bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Propensity in the C‐terminal Domain of the Albino3 Translocase in Thylakoids
Authors: Furr M. , Okoto P. , Baucom D. , Kumar V. , Moradi M. , Heyes C. , Henry R. L. , Kumar T. K. S. 
Published: April 2020

The c terminus of the Albino3 (Alb3) translocase in chloroplasts is a region fundamental to the integration of LHCPs into the thylakoid membrane in cooperation with the cpSRP. Alb3 is an integral membrane protein containing five transmembrane helices in an N‐in, C‐out orientation in the thylakoid membrane. The c terminus of Alb3 is responsible for recruiting the cpSRP43 subunit of cpSRP to the thylakoid membrane for successful integration of LHCPs. This region of Alb3 has previously been described as intrinsically disordered. In more recent developments, intrinsically disordered proteins have been shown to carry out vital functions within cells. While our findings show that cAlb is predominately disordered, we have discovered a region in this protein which has a high propensity towards an ordered structure. This region was first isolated by secondary structural sequence analysis using multiple prediction software. Single point mutations which would contribute to the displacement of structure were made within this region, followed by structural characteristic analysis. Results derived from Far‐UV‐Circular Dichroism, intrinsic fluorescence, and trypsin digestion reveal that the mutations show a decrease in overall structure. Inter‐residue distance and energetics were acquired from smFRET analysis of this region, in its native state and upon denaturation. Differences in smFRET histograms show the region is able to be unfolded with increasing denaturant. Additionally, computational methods show a structural propensity in this region and agree with the smFRET results. These combined biophysical and computational results reveal a region of structure in the N‐terminal of cAlb. The local structure found in this study may prove to be important to the binding event with the cpSRP43 subunit of cpSRPSupport or Funding InformationDepartment of Energy (DOE)
N‐Terminal Variation in Iron Dependent Regulators
Authors: Myers Zachary , Wilson Christopher , Novak Walter R.P. 
Published: April 2020

Iron plays an integral role in a variety of physiological processes, and many pathogenic bacteria rely on iron‐dependent regulators (IDeRs) to sense cellular iron levels and regulate the transcription of iron storage and scavenging genes. The N‐terminal alpha‐helices of IDeRs are thought to play a significant role in dimerization and DNA‐binding. Accordingly, the length of the N‐termini in IDeR proteins varies little across a wide variety of species. Intriguingly, Actinomyces coleocanis possesses two IDeR open reading frames; one is the canonical length (“short”), while the second encodes an additional 39 amino acids at the N‐terminus (“long”). Here we present the expression, purification, and initial DNA‐binding studies of the short and long IDeR forms from the gram‐positive bacteria Actinomyces coleocanis.Support or Funding InformationThis work was supported by the Haines Biochemistry Fund and the John Beeson Student Research in Chemistry Fund.
The Role of Scarpa’s Fascia in the Human Body Wall
Authors: Hall Margaret I. , Plochocki Jeffrey H. , Suarez-Venot Ana , Rodriguez-Sosa Jose R. , Voegele Gina M. 
Published: April 2020

Humans have lost a cutaneous muscle layer called the panniculus carnosus that is found in a large survey of mammals and many non‐human primates. Humans have a tough fascial layer called Scarpa’s fascia in the same position as panniculus carnosus, which some researchers suggest is the remnant of this cutaneous muscle layer. In other mammals, panniculus carnosus shakes the skin, possibly to dislodge ectoparasites. However, in humans, it is possible that a tough fascia is more adaptive for passively maintaining the human orthograde posture than an isotonic muscle layer, and the loss of fur on the skin may require less movement to combat parasites. Interestingly, panniculus carnosus is generally associated with a fatty fascial layer termed panniculus adiposus. Traditionally, Camper’s fascia, a fatty layer just deep to the skin and superficial to Scarpa’s fascia has been assumed to be the representative of panniculus adiposus in human. However, our dissections demonstrate an undescribed fatty layer deep to Scarpa’s fascia. Based on comparative anatomy of the anterior abdominal wall across several groups of mammals, and the continuity of this fatty layer into the human perineum from the abdomen, we suggest that this undescribed deeper layer may be the evolutionarily conserved panniculus adiposus and not Camper’s fascia.Support or Funding InformationFunded by Midwestern University, University of Central Florida, and the American Association for Anatomy
Two Decades Longitudinal Study of VO□ Max and Physical Fitness in a College Population
Authors: Spehlmann Eric , Wright Brian , Ball Thomas , Babington James , Lafontant Pascal J. 
Published: April 2020

