bluuebunny/crossref_metadata_2025 huggingface dataset:
Radil is a KRas‐interacting protein involved in regulating cell adhesion and migration
Authors: Choi Byeong Hyeok , Kou Ziyue , Colon Tania , Lee Eunji , Dai Wei
Published: April 2020

Ras proteins regulate a variety of cellular processes including cell survival, proliferation, and differentiation. Ras genes are among the most frequently mutated oncogenes in human malignancies. To date, there are no successful anti‐cancer drugs in the clinic that target Ras proteins or their pathways. Therefore, it is imperative to identify and characterize new components that regulate Ras activities or mediate their downstream signaling pathways. Recently, we have identified a series of proteins that are associated with KRas using a combination of immunoprecipitation and mass spectrometry. We have initially focused on Radil, a gene product with Ras‐association (RA) and DIL domains. Radil is known to be a downstream effector of RAP1, inhibiting RhoA signaling in the regulation of cell survival, adhesion, and migration. We have demonstrated that Radil interacted with all three isoforms of Ras including HRas, NRas, and KRas although it exhibits the strongest interaction with the latter. Radil knockdown does not seem to affect MEK phosphorylation induced by KRasV12, suggesting that it may lie upstream of KRas regulatory pathway. Intriguingly, ectopic expression of Radil only transiently activates MEK and ERK whereas high levels of Radil are correlated with downregulation of KRas downstream components, suggesting a biphasic regulation of KRas signaling. Moreover, Radil knockdown greatly reduced the number of adhesion foci and depolymerized actin filaments, which was associated with reduced FAK activity, deceased cell proliferation, and downregulation of Vimentin and Snail (two mesenchymal cell markers). Taken together, our current studies strongly suggest that Radil may be important for regulating Ras signaling and the epithelial‐mesenchymal transition.
Perception of overall health, weight status and gaining weight in relationship with BMI among high school students
Authors: Khanna Deepesh , Kahar Payal
Published: April 2020

Introduction

Correct perception of weight status and gaining weight are important motivational factors for physical activity among overweight and obese children. However, misperception is common.

Methods

A face‐to‐face validated survey based study was conducted among high school students. Body Mass Index (BMI) was calculated based on the self‐reported height and weight to compare with perceived weight status based on a question: “Do you consider yourself to be underweight, overweight, about right or obese”? Participants were asked to rate their health and how much they worry about gaining weight. Descriptive and Chi‐Square tests were used for analysis. Level of significance was 0.05.

Results

Males who reported normal BMI, 23.1% considered themselves underweight and 11.5% considered overweight (c2 = 20.305, P=0.016). Females who reported normal BMI, 6.9% considered themselves underweight and 17.2% considered overweight (c2 = 31.613, p=0.000). Males who considered themselves overweight, 40% think they are not obese and 35% think they are obese (c2 = 30.322, p=0.000). Females who considered themselves overweight, 55.6% think they are not obese and 29.6% think they are obese (c2 = 11.415, p=0.076). Only 6% of students in the overweight BMI range, reported their overall health is poor (c2 = 21.266, P=0.047). In the BMI range of overweight, 40% of females and 6.3% of males worry all the time of gaining weight (c2 = 13.470, p=0.336).

Conclusion

Female students being more likely to perceive themselves overweight and worried about gaining weight. A low percentage of male and female students rate their overall health poor.
Neonatal Inflammation Sex‐dependently Impairs Adult, Spinal Microglia
Authors: Beyeler Sarah A. , Plunkett Deanna L. , Huxtable Adrianne G.
Published: April 2020

