that RecF may have an unanticipated function at the replication fork. Present understanding has been achieved by using a variety of techniques including biochemical assays, phenotypic characterization and single molecule imaging microscopy, all of which confirm and build upon the hypothesis that RecF has a unique and novel role independent of RecOR in gap repair. To further investigate this hypothesis, we use a combination of overexpression and deletion mutant experiments. We hope to further distinguish RecF’s role in gap formation by working with the RecA V79L mutant, which possesses an enhanced ability to bind and remove SSB from the ssDNA.
Intralaryngeal application of ATP evokes apneic response mainly via acting on P2X3/P2X2/3 receptors of the superior laryngeal nerve (SLN) in postnatal rats
Authors: Zhuang Jianguo , Gao Xiuping , Wei Wan , Xu Fadi
Published: April 2020

ATP is reportedly capable of stimulating bronchopulmonary C‐ and/or Aδ‐fibers to evoke apnea, bronchoconstriction, and bradycardia via acting on P2X (P2X2/3) receptors. Stimulation of the SLN evokes apnea, bradycardia, and hypertension, especially in early life. Although P2X3/P2X2/3 receptors are present in the nodose and jugular (N/J) ganglion neurons, it is undetermined whether ATP applied onto the larynx mucosa can elicit the cardiorespiratory responses via activation of P2X3/P2X2/3 receptors of the SLN. To address this issue, the cardiorespiratory responses to intralaryngeal perfusion of ATP (a non‐selective P2‐receptor agonist, 5–10 mg/ml) or α,β‐methylene ATP (a selective P2X‐receptor agonist, 2.5–5 mg/ml) were recorded in anesthetized rat pups (P14‐P18) under the following conditions: 1) before and after bilateral SLN section and 2) immediately after 5 min intralaryngeal pretreatment with saline (Ctrl) or saline containing A‐317491 (a P2X3/P2X2/3 receptor antagonist, 2.5–5 mg/ml). The immunoreactivity (IR) of P2X3 receptor was then identified in N/J ganglion neurons retrogradely traced by DiI that was previously injected into the SLN 10 days ago. Whole‐cell patch clamp recording was used to determine the ATP‐ or α,β‐methylene ATP‐induced currents of N/J neurons in primary culture, particularly those labeled by DiI. We found that intralaryngeal perfusion of ATP and α,β‐methylene ATP induced similar apnea (latency < 1.0 s), hypertension, and bradycardia, which were eliminated after bilateral section of the SLN. A‐317491 but not saline pretreatment attenuated the apneic response to ATP and α,β‐methylene ATP by 63% and 76% respectively and failed to affect the evoked hypertension and bradycardia. P2X3 receptor‐IR was identified in ~35% of both N/J ganglion neurons. 12% of these neurons were labeled by DiI, ~60% of which expressed P2X3 receptor‐IR. Both ATP‐ and α,β‐methylene ATP‐induced currents were recorded in DiI‐labeled neurons. The former was reduced by 75% and the latter abolished by A‐317491. We conclude that ATP and α,β‐methylene ATP applied onto the laryngeal mucosa elicit an apnea predominately through activation of P2X3/P2X2/3 receptors of the SLN.
Molecular Viscometer Identified a Population of Mucin Granules of Higher Viscosity in Cystic Fibrosis Cells
Authors: Ponomarchuk Olga , Boudreault Francis , Brochiero Emmanuelle , Gryczynski Ignacy , Dzyuba Sergei V. , Fudala Rafal , Gryczynski Zygmunt , Grygorczyk Ryszard
Published: April 2020

The cystic fibrosis (CF) disease is characterized by abnormally thick and sticky mucus. Normal airway mucus has bulk viscosity (macroviscosity) in the range of 12–15×103 cP increasing to 14–110×103 cP for CF patients. At micro‐length scale (≤1 μm), due to the presence of fluid‐filled spaces between mucin fibers, mucus microrheological properties are similar to that of water (~1 cP). However, they are only slightly higher in CF mucus (~3 cP). Our study was designed to verify if similar viscosity differences exist between normal and CF mucin granules and secreted mucus.