Data from the Centers for Disease Control and Prevention show that 53% of adults age 18 and older met the Physical Activity Guidelines of the Health and Human Services. Previously published research has established a decreasing level of physical fitness in college students. While VO□ max is an accurate predictor of aerobic fitness, due to the relative difficulty of performing the VO□ max, the data collected on student physical activity is often comprised of indirect measurements of fitness including BMI, self‐reported activity level, and estimates of aerobic fitness using VO□ max estimations. We hypothesize that fitness of college students reflects that of general population. Therefore, a comprehensive database was developed that comprised of VO□ max values of college students in a small Midwestern liberal arts institution to assess the physical fitness level of college students. VO□ max were measured in over 500 student‐athletes and non‐athletes over two decades. In addition, other measures of fitness such BM, maximum heart rate were collected. For our studies, we extracted data for analysis using a Python script. Our preliminary observations on a small sample subset suggest that fitness level remained constant over the two decades contrary to general population trends. Further analysis accounting for athletes versus non athlete may explain the apparent steadiness of fitness level. Our study suggests that VO□ Max of college students can provide insight into longitudinal changes in physical fitness.Support or Funding InformationDePauw University
Targeting the Prokaryotic‐Specific GTP Cyclohydrolase‐IB with Guanosine Analogs Toward a New Class of Antibiotics
Authors: Samaan Geroge N. , Paranagama Naduni , Haque Ayesha , Hecht David A. , Purse Byron W. , Swairjo Manal A. 
Published: April 2020

GTP cyclohydrolase I (GCYH‐I) catalyzes the first step in the de novo biosynthesis pathway to folic acid in bacteria, biopterin synthesis in mammals, and the biosynthesis of 7‐deazaguanine modified nucleosides of tRNA in bacteria and archaea. The type IB GTP cyclohydrolase (GCYH‐IB) is a prokaryotic‐specific, essential enzyme found in several pathogens, including N. gonorrhoeae, and is structurally distinct from its human homologue GCYH‐IA involved in biopterin biosynthesis and thus has been proposed as a novel antibacterial drug target. Comparison of the crystal structures of the two enzymes bound to the substrate analog inhibitor 8‐oxo‐GTP shows that the active site of GCYH‐IB is larger and imposes a different conformation of the inhibitor. Based on this structural information, we designed, synthesized and tested a small set of 8‐oxo‐G derivatives with ether linkages expected to occupy water‐filled pockets present only in the active site of GCYH‐IB. The half maximal inhibitory concentrations, measured using standard GCYH‐I activity assays, are indicative of modest potency of these compounds. However, the most potent of these compounds, G3, is selective for GCYH‐IB. In silico molecular modeling of G3 in the active sites of both enzymes supports the observed preferential inhibition of the N. gonorrhoeaea enzyme by G3 on the grounds that it is too large to be accommodated by the active site of human GCYH‐IA. These results support the premise that potent and selective inhibitors of GCYH‐IB could constitute a new class of small molecule antibiotics.Support or Funding InformationNIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).
Using a Modified Delphi Method in the Creation of an Anatomy Readiness Assessment
Authors: Roth Alex Robert , Notebaert Andrew , Dehon Erin , Robertson Ellen 
Published: April 2020