Neonatal inflammation is common and disrupts adult respiratory control, yet the mechanism(s) underlying the impairments are not well understood. Since microglia are key mediators of central inflammation and are important in the lasting effects of neonatal inflammation in non‐respiratory brain regions, we hypothesized lasting dysfunction in microglia underlie impairments in adult respiratory control. Specifically, we hypothesized neonatal inflammation would increase the number of and inflammatory gene expression in adult microglia. Since adult respiratory motor plasticity in the cervical spinal cord is impaired after neonatal inflammation, we analyzed immunomagnetically isolated microglia from the ventral cervical spinal cord. Interestingly, the number of microglia measured by flow cytometry calibrated with counting beads from adults after neonatal inflammation (LPS; 1 mg/kg, i.p.; postnatal day 4) were unchanged in males (LPS: 14,488 ± 4000, n=4; saline: 12,784 ± 4000, n=4, p=0.97) or females (LPS: 14,611 ± 4500, n=4; saline: 9,062 ± 4500, n=5, p=0.79). Additionally, the number of microglia was not significantly different between males and females (p>0.05). Despite no changes in microglia number, we hypothesized microglia function was impaired by neonatal inflammation. Preliminary transcriptomic analysis suggests neonatal inflammation in adult females (n=2) up‐regulated 7 genes and down‐regulated 1 gene, and in adult males (n=3) down‐regulated 1 gene. Gene ontology revealed these differentially expressed genes are associated with inflammatory signaling. While neonatal inflammation has no impact on the number of microglia, it sex‐dependently increases inflammatory‐related gene expression in females with surprisingly few changes in adult males. Thus, adult, female microglia likely contribute to the lasting impairments in respiratory control following neonatal inflammation. Further, the lasting impairments in adult males are likely mediated by a sex‐specific distinct mechanism. Understanding the sex‐specific inflammatory mechanisms impairing respiratory control is crucial to developing treatment strategies to promote breathing during ventilatory control disorders.

Support or Funding Information

Supported by NIH HL141249 (AGH)
Solution Structural Behavior of UBAN Domains Revealed by Coarse‐Grained Computer Simulations
Authors: Wilderman Ross
Published: April 2020

Proteins containing a Ubiquitin‐binding domain in ABIN and NEMO (UBAN) have a broad range of biological roles. Much of the control of ubiquitin recognition by individual UBAN‐domain containing proteins is based on protein structure. Ubiquitin recognition is further modulated by post‐translational modifications. X‐ray crystal structures of UBAN domains display coiled‐coil dimers in the absence or presence of M1‐linked ubiquitin. However, the solution structural behavior of these UBAN domains is not well understood. Extensive coarse‐grained molecular dynamics simulations of UBAN domains from NEMO, optineurin (OPTN), CEP55, and ABIN1 using a predictive energy landscape based model reveal unique signatures of solution structural conformational heterogeneity in NEMO and ABIN1 but a much more homogeneous distribution of highly extended OPTN conformations. Simulating phosphorylation of OPTN S473, a known post‐translational modification that increases M1‐linked ubiquitin affinity for OPTN, greatly increases OPTN conformational heterogeneity. Furthermore, phosphorylation of ABIN1 S442, a likely post‐translational modification in vivo, shifts the ABIN1 conformational distribution. These results indicate the conformational distribution of UBAN domains provides a means to tailor cellular response in specific biological roles, and post‐translational modifications in combination with conformational heterogeneity provide a rheostat‐like means to control cellular signaling pathways.

Support or Funding Information

This work was supported by internal funding from the University of Connecticut to James Halpert.
Fibularis Longus with a Brevis Slip
Authors: Hurley Mary
Published: April 2020

Fibularis longus is the superficial muscle of the lateral compartment, overlying the fibularis brevis muscle. Fibularis tertius is a muscle of the anterior compartment.

The left leg of one of the cadavers of this semester’s gross anatomy course was discovered to have a slip on the anterior side of the fibularis longus muscle break off the muscle and attach to the tuberosity of metatarsal V immediately superior to the tendon of fibularis brevis. No other cadaver in this group was seen to have this version of the muscles.
Development of RFLP and qPCR Assays to Detect the Human C/T‐13910 SNP, which Is Associated with Lactase Persistence
Authors: Carpenter Susanne M. , Weisblatt Alyssa N. , Dahlquist Kam D.
Published: April 2020