We used a custom‐designed membrane‐permeant molecular viscometer (phenyl‐BODIPY) and fluorescence lifetime imaging microscopy (FLIM) to study the microviscosity of mucin granules and secreted mucus. FLIM microviscosity measurements were validated in calibration experiments with sucrose (low microviscosity range), glycerol (high microviscosity range), agarose (gel‐like structure) and reconstituted mucus of isolated bovine mucin proteins in solution. Human bronchial epithelial (HBE) cells, isolated from tissues of 4 normal and 5 CF donors, were seeded on filters and cultured at the air‐liquid interface.

FLIM measurements revealed that median values of microviscosity for normal granules are 41±7 cP (nfilters=30) and 49±11 cP (nfilters=31) for CF granules (p<0.001, Mann–Whitney U test). Next, we classified mucin granules in three categories based on their microviscosity: low (1–30 cP), medium (30–60 cP) and high (>60 cP). The quantity of granules with medium microviscosity does not differ between normal (74%) and CF (77%) donors. However, normal phenotype was characterized by the presence of a larger fraction of low microviscosity mucin granules (19%) compared to CF (4%), while CF phenotype displayed a larger proportion of high microviscosity granules (19%) compared to normal (7%). Raising the pH inside granules by blocking H+‐ATPase with 10 nM bafilomycin lowered microviscosity values of CF granules towards normal phenotype. Microviscosity of secreted mucus covering HBE cells was measured in situ and reached values of 147±54 cP (nfilters=7) for normal donors and 198±75 cP (nfilters=7) for CF donors.

Our study demonstrates the presence of a subpopulation of mucin granules with higher microviscosity in CF that is less pronounced in normal patients. FLIM microviscosity measurements of secreted mucus show much higher values than expected for a water‐filled interfiber spaces in a gel. This may indicate that mucus covering HBE cells resembles an amorphous colloid rather than a well‐structured hydrogel. Relative differences in mucus microviscosity between normal and CF, however, are much smaller than those reported for a bulk viscosity of mucus collected from patients.

Support or Funding Information

Supported by the Cystic Fibrosis Canada (R.G.) and CIHR PJT148593 (E.B.).
The Horse & Rider: A Dynamic 3D Model for Studying the Role of Chronic Postural Asymmetries in Degenerative Osteoarthritis
Authors: Lee Alexander K. K. , Uhl Elizabeth W. , Osborn Michelle L.
Published: April 2020

Chronic lamenesses in horses are often managed but not effectively treated. We hypothesize that (1) a causal relationship exists between the forces created by aberrant biomechanical positions and movements, including those caused by the rider, and degenerative joint diseases; and (2) adjusting the aberrant biomechanics will be therapeutic. To test our hypotheses, we constructed a dynamic 3D computer model using equine CT data and human CT data to model full body positions of horses with riders that can then be related to sites of tissue damage in equine lamenesses. The bones of the horse and human were reconstructed from CT data using the 3D visualization and analysis software, Avizo®. The individual bones were then imported into Autodesk® Maya® for re‐positioning into their respective organism, creating a 3D human model and a 3D equine model. A 3D rig was developed for each model separately to allow for accurate positioning and animation for each. The ribbon spine technique was used for the neck and spine on both models for increased accuracy in positioning and movement with the ability to rotate and bend the spines and necks into natural positions. The rigging used for both the horse and human were adopted to be manipulated using controls for quicker and easier positioning. Once the two rigs were completed, they were imported into a single file. The human rig and its matching skeleton were scaled in reference to the equine rig and its matching skeleton; and the human rigged model was positioned atop the rigged equine model to simulate a rider on its horse. The combined model was used to analyze the chronic postural abnormalities of a horse with cervical osteoarthritis using information from photographs of the horse being ridden at the trot. Findings included a markedly asymmetric stride, a dorsiflexed lower limb that indicates overloading of the front legs, and over‐flexion of the neck that correlated with sites of compressive degeneration in the cervical spine. Our long term goal is to expand the models for biomechanical analysis and to further develop the rigging protocol in order to expand its usefulness for both animal and human studies.