Human gross anatomy is a foundational course that is consistently covered in the first year of all major health professional education programs. Even though human gross anatomy is a major foundational course, it is not required by all health professional education programs. Most occupational therapy and physical therapy programs do require gross anatomy as a prerequisite course before admission, but often medical and dental schools do not. This leads to incoming students having a variety of exposure levels to the content of human gross anatomy, with many of the students having no exposure at all. It is easy to assess the amount of anatomy exposure that a student has had just by looking at their transcript, the major difference is the quality of the exposure that they have had.In order to assess the quality of anatomy exposure that students have had in the past we needed to create an assessment that covered the foundational information in anatomy. To create this assessment a survey was conducted asking anatomy course instructors what textbooks they used for the health professional education program that they teach. The four most commonly used textbooks were then selected as the major source of information that incoming health professional students will receive in their gross anatomy course. The introductory chapters of these books were reviewed as they contain foundational anatomy information that will be built upon throughout the rest of the human gross anatomy course. A master list of all the foundational structures, terms, and concepts was created from the first chapter of these textbooks and evaluated using a Modified Delphi method. This master list was sent to experts (health professional human gross anatomy course directors) from multiple institutions for validation. After two rounds of the Modified Delphi method any of these topics with a Content Validity Index above 80% agreement were then considered validated foundational information. This new validated list will then be used to create an Anatomy Readiness Assessment.The Anatomy Readiness Assessment is designed to be given to health professional students prior to starting their gross anatomy course. This will assess their understanding of foundational information for anatomy prior to starting the course and may be used to identify students at risk prior to the first examination.
Linking Energy Metabolism to Cell Fate Decision During Kidney Development
Authors: Tortelote Giovane G. , Hillard Sylvia A. , Colon-Leyva Mariel , Hughes Jordan B. , El-Dahr Samir S. , Saifudeen Zubaida 
Published: April 2020

ObjectiveMetabolic flux switching accompanies cell fate changes in several organisms and cell types. We recently showed that glycolysis inhibition drives differentiation of nephron progenitor cell (NPC) in embryonic (E) kidney in organ culture experiments. We utilized a transcriptomics approach to uncover renewal vs differentiation genes in pathways targeted by glycolysis vs Wnt‐mediated differentiation, as canonical Wnt signaling is an established inducer of nephrogenesis.MethodsBulk RNASeq was performed on cultured NPCs isolated from E13.5 kidneys after 24h treatment with either the glycolysis inhibitor YN1 (20uM) or GSK3b inhibitor CHIR (3uM) to activate Wnt signaling. NPCs were isolated from 3 separate litters and pooled per litter. Differential expression (DE) thresholds: p‐value<0.05, fold‐change 0.6.We have also carried out organ culture experiments on E12.5 kidneys; paired kidneys were fixed after 24h Pravastatin (10uM) or vehicle treatment for immunostaining to identify NPCs (Six2+) or nascent nephrons (Lhx1+).ResultsCompared to renewing NPCs, approximately 550/15,300 genes were differentially expressed after CHIR or YN1 treatment, respectively. Pathway analysis revealed the common top over‐represented pathways as Cholesterol biosynthesis, Wnt and Calcium signaling. Fatty acid metabolism related pathways dominated in YN1‐treated NPCs and amino acid biosynthesis pathways in CHIR‐treated NPCs. Furthermore, to determine whether the inhibition of cholesterol biosynthesis is a requisite for nephrogenesis or a result of the differentiated state, E12.5 kidneys were treated with HMG‐CoA reductase inhibitor, Pravastatin, resulting in a significant increase in nephrogenesis marker staining (Lhx1+) and decrease in the self‐renewing progenitor pool (Six2+).ConclusionsOur data suggest glycolysis is required to maintain the cholesterol biosynthesis pathway and NPC self‐renewal and that the inhibition of either pathway directs the NPC toward differentiation.Support or Funding InformationThis work was supported by the National Institutes of Health: NIH/NIDDK R01 DK118231
The deubiquitinase USP40 promotes microvascular endothelial cell integrity and protects against lung injury
Authors: Miao Jiaxing , Wei Jianxin , Taleb Sarah , Mallampalli Rama , Zhao Yutong , Zhao Jing 
Published: April 2020