Human infants, like those of other mammals, express the lactase enzyme to digest the lactose sugar in mother’s milk. After weaning, the lactase‐phlorizin hydrolase gene (LCT) ceases to produce lactase, resulting in the lactase non‐persistence (LNP) phenotype. However, in 35% of the world’s population of humans, expression of the lactase enzyme continues into adulthood, known as lactase persistence (LP). The LP phenotype occurs when an individual has one of more than 20 single nucleotide polymorphisms (SNPs) located near the lactase gene. The most commonly studied SNP is the “European” SNP, C/T‐13910, which occurs in an enhancer region upstream of the LCT gene in intron 13 of the neighboring MCM6 gene. The T allele correlates with LP, as it results in a higher expression of lactase due to increased binding of the Oct‐1 regulatory transcription factor and/or prevention of gene silencing. Several studies have been conducted in non‐European, isolated populations to determine the frequency of LP within the ethnic group. However, fewer studies have been conducted in ethnically diverse locations such as the US. To lay the groundwork for such a study, we have successfully implemented a restriction fragment length polymorphism (RFLP) assay reported in Morales et al. (2011, doi:10.1136/bmjopen‐2011‐000125) to detect the C/T‐13910 SNP in human cheek cell lysate, confirming the results by direct sequencing of the DNA. We used the TOPO cloning technique to generate control plasmids containing inserts with either the “C” or “T” genotype to use as positive controls in future assays. We confirmed that the sequence of the control plasmid inserts match the original target sequence. We are using the control plasmids to optimize a SYBR green quantitative polymerase chain reaction (qPCR) genotyping assay based on Weinlander et al. (2010, doi:10.1002/bmb.20357) as an alternate method of genotyping. Finally, we have curated a list of SNPs from the literature and public databases that are known to affect the LP phenotype in different world populations.

Support or Funding Information

Loyola Marymount University Summer Undergraduate Research Program (S.M.C., A.N.W.), Loyola Marymount University Honors Summer Research Fellowship (S.M.C.)
Call for Educational Consistency: The Need to Establish Gross Anatomy Learning Objectives for the Entry Level Physical Therapist
Authors: Carroll Melissa A. , Tracy-Bee Mary , McKenzie Alison
Published: April 2020

Background and Objectives

Gross anatomy guidelines and recommendations are available for pre‐clinical medical faculty, however there is no consensus regarding anatomy student learning objectives for physical therapist education (PTE) programs. The Commission on Accreditation in Physical Therapy Education (CAPTE) requirements for gross anatomic content are vague and leave room for misinterpretation. This results in varied anatomical curricular content among PTE programs at different institutions. Faculty, whether seasoned or new, can benefit from guidelines and recommendations when making decisions during curricular reform or when prioritizing and emphasizing anatomic content for the curriculum. As such, developing recommended anatomy objectives to serve as a curricular guide may foster more consistent student outcomes, while preserving instructional autonomy.

Position and Rationale

Anatomy faculty in PTE, who may or may not be physical therapists, would benefit from evidence‐based, objective, and consensus‐driven curricular recommendations to ensure that the most appropriate and clinically relevant anatomy is taught. Anatomy course content is often chosen as the material core faculty (which may or may not include anatomists) deem most important. Importance may be determined due to prior training, personal experience, and pedagogical views of anatomy content. Since all students must pass the same licensure exam, determining the key anatomy content for PTE will promote consistency and reduce ambiguity among faculty. Recommendations must also allow for instructional and programmatic autonomy.

Method

Similar to the approach used to create anatomy learning objectives published by the Education Affairs Committee of AAA for pre‐clinical medical students, we suggest that anatomic content be identified, collaboratively vetted by basic science faculty and clinicians, affirmed, and recommended for adoption. Involvement of members in key professional organizations, such as the Anatomy Education Special Interest Group of the American Physical Therapy’s Academy of Physical Therapist Education, the American Association for Anatomy, and the American Association of Clinical Anatomists is paramount.

Discussion and Conclusion

We are advocating for collaboration amongst anatomy faculty in PTE to create specific and measurable anatomy learning objectives, guidelines, and recommendations that are peer‐reviewed and evaluated by both educators and clinicians, and that reflect a global consensus.
Pre‐Steady State Kinetic Analysis of DnaE Polymerase Illuminates the Limited (But Essential) Role It Performs in Gram‐Positive DNA Replication
Authors: Fagan Sean , Pata Janice
Published: April 2020