Support or Funding Information

Louisiana State University School of Veterinary Medicine Charles V. Cusimano Equine Health Studies Program
Bioprospecting for Novel Natural Products in Ancient Non‐Actinobacteria
Authors: Hamrick Grayson S. , Hoang June , Kokona Bashkim , Charkoudian Louise K.
Published: April 2020

Polyketides are a particularly important category of secondary metabolite, and alone comprise 20% of the top‐selling drugs around the world.
1 Polyaromatic (type II) polyketides incorporate aromatic rings into their structures and are synthesized in vivo by a cohort of enzymes called a synthase. Three enzymes remain constant in all type II polyketide synthases: ketosynthase (KS), chain length factor (CLF), and acyl carrier protein (ACP). These three enzymes are known as the minimal polyketide synthase (PKS) and form the basis for the production of all type II polyketides. The most well‐studied of these polyketides are produced by a phylum known as Actinobacteria. Some of the polyketides that these bacteria produce include tetracycline, erythromycin, and lovastatin. Unfortunately, Actinobacteria are difficult to work with in the lab and researchers have historically been unsuccessful in expressing their KS‐CLF complexes in robust heterologous hosts such as E. coli. In 2015, a study by the Charkoudian and Hillenmeyer labs showed that the gene clusters responsible for the transcription and translation of the minimal PKS are present in some anciently diverged strains of non‐Actinobacteria.
2 These strains are known as orphan strains; the sequences of the genes are known but the enzymes and the product they manufacture remain uncharacterized. Many of the uncharacterized products include those made by non‐Actinobacterial species. Additionally, many of the identified non‐Actinobacterial species originate in phyla such as the Firmicutes and Proteobacteria, which means that their gene products could be expressed in tractable, non‐Actinobacterial heterologous hosts, such as S. pyogenes (a Firmicute) or E. coli (a Proteobacteria). Thus, non‐Actinobacterial ancient strains seemed ripe for bioprospecting.

One of the first non‐Actinobacterial species that this research considered was the strain Gloeocapsa sp. PCC 7428, a cyanobacteria whose genome was found to contain a putative PKS gene cluster in the original 2015 study. Herein we show that the Gloeocapsa KS‐CLF can be expressed and purified in E. coli in high titers. Further characterization has shown this KS‐CLF to have several valuable qualities. The successful expression of such a protein may open doors to solutions to the antibiotic resistance problem that were previously closed.

Support or Funding Information

1. This work was supported by the Arnold & Mabel Beckman Foundation Scholar Award to G.S.H. and NSF CAREER grant number CHE‐1652424 to L.K.C.
Characterization of an Acyl‐CoA:retinol Acyltransferase (ARAT) Activity in the Hepatopancreas of the American Lobster (Homarus americanus)
Authors: Dew Stephanie E. , Wells Collin K. , Honaker Olivia M. , Li Yuzhang
Published: April 2020

Vitamin A (retinol) is a necessary nutrient in higher animals. In mammals, the pathways by which retinol is transported and metabolized are well described. One important reaction in these pathways is the conversion of retinol into retinyl esters, which occurs in several tissues, including the small intestine, liver, and eye. Little is known about the conversion of retinol into retinyl esters in invertebrates. We have identified an acyl‐CoA:retinol acyltransferase (ARAT) activity in the hepatopancreas of the American lobster (Homarus americanus). This activity shows a linear dependence on time and microsome concentration. It shows temperature dependence and an optimal pH of 6.5. We have characterized its acyl‐CoA preference and determined Km and Vmax values for both retinol and acyl‐CoA.

Support or Funding Information

Centre College