Endothelial cell (EC) barrier disruption and inflammation are the pathological hallmarks for inflammatory diseases, such as acute lung injury. Ubiquitination is a post‐translational modification. Ubiquitin proteins covalently link to substrate proteins, thereby regulating protein stability and enzyme activity. Deubiquitinating enzymes (DUBs) reserve this process. USP40, a newly recognized DUB, has been shown to regulate glomerular permeability in zebrafish. Our goal is to understand the role of USP40 in microvascular EC integrity. NF‐κB pathway plays a central role in the upregulation of EC activity. V5‐tagged USP40 (USP40‐V5) overexpression attenuated lipopolysaccharide (LPS)‐induced phosphorylation of I‐κB in human lung microvascular endothelial cells (HLMVECs). TNFα treatment increased NF‐κBp65 nuclear translocation, while the effect was attenuated in USP40‐overexpressing cells. Overexpression of USP40 significantly attenuated LPS‐induced ICAM1 expression, while depletion of USP40 promoted ICAM1 levels. RhoA‐mediated phosphorylation of myosin light chain (MLC) promotes EC barrier dysfunction. USP40‐V5 attenuated LPS‐induced activation of RhoA. Further, LPS treatment of HLMVECs increased phosphorylation of MLC, and it was reduced by USP40 overexpression. Overexpression of USP40 attenuated LPS‐reduced transendothelial electrical resistance (TEER), while knockdown of USP40 by USP40 siRNA transfection increased LPS‐induced EC permeability in a transwell leakage assay. Immunostaining with VE‐cadherin showed that USP40 siRNA promoted LPS‐induced gap formation in HLMVECs. We generated USP40 deficient (USP40−/−) mice by CRISPR/Cas9 system. USP40−/− mice showed an increase in ICAM1 in lung tissues and BAL protein levels in intratracheal (IT) LPS (2 mg/kg, 24 h)‐induced murine model of acute lung injury (ALI). To investigate if increases in USP40 levels protect against LPS‐induced ALI, lentiviral‐driven overexpression of USP40 in vessels of mice was performed. LPS increased Evans blue leakage, BAL protein levels, and neutrophil influx, which were significantly decreased in lenti‐USP40 delivered mice. Our observations are the first to reveal that USP40 reduces the severity of ALI through diminishing the activation of NF‐κB‐and RhoA‐mediated inflammation and EC permeability.Support or Funding InformationThis study is supported by NIH R01 grants
Integrating Inclusive Pedagogy into an undergraduate introductory Physiology course
Authors: Rhinehart Erin 
Published: April 2020

We are constantly bombarded with images about what type of person “does science”, and these images usually convey a very narrow view of science and scientists. In most science courses and science textbooks, the people highlighted for making most of the significant contributions are white males. Whether or not this lack of diverse representations of scientists registers consciously, the impact of this messaging about the types of people who work in science‐related careers is clear and consistent. Therefore, it is important that faculty recognize the impact of this messaging and act to combat it, especially in light of the increasingly diverse array of postsecondary students interested in STEM majors. One way to enhance student belonging in STEM is to provide assignments that allow students to recognize the contributions of non‐stereotypical scientists. To accomplish this, we added a biographical presentation assignment to an undergraduate Introduction to Physiology course with the objective of providing multiple of examples throughout the course of the semester that counter scientist stereotypes. For the assignment, students selected a scientist to research from a curated list that includes only women and minority physiologists. The assignment required that they write a paper and give an oral presentation to the class about the selected physiologist. The content of the presentation and the paper included a description of the scientist’s career trajectory and scientific contributions as well as information about their personal life and a discussion about which things the student found most relatable to their own life. To assess the impact of the assignment on student notions of scientist stereotypes and overall scientist relatability, we conducted a pre‐ and post‐course survey, which indicated a significant shift in student biases regarding scientists being predominantly white and male. In addition, a focus group assessment revealed themes indicating that the assignment also improved student’s ability to identify with people in science‐related careers. Therefore, this pedagogical intervention appears to be beneficial as a step toward creating a more inclusive STEM educational environment.Support or Funding InformationNSF CAREER 1350448
125‐I‐Sarcosine(1), D‐Alanine(8) Angiotensin II as a Biased Radioligand for AT1 Angiotensin II Receptors
Authors: Speth Robert Charles , Desai Nisha , Chabbra Alesa , Gnozzo Giovanna , Stoyell-Conti Filipe F. , West Crystal A. 
Published: April 2020