PolC and DnaE are the essential C‐family replicative polymerases in the Gram‐positive pathogen Staphylococcus aureus. Both PolC and DnaE have fast catalytic rates on DNA (PolC kpol, 500–1000 s−1; DnaE kpol, 300–400 s−1) however, we know PolC performs the bulk of genome replication. PolC is unable to initiate synthesis from RNA primers and relies on DnaE to perform this role before taking over replication. What remains to be determined is where and how this polymerase switching occurs. Using rapid quench‐flow we have performed detailed pre‐steady state kinetic analysis of DnaE on both a DNA‐ and RNA‐primed substrate. We have determined key kinetic properties that explain why DnaE has been confined to its role of replication initiation. DnaE shows a comparable affinity for both DNA‐ and RNA‐primed templates (Kd: 7 nM vs. 18 nM), but DnaE falls off an RNA‐primed template 50‐fold faster than a DNA‐primed template (koff: 5.34 s−1 vs. 0.11 s−1). Additionally, when incorporating a nucleotide on an RNA‐primed template, DnaE demonstrates a slower catalytic rate (kpol: 120 s−1), reversible chemistry, and a rate‐limiting pyrophosphate release rate (kPPi: ~20 s−1), which is not observed on a DNA‐primed template. These critical kinetic characteristics explain the limited role DnaE plays in genome replication. When extending from an RNA‐primed substrate DnaE is slow and falls off the substrate rapidly after adding a few nucleotides. This provides a critical juncture for PolC to compete for binding and finish replicating the template.

Support or Funding Information

National Institute of General Medical Sciences

Project Number: 5R01GM080573‐09
Essential Anatomy for General Pediatrics in the Undergraduate Medical Curriculum
Authors: Cotter Meghan , Lopez Lisa , Carpenetti Tiffany , Downs Mary Beth , Keim Sarah , Hankin Mark , Marzban Hassan , Kirera Francis , Topping Daniel , Mork Amy , Wineski Lawrence , Brooks William , Latacha Kimberly , Zumwalt Ann , Harrell Kelly , Sanky Charles , Laitman Jeffrey , Reidenberg Joy S. , Lewis Steven , Farias Anna , Granite Guinevere , McWhorter David , Royer Danielle , Martindale Jim , Harmon Derek
Published: April 2020

Introduction

To prepare medical students for clinical training and practice, it is critical to understand the anatomical knowledge considered most important for different clinical specialties.

Aim

To address this issue, a consortium of anatomists in the US and Canada is collecting data from clinical educators in Pediatrics clerkships and electives to identify the anatomy they consider essential.

Methods

An IRB‐approved, online survey (Qualtrics, Seattle, WA) was used to assess the importance of 98 anatomical topics in seven body regions. The study first examined the percentage of Pediatrics clinical educators (clerkship/elective directors and attending physicians) that considered each anatomical region important to their specialty. Second, the study examined the rank assigned to each anatomical topic using an ordinal scale from 1 (not important) to 4 (essential).

Results

At the time of abstract submission, data had been collected from 66 Pediatrics clinical educators at 20 medical schools. The percentage of Pediatrics clinical educators that considered each anatomical region important to their specialty were (highest‐to‐lowest): Abdomen (100%), Head & Neck (98%), Lower Limb (96%), Thorax (96%), Pelvis & Perineum (94%), Upper Limb (93%), and Back (83%). Further data analysis has identified the highest ranked anatomical topics in each body region for the Pediatrics clerkship/elective.

Discussion and Conclusion

This database provides detailed information regarding the most clinically relevant anatomical topics as identified by Pediatrics clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.
Anti‐inflammatory Effect of Hydrolyzed Jujube Ethanolic Extract
Authors: Kim Yoonsu , Oh Jisun , Kim Jong-Sang
Published: April 2020

Air pollutants, commonly derived from combustion reaction, are correlated with systematic inflammation, causing bronchial and asthmatic disorders. Therefore, it is very important to develop the ingredients which protect people from pulmonary damage caused by air pollutants. This study aimed at examining the anti‐inflammatory activity of Zizyphus jujube extract. Jujube fruit harvested from Gyeongsan region, Republic of Korea was ground, heated, and extracted in aqueous ethanol with or without enzymatic hydrolysis using Viscozyme® L (Novozyme, Bagsvaerd, Denmark). The jujube ethanolic extract prepared after treatment with heat and the enzyme (HJE) was found to effectively scavenge radicals as well as contain the considerable levels of total phenolics and flavonoids compared to non‐hydrolyzed extract (NHJE). In vitro studies using various types of cell lines demonstrated that HJE (1) decreased nitric oxide (NO) production from lipopolysaccharide (LPS)‐treated Raw 264.7 murine macrophages in a concentration‐dependent manner, (2) lowered secretions of pro‐inflammatory cytokines including tumor necrosis factor α (TNFα), interleukin‐6 (IL‐6) and interleukin‐1β (IL‐1β) from O‐tetradecanoylphorbol‐13‐acetate (TPA)‐treated THP‐1 human monocytes, and (3) reduced expressions of inflammation‐associated proteins, such as cytoplasmic COX‐2, iNOS, and nuclear NF‐κB in A549 human lung epithelial cells. To further examine the anti‐inflammatory effect of dietary HJE in animal, C57BL/6J mice were orally fed HJE for 2 weeks, followed by induction of lung inflammation by i.p. administration of benzo(a)pyrene (50 mg/kg B.W). A total of forty eight C57BL/6J mice (6‐week old male, 20–25 g) were divided into 6 groups (n = 8) as follows: (1) control, (2) B(a)P alone, (3) B(a)P+NHJE‐L (0.75 g/Kg B.W), (4) B(a)P+NHJE‐H (1.5 g/Kg B.W), (5) B(a)P+HJE‐L (0.75 g/Kg B.W), and (6) B(a)P+HJE‐H (1.5 g/kg B.W). Oral supplementation of HJE notably diminished the B(a)P‐induced elevation of pro‐inflammatory cytokine levels in the plasma and attenuated B(a)P‐induced pulmonary inflammation as evaluated by histological observation. These findings suggest that HJE can protect the lung epithelial cells from B(a)P‐induced injury presumably through attenuating macrophage‐associated inflammatory response and could be developed into the agent that suppresses air pollutant‐induced lung inflammation.

Support or Funding Information

This study was funded by the Forest Resources Development Institute of Gyeongsangbuk‐do, and the National Research Foundation of Korea grant (Grant No. 2017R1A2B4005087) by the Ministry of Science and ICT, Republic of Korea.
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 cpSRP

Support or Funding Information

Department of Energy (DOE)
Breaking the Barrier Between Science and Community
Authors: Karns Taylor J. , Rohlman Christopher E. , Streu Craig N.
Published: April 2020

The Albion College Biochemistry Club, a student chapter of ASBMB, was created in the fall of 2016 to create a community for like‐minded students and integrate the Albion community with Albion College. This follows the Albion‐Marshall School District merger in 2014 along with the increased requirement of K‐12 science education in the State of Michigan. The need for science exposure and cross‐community interaction between the towns of Marshall and Albion lent itself to an opportunity for the students at Albion College. With many of our current students at Albion College experiencing the merger firsthand, we understood the potential of our impact upon younger generations that had not previously had meaningful interactions with local college students. The Albion College Biochemistry Club has therefore been designing and executing programming to generate excitement for science in younger generations within the local school district. In a short amount of time, the Biochemistry Club has participated in judging local science fairs, providing demonstrations across varying ages, and supporting local vendors, while instilling a sense of greater purpose within college students. Focusing first on sharing science in the classroom has encouraged our students to explain science in a way that people of all ages can understand. The success of this programing from the Albion College Biochemistry Club has resulted in an influx of member attendance and a feeling of excitement to learn about science from the classrooms we visit. The work completed by the Albion College Biochemistry Club has been monumental in bridging science through both the college student and the community of youth, but there is always more work to be done.
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 Information

This 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 Information

Funded 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 Information

DePauw 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 Information

NIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).
The role of mobility in flavivirus protease substrate selection
Authors: Hardy Jeanne
Published: April 2020