The concept of biased agonism of G protein‐coupled receptors arose from studies of the AT1 subtype of angiotensin (Ang) II receptor. A putative antagonist ligand, sarcosine1, isoleucine4, isoleucine8 Ang II (SII) showed negligible ability to activate G protein signaling pathways of the AT1 receptor, but was able to cause AT1 receptor internalization and mitogen activated protein (MAP) kinase activation at micromolar concentrations. Subsequently, a different Ang II congener; sarcosine1, D‐alanine8 Ang II was developed as a potential therapeutic (TRV‐027; TRV120027) for treatment of acute heart failure. While clinical trials did not support a therapeutic benefit of sarcosine1, D‐alanine8 Ang II for treatment of heart failure, it still promises to be a better biased agonist than SII, which acts only at micromolar concentrations. To assess the potential use of sarcosine1, D‐alanine8 Ang II as a radioligand for quantification of Ang II receptor binding, we radiolabeled sarcosine1, D‐alanine8 Ang II with 125iodine and compared its binding to that of 125I‐sarcosine1 isoleucine8 Ang II in rat liver membranes, which express high levels of AT1 receptors and negligible levels of AT2 receptors. At concentrations up to 5.6 nM, 125I‐sarcosine1, D‐alanine8 Ang II showed little evidence of saturable binding to AT1 receptors, in contrast to 125I‐sarcosine1 isoleucine8 Ang II, which showed a KD of 0.6 nM. At 5.6 nM 125I‐sarcosine1 isoleucine8 Ang II binding to AT1 receptors was 8.5 times greater than 125I‐sarcosine1, D‐alanine8 Ang II. To verify that sarcosine1, D‐alanine8 Ang II binds to liver membrane AT1 receptors, we compared the ability of sarcosine1, D‐alanine8 Ang II and Ang II to compete for 125I‐sarcosine1 isoleucine8 Ang II binding. Sarcosine1 isoleucine8 Ang II and Ang II were similar in their ability to compete for AT1 receptor binding: 6.7±0.7 nM and 15.6±0.8 nM and 84.3±1.1 and 86.7±0.3 % of total binding (IC50, n=2±SD), respectively. This suggests that iodination of tyr4 of sarcosine1, D‐alanine8 Ang II alters its ability to bind to AT1 receptors and potentially act as a biased agonist. Continuing studies will examine the ability of higher concentrations of 125/127I‐sarcosine1, D‐alanine8 Ang II to interact with Ang II receptors.Support or Funding InformationNova Southeastern University President’s Faculty Research Development Grant, Cardiovascular Neuroscience Research Fund
Cofactor Activity in Placental Microvilli for the Cleavage of Complement Component 3 by Factor I
Authors: Vanderpuye Oluseyi A. 
Published: April 2020

Human placental microvilli are in direct contact with maternal blood and the activity of complement components of the immune system must be regulated on this plasma membrane to prevent adverse effects to the fetus. A key point for regulation of the complement cascade is the proteolytic inactivation of complement component 3 which has become fixed on cell membranes by Factor I which requires a membrane bound cofactor. CD46 is the most widely distributed integral membrane protein cofactor for Factor I and is abundant in placental microvilli. An assay was developed for cofactor activity for Factor I that involved coupling of biotinylated methylamine‐ activated C3 (C3ma) to thiolpropyl agarose as a substrate. Microvilli from the surface of placental trophoblast cells are readily isolated by subcellular fractionation. The immobilized C3ma substrate was incubated with Triton X‐100 solubilized microvilli or other potential cofactor sources in the presence of Factor I. Cleavage of the C3ma was detected by SDS gel electrophoresis followed by electrophoretic transfer to nitrocellulose, blotting with alkaline phosphatase‐conjugated avidin and detection with an alkaline phosphatase substrate. It was found that cofactor activity was detectable in Triton X‐100 solubilized placental microvilli and platelets. However, a soluble form of CD46 that was not sedimented by centrifugation at 100,000g was associated with microvilli and also had co‐factor activity. In addition it was found that either CD46 or Factor I could first be bound to the immobilized C3ma and that cleavage could then be initiated upon addition of the missing component. The cofactor activity in placental microvilli could be removed by immunoabsorption with polyclonal or monoclonal antibodies to CD46 suggesting that this was the main source of cofactor activity for intrinsic C3 inactivation on placental microvilli.In conclusion, CD46 was found to be the major if not only cofactor on placental microvilli for Factor I inactivation and existed in membrane bound and soluble forms associated with microvilli. Either of Factor I or its cofactor CD46 can bind to C3ma and the missing component can be added to form an active ternary complex.