Flaviviruses, including Zika virus and dengue virus, continue to threaten public health. The associated viral proteases, known as NS2B‐NS3pro, are essential to the viral life cycles and can interfere with host responses, making the proteases drug targets of considerable interest. Although the viruses are highly homologous, Zika virus is capable of severely damaging developing fetuses as well as causing neurological abnormalities in adults whereas dengue causes a sometime fatal hemorrhagic fever. While the proteases are highly identical, the catalytic efficiency and the substrates selection between Zika and dengue proteases are highly divergent, leading us to hypothesize that unique properties in the viral proteases may contribute the disparate viral pathologies. This work has made significant progress towards uncovering the role of conformation and flexibility in NS2B‐NS3pro from the function of these two flaviviruses on multiple fronts. First, NS2B‐NS3 proteases transitions between two widely different conformational states: an ‘open’ (inactive) conformation and a ‘closed’ (active) conformation. We developed versions of NS2B‐NS3pro that allow us to trap the enzyme in distinct conformations. Our data suggests that the enzymatic activity is dependent not only on the movement of NS2B, but may also rely on the flexibility of the protease core. Locking the enzyme into the ‘closed’ conformation dramatically increased activity. Second, to elucidate how Zika virus protease affects host cellular pathways and consequent pathologies, we used unbiased N‐terminomics to identify 31 human proteins cleaved by the NS2B‐NS3 protease. In particular, autophagy‐related protein 16‐1 (ATG16L1) and eukaryotic translation initiation factor 4 gamma (eIF4G1) are dramatically depleted during Zika virus infection. ATG16L1 and eIF4G1 mediate type‐II interferon production and host‐cell translation, respectively, likely aiding immune system evasion and driving the Zika life cycle. Intriguingly, the NS2B co‐factor region from Zika virus protease is essential for recognition of host cell substrates. Replacing the NS2B region in another flavivirus protease enabled recognition of novel Zika‐specific substrates by hybrid proteases, suggesting that the co‐factor is the principal determinant in ZVP substrate selection.
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

Objective

Metabolic 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.

Methods

Bulk 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+).

Results

Compared 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+).

Conclusions

Our 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 Information

This 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 Information

This study is supported by NIH R01 grants
Binge drinking effects on markers of skeletal muscle protein synthesis and degradation
Authors: Reed Carter H.
Published: April 2020

Rates of binge drinking in the United States and around the world are increasing despite much data on the detrimental effects associated with this behavior. The deleterious effects of alcohol on brain, liver, and cardiac muscle have been well characterized, but there is still a gap in our understanding of the reduction in the net protein balance in skeletal muscle. Prior work has demonstrated dysregulation in muscle protein synthesis in response to chronic alcohol exposure; however, pathways involved in protein breakdown have been largely unstudied, and studies have been limited to work almost exclusively in males. The aim of the current study was to investigate the effects of binge drinking on markers of muscle protein synthesis and degradation, and to examine the role of exercise in this context. 36 female C57BL/6 mice (7‐weeks of age) were randomized to the following conditions: half of the mice were allowed access to a running wheel for 2.5 hours/day for 5 weeks (EX) and half remained sedentary (SED). During the final week, half of the mice from each condition underwent a binge drinking protocol, in which mice were allowed restricted alcohol access for 2 hours for 4 consecutive days and on the fifth day allowed to drink ad libitum for 4 hours. This experimental paradigm resulted in the following four groups: SED water, SED alcohol, EX water, and EX alcohol. Gastrocnemius gene expression of the muscle enriched ubiquitin ligases MuRF1 and MAFbx, was assessed via qPCR. Phosphorylation of p70S6K was examined by Western blot as an indicator of anabolic signaling. There were no differences in food intake or body weight among groups. MuRF1 was significantly decreased with exercise (p = 0.03), but unaffected by alcohol. There was a significant interaction between alcohol and exercise (p = 0.04), and a main effect of exercise (p < 0.001) on MuRF1. In sedentary mice, alcohol tended to increase MAFbx mRNA (p=0.07); whereas EX alcohol mice had reduced MAFbx compared to SED alcohol (p < 0.001). There was a tendency of exercise to increase phosphorylation of p70S6k (p = 0.065). Our results suggest this model of binge drinking may be useful for determining the effects of binge drinking on the skeletal muscle protein balance, and alcohol may promote expression of atrogenes. In addition, daily exercise could mitigate the dysregulation in skeletal muscle homeostasis caused by alcohol.
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 Information

NSF 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 Information

Nova Southeastern University President’s Faculty Research Development Grant, Cardiovascular Neuroscience Research Fund